U.S. patent application number 10/161791 was filed with the patent office on 2003-10-02 for nck sh3 binding peptides.
Invention is credited to Der, Channing J., Fowlkes, Dana M., Kay, Brian K., Quilliam, Lawrence A., Rider, James E., Sparks, Andrew B., Thorn, Judith M..
Application Number | 20030186863 10/161791 |
Document ID | / |
Family ID | 24413658 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186863 |
Kind Code |
A1 |
Sparks, Andrew B. ; et
al. |
October 2, 2003 |
Nck SH3 binding peptides
Abstract
Peptides having general and specific binding affinities for the
Src homology region 3 (SH3) domains of proteins are disclosed in
the present invention. In particular, SH3 binding peptides have
been isolated from phage-displayed random peptide libraries which
had been screened for isolates that bind to bacterial fusion
proteins comprising SH3 and glutathione S-transferase (GST).
Preferred peptides are disclosed which comprise a core 7-mer
sequence (preferably, a consensus motif) and two or more,
preferably at least six, additional amino acid residues flanking
the core sequence, for a total length of 9, preferably at least 13,
amino acid residues and no more than about 45 amino acid residues.
Such peptides manifest preferential binding affinities for certain
SH3 domains. The preferred peptides exhibit specific binding
affinities for the Src-family of proteins. In vitro and in vivo
results are presented which demonstrate the biochemical activity of
such peptides.
Inventors: |
Sparks, Andrew B.;
(Pikesville, MD) ; Kay, Brian K.; (Chapel Hill,
NC) ; Thorn, Judith M.; (Carrboro, NC) ;
Quilliam, Lawrence A.; (Indianapolis, IN) ; Der,
Channing J.; (Chapel Hill, NC) ; Fowlkes, Dana
M.; (Chapel Hill, NC) ; Rider, James E.;
(Carrboro, NC) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
345 Park Avenue
New York
NY
10154-0053
US
|
Family ID: |
24413658 |
Appl. No.: |
10/161791 |
Filed: |
May 31, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10161791 |
May 31, 2002 |
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09500124 |
Feb 8, 2000 |
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6432920 |
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09500124 |
Feb 8, 2000 |
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08602999 |
Feb 16, 1996 |
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6184205 |
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08602999 |
Feb 16, 1996 |
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08483555 |
Jun 7, 1995 |
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08483555 |
Jun 7, 1995 |
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08278865 |
Jul 22, 1994 |
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6303574 |
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Current U.S.
Class: |
514/44R ;
514/21.5; 530/324; 530/325; 530/326; 530/327 |
Current CPC
Class: |
G01N 33/573 20130101;
G01N 33/68 20130101; A61K 47/62 20170801; G01N 33/5041 20130101;
C40B 30/04 20130101; C07K 14/435 20130101; G01N 33/5011 20130101;
G01N 33/6845 20130101; C07K 2319/00 20130101; C07K 7/06 20130101;
G01N 33/5008 20130101; C07K 14/001 20130101; A61P 35/00 20180101;
C07K 7/08 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ; 514/13;
514/14; 514/15; 530/324; 530/325; 530/326; 530/327 |
International
Class: |
A61K 038/16; A61K
038/10; A61K 038/08; C07K 007/08; C07K 007/06 |
Claims
What is claimed is:
1. A peptide having at least nine and up to forty-five amino acid
residues, including an amino acid sequence of the formula,
R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the
peptide, in which each number represents an amino acid residue,
such that 2 represents any amino acid residue except cysteine, 5
and 6 each represents a hydrophobic amino acid residue, 8
represents any amino acid residue except cysteine, and 9 represents
a hydrophilic amino acid residue except cysteine, each letter being
the standard one-letter symbol for the corresponding amino acid,
said peptide exhibiting a binding affinity for the SH3 domain of
Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID
NO:11).
2. The peptide of claim 1 in which 2 is a P, R, A, L, Q, E or
S.
3. The peptide of claim 1 in which 5 is a P, M, I or L.
4. The peptide of claim 1 in which 6 is a P, L, I or V.
5. The peptide of claim 1 in which 8 is a T, R, P, I, N, E, V, S,
A, G or L.
6. The peptide of claim 1 in which 9 is a T, R, S, H or D.
7. The peptide of claim 1 which further comprises a
C-terminal-flanking amino acid sequence of the formula 10, 10-11,
10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID
NO:13), in which each number represents any amino acid residue
except cysteine, such that 10 is bound to 9 by a peptide bond.
8. The peptide of claim 7 in which 10 is T, R, L, S, D, P, A or
N.
9. The peptide of claim 7 in which 11 is R, P, A, Q, S or T.
10. The peptide of claim 7 in which 12 is P, S, R or T.
11. The peptide of claim 7 in which 13 is P, S, R, F, H or T.
12. The peptide of claim 7 in which 14 is S, R, G or T.
13. The peptide of claim 1 which further comprises an
N-terminal-flanking amino acid sequence of the formula 1', 2'-1',
3'-2'-1' or 4'-3'-2'-1' (SEQ ID NO:14) in which each number
represents any amino acid residue except cysteine, such that 1' is
bound to R by a peptide bond.
14. The peptide of claim 13 in which 1' is T. P, S, N, F, W, K, H,
Q or G.
15. The peptide of claim 13 in which 2' is S, T, G, P, R, Q, L, A
or H.
16. The peptide of claim 13 in which 3' is R, S, P, G, A, V, Y or
L.
17. The peptide of claim 13 in which 4' is R, S, V, T, G, L or
F.
18. A peptide having at least thirteen and up to forty-five amino
acid residues, including an amino acid sequence of the formula,
3'-2'-1'-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere
along the peptide, in which each number represents an amino acid
residue, such that 3', 2', 1', 2, 8, and 10 each represents any
amino acid residue except cysteine, 5 and 6 each represents a
hydrophobic amino acid residue, and 9 represents a hydrophilic
amino acid residue except cysteine, each letter being the standard
one-letter symbol for the corresponding amino acid, said peptide
exhibiting a binding affinity for the SH3 domain of Src.
19. The peptide of claim 18 in which 5 is a P or M.
20. The peptide of claim 18 in which 1' is T, P, S or N.
21. The peptide of claim 18 in which 2' is S or T.
22. The peptide of claim 18 in which 3' is R or S.
23. The peptide of claim 18 in which 10 is T or R.
24. The peptide of claim 1 the binding affinity of which is at
least three-fold greater than that exhibited by the peptide RPLPPLP
for the SH3 domain of Src.
25. The peptide of claim 18 the binding affinity of which is at
least three-fold greater than that exhibited by the peptide RPLPPLP
(SEQ ID NO:9) for the SH3 domain of Src.
26. The peptide of claim 1 the binding affinity of which is at
least four-fold greater than that exhibited by the peptide RPLPPLP
(SEQ ID NO:9) for the SH3 domain of Src.
27. The peptide of claim 18 the binding affinity of which is at
least four-fold greater than that exhibited by the peptide RPLPPLP
(SEQ ID NO:9) for the SH3 domain of Src.
28. The peptide of claim 1 which further exhibits a general binding
affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras GAP,
Nck, p85 PI-3'Kinase, and proteins related thereto.
29. The peptide of claim 1 which exhibits a selective binding
affinity for the SH3 domain of Src and Src-related proteins.
30. The peptide of claim 18 which further exhibits a general
binding affinity for the SH3 domain of Abl, Grb2, PLC- , PLC- , Ras
GAP, Nck, p85 PI-3'Kinase, and proteins related thereto.
31. The peptide of claim 18 which exhibits a selective binding
affinity for the SH3 domain of Src and Src-related proteins.
32. A peptide having the amino acid sequence RSTPRPLPMLPTTR (SEQ ID
NO:62).
33. A peptide having the amino acid sequence RSTPRPLPPLPTTR (SEQ ID
NO:67).
34. A peptide having the amino acid sequence GILAPPVPPRNTR (SEQ ID
NO:63).
35. A peptide having the amino acid sequence VLKRPLPIPPVTR (SEQ ID
NO:64).
36. A peptide having the amino acid sequence GPHRRLPPTPATR (SEQ ID
NO:65).
37. A peptide having the amino acid sequence ANPSPATRPLPTR (SEQ ID
NO:66).
38. A peptide having an amino acid sequence selected from the group
consisting of RSTRPLPILPRTT, STPRPLPMLPTTR, STNRPLPMIPTTR,
RSTRPLPSLPITT, STSRPLPSLPTTR, RSTRSLPPLPPTT, RSTRQLPIPPTTT,
STPRPLPLIPTTP, RSTRPLPPTPLTT, and RSTRPQPPPPITT (SEQ ID
NOS:85-94).
39. A peptide having the amino acid sequence selected from the
group consisting of VLKRPLPIPPVTR (SEQ ID NO:64), YSTRPVPPITRPS
(SEQ ID NO:76), SHKSRLPPLPTRP (SEQ ID NO:77), GPHRRLPPTPATR (SEQ ID
NO:65), PATRPLPTRPSRT (SEQ ID NO:81), and SGGILAPPVPPRN (SEQ ID
NO:84).
40. A peptide having the amino acid sequence selected from the
group consisting of PPNSPLPPLPTHL (SEQ ID NO:72), TGRGPLPPLPNDS
(SEQ ID NO:74), YRFRALPSPPSAS, LAQRQLPPTPGRD ALQRRLPRTPPPA (SEQ ID
NOS:78-80), YSTRPLPSRPSRT, and XPGRILLLPSEPR (SEQ ID
NOS:82-83).
41. A construct comprising a nucleic acid encoding a peptide of
claim 1 or its complement.
42. The construct of claim 41 which is a DNA polynucleotide.
43. The construct of claim 41 which is a RNA polynucleotide.
44. A construct comprising a nucleic acid encoding a peptide of
claim 18 or its complement.
45. The construct of claim 44 which is a DNA polynucleotide.
46. The construct of claim 44 which is a RNA polynucleotide.
47. The construct of claim 41 which is a transforming vector.
48. The construct of claim 44 which is a transforming vector.
49. A host cell transformed with the vector of claim 47.
50. A host cell transformed with the vector of claim 48.
51. A conjugate comprising a peptide of claim 1 and a second
molecule.
52. The conjugate of claim 51 in which said second molecule is
selected from the group consisting of an amino acid, a peptide, a
protein, a nucleic acid, a nucleoside, a glycosidic residue, a
label, a drug or a small molecule.
53. A diagnostic kit for the detection of SH3 domains comprising an
SH3 domain-binding peptide and a detectable label conjugated to
said peptide directly, indirectly or by complexation, said peptide
comprising: (i) a core sequence motif of the formula
RXLP.phi..phi.P (SEQ ID NO:71), wherein X represents any amino acid
except cysteine and .phi. represents a hydrophobic amino acid
residue, each letter representing the standard one-letter
designation for the corresponding amino acid residue; and (ii) two
or more additional amino acid residues flanking said core sequence
at its C-terminal end, N-terminal end or both.
54. A drug delivery system comprising an SH3 domain-binding peptide
and a drug conjugated to said peptide directly, indirectly or by
complexation, said peptide comprising: (i) a core sequence motif of
the formula RXLP.phi..phi.P (SEQ ID NO:71), wherein X represents
any amino acid except cysteine and .phi. represents a hydrophobic
amino acid residue, each letter representing the standard
one-letter designation for the corresponding amino acid residue;
and (ii) two or more additional amino acid residues flanking said
core sequence at its C-terminal end, N-terminal end or both.
55. The drug delivery system of claim 54 which may be administered
parenterally, orally, enterally, topically or by inhalation.
56. The drug delivery system of claim 54 which may be administered
intranasally, opthalmically or intravaginally.
57. The drug delivery system of claim 54 which is in the form of a
solid, gel, liquid or aerosol.
58. A method of modulating the activity of Src or Src-related
proteins comprising administering a composition comprising an
effective amount of a peptide of claim 1 and a carrier.
59. The method of claim 58 which inhibits the activity of Src or
Src-related proteins.
60. The method of claim 58 which activates Src or Src-related
proteins.
61. A method of identifying a peptide having a region that binds to
an SH3 domain comprising: (a) providing an immobilized target
protein comprising an SH3 domain; (b) incubating said immobilized
target protein with an aliquot taken from a random peptide library;
(c) washing unbound peptide from said immobilized target protein;
(d) recovering the peptide bound to said immobilized target
protein; and (e) determining the primary sequence of the SH3
domain-binding peptide.
62. The method of claim 61 in which said library is a displayed
random peptide library.
63. The method of claim 62 in which said library is a
phage-displayed random peptide library.
64. The method of claim 62 in which said library is a
phagemid-displayed random peptide library.
65. The method of claim 61 in which step (c) includes washing
unbound phage from said immobilized target protein; step (d)
includes recovering the phage bound to said immobilized target
protein; and step (e) includes determining the relevant nucleotide
sequence of said binding phage nucleic acid, from which the primary
sequence corresponding to the SH3 domain-binding peptide is
deduced.
66. A method of identifying a peptide having a region that binds to
an SH3 domain comprising: (a) providing an immobilized target
protein comprising an SH3 domain; (b) incubating said immobilized
target protein with an aliquot taken from a phage-displayed random
peptide library, which library includes peptides having a random
sequence of 8 amino acid residues; (c) washing unbound phage from
said immobilized target protein; (d) recovering the phage bound to
said immobilized target protein; and (e) determining the relevant
nucleotide sequence of said binding phage nucleic acid and deducing
the primary sequence corresponding to the SH3 domain-binding
peptide.
67. The method of claim 66 which further comprises amplifying the
titer of the recovered phage.
68. The method of claim 66 which further comprises repeating the
steps of incubation, washing and recovery to provide SH3
domain-binding peptide-enriched phage.
69. A pharmaceutical composition comprising an SH3 domain-binding
peptide and a pharmaceutically acceptable carrier, said peptide
comprising: (i) a 9-mer sequence motif of the formula
RXLP.phi..phi.PX.psi. (SEQ ID NO:10), wherein X represents any
amino acid except cysteine, .phi. represents a hydrophobic amino
acid residue, and wherein .psi. is a hydrophilic amino acid residue
except cysteine, each letter representing the standard one-letter
designation for the corresponding amino acid residue; and,
optionally, (ii) additional amino acid residues flanking said 9-mer
sequence at its C-terminal end, N-terminal end or both, up to a
total of 45 amino acid residues, including said 9-mer sequence.
70. The composition of claim 69 in which at least one additional
amino acid flanks said 9-mer sequence.
71. The composition of claim 69 in which at least two additional
amino acids flank said 9-mer sequence.
72. The composition of claim 69 in which at least three additional
amino acids flank said 9-mer sequence.
73. A method of disrupting protein tyrosine kinase-mediated signal
transduction pathways comprising administering an effective amount
of a peptide of claim 1.
74. A method of regulating the processing, trafficking or
translation of RNA by administering an effective amount of a
peptide of claim 1.
75. A purified peptide that binds to the SH3 domain of Cortactin,
said peptide comprising the amino acid sequence ZPP.phi.PxKPxW (SEQ
ID NO:113), where Z represents K or R; .phi. represents a
hydrophobic amino acid; and x represents any amino acid.
76. A purified peptide that binds to the middle SH3 domain of Nck,
said peptide comprising the amino acid sequence
.phi.xxxxPxPP.phi.RZxSL (SEQ ID NO:127), where Z represents S or T;
.phi. represents a hydrophobic amino acid; and x represents any
amino acid.
77. A purified peptide that binds to the SH3 domain of Abl, said
peptide comprising the amino acid sequence PPxWxPPP.phi.P (SEQ ID
NO:141), where .phi. represents a hydrophobic amino acid; and x
represents any amino acid.
78. A purified peptide that binds to the SH3 domain of Src, said
peptide comprising the amino acid sequence LXXRPLPX.psi.P (SEQ ID
NO:165), where .psi. represents an aliphatic amino acid; and X
represents any amino acid.
79. A purified peptide that binds to the SH3 domain of Cortactin,
said peptide comprising the amino acid sequence +PP.psi.PXKPXWL
(SEQ ID NO:166), where +represents a basic amino acid; .psi.
represents an aliphatic amino acid; and X represents any amino
acid.
80. A purified peptide that binds to the SH3 domain of Abl, said
peptide comprising the amino acid sequence PPX.theta.XPPP.psi.P
(SEQ ID NO:173) , where .theta. represents an aromatic amino acid;
.psi. represents an aliphatic amino acid; and X represents any
amino acid.
81. A purified peptide that binds to the SH3 domain of PLC said
peptide comprising the amino acid sequence PPVPPRPXXTL (SEQ ID
NO:175), where X represents any amino acid.
82. A purified peptide that binds to the SH3 domain of p53bp2, said
peptide comprising the amino acid sequence RPX.psi.P.psi.R+SXP (SEQ
ID NO:196), where + represents a basic amino acid; .psi. represents
an aliphatic amino acid; and X represents any amino acid.
83. A purified peptide that binds to the N terminal SH3 domain of
Crk, said peptide comprising the amino acid sequence
.psi.P.psi.P.psi.LP.psi.K (SEQ ID NO:210), where .psi. represents
an aliphatic amino acid; and X represents any amino acid.
84. A purified peptide that binds to the SH3 domain of Yes, said
peptide comprising the amino acid sequence .psi.XXRPLPXLP (SEQ ID
NO:222), where .psi. represents an aliphatic amino acid; and X
represents any amino acid.
85. A purified peptide that binds to the N terminal SH3 domain of
Grb2, said peptide comprising an amino acid sequence selected from
the group consisting of: +.theta.DXPLPXLP (SEQ ID NO:223),
Y.theta.X+PLPXLP (SEQ ID NO:238), and 6DPLPXLP (SEQ ID NO:243),
where .theta. represent an aromatic amino acid; +represents a basic
amino acid; .psi. represents an aliphatic amino acid; and X
represents any amino acid.
86. A purified peptide that binds to the SH3 domain of Cortactin,
said peptide comprising an amino acid sequence selected from the
group consisting of:
18 LTPQSKPPLPPKPSAV; (a portion of SEQ ID NO:112) SSHNSRPPLPEKPSWL;
(a portion of SEQ ID NO:111) PVKPPLPAKPWWLPPL; (SEQ ID NO:167)
TERPPLPQRPDWLSYS; (a portion of SEQ ID NO:109) LGEFSKPPIPQKPTWM; (a
portion of SEQ ID NO:108) YPQFRPPVPPKPSLMQ; (SEQ ID NO:168)
VTRPPLPPKPGHMADF; (SEQ ID NO:169) VSLGLKPPVPPKPMQL; (SEQ ID NO:170)
LLGPPVPPKPQTLFSF; (a portion of SEQ ID NO:107) YKPEVPARPIWLSEL;
(SEQ ID NO:171) GAGAARPLVPKKPLFL; and (SEQ ID NO:172)
SREPDWLCPNCPLLLRSDSR. (SEQ ID NO:110)
87. A purified peptide that binds to the middle SH3 domain of Nck,
said peptide comprising an amino acid sequence selected from the
group consisting of:
19 SSLGVGWKPLPPMRTASLSR; (SEQ ID NO:114) SSVGFADRPRPPLRVESLSR; (SEQ
ID NO:115) SSAGILRPPEKPXRSFSLSR; (SEQ ID NO:116)
SSPYTGDVPIPPLRGASLSR; (SEQ ID NO:117) SSLMGSWPPVPPLRSDSLSR; (SEQ ID
NO:118) SSIGEDTPPSPPTRRASLSR; (SEQ ID NO:119) SRSLSEVSPKPPIRSVSLSR;
(SEQ ID NO:120) SSVSEGYSPPLPPRSTSLSR; (SEQ ID NO:121)
SSSFTLAAPTPPTRSLSLSR; (SEQ ID NO:122) SSPPYELPPRPPNRTVSLSR; (SEQ ID
NO:123) SRVVDGLAPPPPVRLSSLSR; (SEQ ID NO:124) SSLGYSGAPVPPHRxSSLSR;
and (SEQ ID NO:125) SSISDYSRPPPPVRTLSLSR. (SEQ ID NO:126)
88. A purified peptide that binds to the SH3 domain of Abl, said
peptide comprising an amino acid sequence selected from the group
consisting of:
20 PPWWAPPPIPNSPQVL; (SEQ ID NO:174); PPKFSPPPPPYWQLHA; (a portion
of SEQ ID NO:132) PPHWAPPAPPAMSPPI; (a portion of SEQ ID NO:130)
PPTWTPPKPPGWGVVF; (a portion of SEQ ID NO:137) PPSFAPPAAPPRHSFG; (a
portion of SEQ ID NO:133) PTYPPPPPPPDTAKGA; (a portion of SEQ ID
NO:135) GPRWSPPPVPLPTSLD; (a portion of SEQ ID NO:128)
APTWSPPALPNVAKYK; (a portion of SEQ ID NO:138) PPDYAAPAIPSSLWVD; (a
portion of SEQ ID NO:129) IKGPRFPVPPVPLNGV; (a portion of SEQ ID
NO:139) PPAWSPPHRPVAFGST; (a portion of SEQ ID NO:140)
APKKPAPPVPMMAHVM; (a portion of SEQ ID NO:134)
SSDRCWECPPWPAGGQRGSR; (SEQ ID NO:131) and SSPPXXXPPPIPNSPQVLSR.
(SEQ ID NO:136)
89. A purified peptide that binds to the SH3 domain of PLC said
peptide comprising an amino acid sequence selected from the group
consisting of:
21 MPPPVPPRPPGTLQVA; (SEQ ID NO:176) LSYSPPPVPPRPDSTL; (SEQ ID
NO:177) VLAPPVPPRPGNTFFT; (SEQ ID NO:178) YRPPVAPRPPSSLSVD; (SEQ ID
NO:179) LQCPDCPRVPPRPIPI; (SEQ ID NO:180) VPPLVAPRPPSTLNSL; (a
portion of SEQ ID NO:143) LTPPPFPKRPRWTLPE; (SEQ ID NO:181)
YWPHRPPLAPPQTTLG; (SEQ ID NO:182) SSMKVHNFPLPPLPSYETSR; (SEQ ID
NO:142) SSLYWQHGPDPPVGAPQLSR; (SEQ ID NO:144) and
SSHPLNSWPGGPFRHNLSSR. (SEQ ID NO:145)
90. A purified peptide that binds to the SH3 domain of Src, said
peptide comprising an amino acid sequence selected from the group
consisting of:
22 LASRPLPLLPNSAPGQ; (a portion of SEQ ID NO:155) LTGRPLPALPPPFSDF;
(a portion of SEQ ID NO:152) PAYRPLPRLPDLSVIY; (a portion of SEQ ID
NO:150) RALRVRPLPPVPGTSL; (a portion of SEQ ID NO:146)
DAPGSLPFRPLPPVPT; (a portion of SEQ ID NO:148) LKWRALPPLPETDTPY; (a
portion of SEQ ID NO:157) ISQRALPPLPLMSDPA; (a portion of SEQ ID
NO:149) LTSRPLPDIPVRPSKS; (a portion of SEQ ID NO:156)
NTNRPLPPTPDGLDVR; (a portion of SEQ ID NO:158) MKDRVLPPIPTVESAV; (a
portion of SEQ ID NO:153) LQSRPLPLPPQSSYPI; (a portion of SEQ ID
NO:159) FINRRLPALPPDNSLL; (a portion of SEQ ID NO:151)
FRALPLPPTPDNPFAG; and (a portion of SEQ ID NO:147)
LYSAIAPDPPPRNSSS. (a portion of SEQ ID NO:154)
91. A purified peptide that binds to the SH3 domain of p53bp2, said
peptide comprising an amino acid sequence selected from the group
consisting of:
23 YDASSAPQRPPLPVRKSRP; (SEQ ID NO:183) EYVNASPERPPIPGRKSRP; (SEQ
ID NO:184) WNGIAIPGRPEIPPRASRP; (SEQ ID NO:185)
SMIFIYPERPSPPPRFSRP; (SEQ ID NO:186) GVEEWNPERPQIPLRLSRP; (SEQ ID
NO:187) WVVDSRPDIPLRRSLP; (SEQ ID NO:188) VVPLGRPEIPLRKSLP; (SEQ ID
NO:189) GGTVGRPPIPERKSVD; (SEQ ID NO:190) YSHAGRPEVPPRQSKP; (SEQ ID
NO:191) FSAAARPDIPSRASTP; (SEQ ID NO:192) LYIPKRPEVPPRRHEA; (SEQ ID
NO:193) NNISARPPLPSRQNPP; and (SEQ ID NO:194) MAGTPRPAVPQRMNPP.
(SEQ ID NO:195)
92. A purified peptide that binds to the N terminal SH3 domain of
Crk, said peptide comprising an amino acid sequence selected from
the group consisting of:
24 GQPAGDPDPPPLPAKF; (SEQ ID NO:197) FEQTGVPLLPPKSFKY; (SEQ ID
NO:198) IFGDPPPPIPMKGRSL; (SEQ ID NO:199) SNQGSIPVLPIKRVQY; (SEQ ID
NO:200) NYVNALPPGPPLPAKN; (SEQ ID NO:201) SSDPERPVLPPKLWSV; (SEQ ID
NO:202) HFGPSKPPLPIKTRIT; (SEQ ID NO:203) DWKVPEPPVPKLPLKQ; (SEQ ID
NO:204) ATSEGLPILPSKVGSY; (SEQ ID NO:205) NANVSAPRAPAFPVKT; (SEQ ID
NO:206) EMVLGPPVPPKRGTVV; (SEQ ID NO:207) AGSRHPPTLPPKESGG; and
(SEQ ID NO:208) SVAADPPRLPAKSRPQ. (SEQ ID NO:209)
93. A purified peptide that binds to the SH3 domain of Yes, said
peptide comprising an amino acid sequence selected from the group
consisting of:
25 ITMRPLPALPGHGQIH; (SEQ ID NO:211) LPRRPLPDLPMAAGKG; (SEQ ID
NO:212) LGSRPLPPTPRQWPEV; (SEQ ID NO:213) STIRPLPAIPRDTLLT; (SEQ ID
NO:214) RSGRPLPPIPEVGHNV; (SEQ ID NO:215) IGSRPLPWTPDDLGSA; (SEQ ID
NO:216) LAQRELPGLPAGAGVS; (SEQ ID NO:217) IPGRALPELPPQRALP; (SEQ ID
NO:218) FVGRELPPTPRTVIPW; (SEQ ID NO:219) DPRSALPALPLTPLQT; and
(SEQ ID NO:220) SPHDVLPALPDSHSKS. (SEQ ID NO:221)
94. A purified peptide that binds to the N terminal SH3 domain of
Grb2, said peptide comprising an amino acid sequence selected from
the group consisting of:
26 KWDSLLPALPPAFTVE; (SEQ ID NO:224) RWDQVLPELPTSKGQI; (SEQ ID
NO:225) RFDFPLPTHPNLQKAH; (SEQ ID NO:226) RLDSPLPALPPTVMQN; (SEQ ID
NO:227) RWGAPLPPLPEYSWST; (SEQ ID NO:228) YWDMPLPRLPGEEPSL; (SEQ ID
NO:229) RFDYNLPDVPLSLGTA; (SEQ ID NO:230) TKKPNAPLPPLPAYMG; (SEQ ID
NO:231) KWDLDLPPEPMSLGNY; (SEQ ID NO:232) YYQRPLPPLPLSHFES; (SEQ ID
NO:234) YYRKPLPNLPRGQTDD; (SEQ ID NO:235) YFDKPLPESPGALMSL; (SEQ ID
NO:236) YFSRALPGLPERQEAH; (SEQ ID NO:237) SLWDPLPPIPQSKTSV; (SEQ ID
NO:239) SYYDPLPKLPDPGDLG; (SEQ ID NO:240) KLYYPLPPVPFKDTKH; and
(SEQ ID NO:241) DPYDALPETPSMKASQ. (SEQ ID NO:242)
95. A purified peptide having an amino acid sequence selected from
the group consisting of: SEQ ID NOs: 250-252, 254, 256-259, 261,
262, 264-266, 269-272, 275, 280, 281, 286-289, 291, 294, and
295.
96. A purified peptide having an amino acid sequence selected from
the group consisting of: SEQ ID NOs: 296-453.
97. A method of identifying an inhibitor of the binding between a
first molecule comprising an SH3 domain and a second molecule that
binds to the SH3 domain comprising incubating one or more compounds
from which it is desired to select such an inhibitor with the first
molecule and the second molecule under conditions conducive to
binding and detecting the one or more compounds that inhibit
binding of the first molecule to the second molecule
98. The method of claim 97 where the second molecule is obtained
by: (i) screening a peptide library with the SH3 domain to obtain
peptides that bind the SH3 domain; (ii) determining a consensus
sequence for the peptides obtained in step (i); (iii) producing a
peptide comprising the consensus sequence; wherein the second
molecule comprises the peptide comprising the consensus
sequence.
99. The method of claim 97 where the second molecule is obtained
by: (i) screening a peptide library with the SH3 domain to obtain
peptides that bind the SH3 domain; (ii) determining a consensus
sequence for the peptides obtained in step (i); (iii) searching a
database to identify amino acid sequences that resemble the
consensus sequence of step (ii); (iv) producing a peptide
comprising an amino acid sequence identified in step (iii); wherein
the second molecule comprises the peptide comprising an amino acid
sequence identified in step (iii).
100. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Cortactin, said peptide comprising
the amino acid sequence ZPP.phi.PxKPxW (SEQ ID NO:113), where Z
represents K or R; + represents a hydrophobic amino acid; and x
represents any amino acid.
101. The method of claim 97 where the second molecule is a peptide
that binds to the middle SH3 domain of Nck, said peptide comprising
the amino acid sequence .phi.xxxxxPxPP.phi.RZxSL (SEQ ID NO:127),
where Z represents S or T; .phi. represents a hydrophobic amino
acid; and x represents any amino acid.
102. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Abl, said peptide comprising the
amino acid sequence PPxWxPPP.phi.P (SEQ ID NO:141), where .phi.
represents a hydrophobic amino acid; and x represents any amino
acid.
103. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Src, said peptide comprising the
amino acid sequence LXXRPLPX.psi.P (SEQ ID NO:165), where .psi.
represents an aliphatic amino acid; and X represents any amino
acid.
104. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Cortactin, said peptide comprising
the amino acid sequence +PP.psi.PXKPXWL (SEQ ID NO:166), where +
represents a basic amino acid; .psi. represents an aliphatic amino
acid; and X represents any amino acid.
105. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Abl, said peptide comprising the
amino acid sequence PPX.theta.XPPP.psi.P (SEQ ID NO:173), where
.theta.represents an aromatic amino acid; .psi. represents an
aliphatic amino acid; and X represents any amino acid.
106. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of PLC.gamma., said peptide comprising
the amino acid sequence PPVPPRPXXTL (SEQ ID NO:175), where X
represents any amino acid.
107. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of p53bp2, said peptide comprising the
amino acid sequence RPX.psi.P.psi.R+SXP (SEQ ID NO:196), where +
represents a basic amino acid; .psi. represents an aliphatic amino
acid; and X represents any amino acid.
108. The method of claim 97 where the second molecule is a peptide
that binds to the N terminal SH3 domain of Crk, said peptide
comprising the amino acid sequence .psi.P.psi.LP.psi.K (SEQ ID
NO:210), where .psi. represents an aliphatic amino acid; and X
represents any amino acid.
109. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Yes, said peptide comprising the
amino acid sequence .psi.XXRPLPXLP (SEQ ID NO:222), where .psi.
represents an aliphatic amino acid; and X represents any amino
acid.
110. The method of claim 97 where the second molecule is a peptide
that binds to the N terminal SH3 domain of Grb2, said peptide
comprising an amino acid sequence selected from the group
consisting of: +.theta.DXPLPXLP (SEQ ID NO:223), Y.theta.X+PLPXLP
(SEQ ID NO:238), and .theta.DPLPXLP (SEQ ID NO:243), where .theta.
represent an aromatic amino acid; +represents a basic amino acid;
.psi. represents an aliphatic amino acid; and X represents any
amino acid.
111. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Cortactin, said peptide comprising
an amino acid sequence selected from the group consisting of:
27 LTPQSKPPLPPKPSAV; (a portion of SEQ ID NO:112) SSHNSRPPLPEKPSWL;
(a portion of SEQ ID NO:111) PVKPPLPAKPWWLPPL; (SEQ ID NO:167)
TERPPLPQRPDWLSYS; (a portion of SEQ ID NO:109) LGEFSKPPIPQKPTWM; (a
portion of SEQ ID NO:108) YPQFRPPVPPKPSLMQ; (SEQ ID NO:168)
VTRPPLPPKPGHMADF; (SEQ ID NO:169) VSLGLKPPVPPKPMQL; (SEQ ID NO:170)
LLGPPVPPKPQTLFSF; (a portion of SEQ ID NO:107) YKPEVPARPIWLSEL;
(SEQ ID NO:171) GAGAARPLVPKKPLFL; and (SEQ ID NO:172)
SREPDWLCPNCPLLLRSDSR. (SEQ ID NO:110)
112. The method of claim 97 where the second molecule is a peptide
that binds to the middle SH3 domain of Nck, said peptide comprising
an amino acid sequence selected from the group consisting of:
28 SSLGVGWKPLPPMRTASLSR; (SEQ ID NO:114) SSVGFADRPRPPLRVESLSR; (SEQ
ID NO:115) SSAGILRPPEKPXRSFSLSR; (SEQ ID NO:116)
SSPYTGDVPIPPLRGASLSR; (SEQ ID NO:117) SSLMGSWPPVPPLRSDSLSR; (SEQ ID
NO:118) SSIGEDTPPSPPTRRASLSR; (SEQ ID NO:119) SRSLSEVSPKPPIRSVSLSR;
(SEQ ID NO:120) SSVSEGYSPPLPPRSTSLSR; (SEQ ID NO:121)
SSSFTLAAPTPPTRSLSLSR; (SEQ ID NO:122) SRVVDGLAPPPPVRLSSLSR; (SEQ ID
NO:123) SRVVDGLAPPPPVRLSSLSR; (SEQ ID NO:124) SSLGYSGAPVPPHRxSSLSR;
and (SEQ ID NO:125) SSISDYSRPPPPVRTLSLSR. (SEQ ID NO:126)
113. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Abl, said peptide comprising an
amino acid sequence selected from the group consisting of:
29 PPWWAPPPIPNSPQVL; (SEQ ID NO:174) PPKFSPPPPPYWQLHA; (a portion
of SEQ ID NO:132) PPHWAPPAPPAMSPPI; (a portion of SEQ ID NO:130)
PPTWTPPKPPGWGVVF; (a portion of SEQ ID NO:137) PPSFAPPAAPPRHSFG; (a
portion of SEQ ID NO:133) PTYPPPPPPDTAKGA; (a portion of SEQ ID
NO:135) GPRWSPPPVPLPTSLD; (a portion of SEQ ID NO:128)
APTWSPPALPNVAKYK; (a portion of SEQ ID NO:138) PPDYAAPAIPSSLWVD; (a
portion of SEQ ID NO:129) IKGPRFPVPPVPLNGV; (a portion of SEQ ID
NO:139) PPAWSPPHRPVAFGST; (a portion of SEQ ID NO:140)
APKKPAPPVPMMAHVM; (a portion of SEQ ID NO:134)
SSDRCWECPPWPAGGQRGSR; (SEQ ID NO:131) and SSPPXXXPPPIPNSPQVLSR.
(SEQ ID NO:136)
114. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of PLC.gamma., said peptide comprising
an amino acid sequence selected from the group consisting of:
30 MPPPVPPRPPGTLQVA; (SEQ ID NO:176) LSYSPPPVPPRPDSTL; (SEQ ID
NO:177) VLAPPVPPRPGNTFFT; (SEQ ID NO:178) YRPPVAPRPPSSLSVD; (SEQ ID
NO:179) LQCPDCPRVPPRPIPI; (SEQ ID NO:180) VPPLVAPRPPSTLNSL; (a
portion of SEQ ID NO:143) LTPPPFPKRPRWTLPE; (SEQ ID NO:181)
YWPHRPPLAPPQTTLG; (SEQ ID NO:182) SSMKVHNFPLPPLPSYETSR; (SEQ ID
NO:142) SSLYWQHGPDPPVGAPQLSR; (SEQ ID NO:144) and
SSHPLNSWPGGPFRHNLSSR. (SEQ ID NO:145)
115. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Src, said peptide comprising an
amino acid sequence selected from the group consisting of:
31 LASRPLPLLPNSAPGQ; (a portion of SEQ ID NO:155) LTGRPLPALPPPFSDF;
(a portion of SEQ ID NO:152) PAYRPLPRLPDLSVIY; (a portion of SEQ ID
NO:150) RALRVRPLPPVPGTSL; (a portion of SEQ ID NO:146)
DAPGSLPFRPLPPVPT; (a portion of SEQ ID NO:148) LKWRALPPLPETDTPY; (a
portion of SEQ ID NO:157) ISQRALPPLPLMSDPA; (a portion of SEQ ID
NO:149) LTSRPLPDIPVRPSKS; (a portion of SEQ ID NO:156)
NTNRPLPPTPDGLDVR; (a portion of SEQ ID NO:158) MKDRVLPPIPTVESAV; (a
portion of SEQ ID NO:153) LQSRPLPLPPQSSYPI; (a portion of SEQ ID
NO:159) FINRRLPALPPDNSLL; (a portion of SEQ ID NO:151)
FRALPLPPTPDNPFAG; and (a portion of SEQ ID NO:147)
LYSAIAPDPPPRNSSS. (a portion of SEQ ID NO:154)
116. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of p53bp2, said peptide comprising an
amino acid sequence selected from the group consisting of:
32 YDASSAPQRPPLPVRKSRP; (SEQ ID NO:183) EYVNASPERPPIPGRKSRP; (SEQ
ID NO:184) WNGIAIPGRPEIPPRASRP; (SEQ ID NO:185)
SMIFIYPERPSPPPRFSRP; (SEQ ID NO:186) GVEEWNPERPQIPLRLSRP; (SEQ ID
NO:187) WVVDSRPDIPLRRSLP; (SEQ ID NO:188) VVPLGRPEIPLRKSLP; (SEQ ID
NO:189) GGTVGRPPIPERKSVD; (SEQ ID NO:190) YSHAGRPEVPPRQSKP; (SEQ ID
NO:191) FSAAARPDIPSRASTP; (SEQ ID NO:192) LYIPKRPEVPPRRHEA; (SEQ ID
NO:193) NNISARPPLPSRQNPP; and (SEQ ID NO:194) MAGTPRPAVPQRMNPP.
(SEQ ID NO:195)
117. The method of claim 97 where the second molecule is a peptide
that binds to the N terminal SH3 domain of Crk, said peptide
comprising an amino acid sequence selected from the group
consisting of:
33 GQPAGDPDPPPLPAKF; (SEQ ID NO:197) FEQTGVPLLPPKSFKY; (SEQ ID
NO:198) IFGDPPPPIPMKGRSL; (SEQ ID NO:199) SNQGSIPVLPIKRVQY; (SEQ ID
NO:200) NYVNALPPGPPLPAKN; (SEQ ID NO:201) SSDPERPVLPPKLWSV; (SEQ ID
NO:202) HFGPSKPPLPIKTRIT; (SEQ ID NO:203) DWKVPEPPVPKLPLKQ; (SEQ ID
NO:204) ATSEGLPILPSKVGSY; (SEQ ID NO:205) NANVSAPRAPAFPVKT; (SEQ ID
NO:206) EMVLGPPVPPKRGTVV; (SEQ ID NO:207) AGSRHPPTLPPKESGG; and
(SEQ ID NO:208) SVAADPPRLPAKSRPQ. (SEQ ID NO:209)
118. The method of claim 97 where the second molecule is a peptide
that binds to the SH3 domain of Yes, said peptide comprising an
amino acid sequence selected from the group consisting of:
34 ITMRPLPALPGHGQIH; (SEQ ID NO:211) LPRRPLPDLPMAAGKG; (SEQ ID
NO:212) LGSRPLPPTPRQWPEV; (SEQ ID NO:213) STIRPLPAIPRDTLLT; (SEQ ID
NO:214) RSGRPLPPIPEVGHNV; (SEQ ID NO:215) IGSRPLPWTPDDLGSA; (SEQ ID
NO:216) LAQRELPGLPAGAGVS; (SEQ ID NO:217) IPGRALPELPPQRALP; (SEQ ID
NO:218) FVGRELPPTPRTVIPW; (SEQ ID NO:219) DPRSALPALPLTPLQT; and
(SEQ ID NO:220) SPHDVLPALPDSHSKS. (SEQ ID NO:221)
119. The method of claim 97 where the second molecule is a peptide
that binds to the N terminal SH3 domain of Grb2, said peptide
comprising an amino acid sequence selected from the group
consisting of:
35 KWDSLLPALPPAFTVE; (SEQ ID NO:224) RWDQVLPELPTSKGQI; (SEQ ID
NO:225) RFDFPLPTHPNLQKAH; (SEQ ID NO:226) RLDSPLPALPPTVMQN; (SEQ ID
NO:227) RWGAPLPPLPEYSWST; (SEQ ID NO:228) YWDMPLPRLPGEEPSL; (SEQ ID
NO:229) RFDYNLPDVPLSLGTA; (SEQ ID NO:230) TKKPNAPLPPLPAYMG; (SEQ ID
NO:231) KWDLDLPPEPMSLGNY; (SEQ ID NO:232) YYQRPLPPLPLSHFES; (SEQ ID
NO:234) YYRKPLPNLPRGQTDD; (SEQ ID NO:235) YFDKPLPESPGALMSL; (SEQ ID
NO:236) YFSRALPGLPERQEAH; (SEQ ID NO:237) SLWDPLPPIPQSKTSV; (SEQ ID
NO:239) SYYDPLPKLPDPGDLG; (SEQ ID NO:240) KLYYPLPPVPFKDTKH; and
(SEQ ID NO:241) DPYDALPETPSMKASQ. (SEQ ID NO:242)
120. The method of claim 97 where the second molecule is a peptide
having an amino acid sequence selected from the group consisting
of: SEQ ID NOs: 250-252, 254, 256-259, 261, 262, 264-266, 269-272,
275, 280, 281, 286-289, 291, 294, and 295.
121. The method of claim 97 where the second molecule is a peptide
having an amino acid sequence selected from the group consisting
of: SEQ ID NOs: 296-453.
122. A method of identifying a compound that affects the binding of
a molecule comprising an SH3 domain and a ligand of the SH3 domain,
the method comprising: (a) contacting the SH3 domain and the ligand
under conditions conducive to binding in the presence of a
candidate compound and measuring the amount of binding between the
SH3 domain and the ligand; (b) comparing the amount of binding in
step (a) with the amount of binding known or determined to occur
between the molecule and the ligand in the absence of the candidate
compound, where a difference in the amount of binding between step
(a) and the amount of binding known or determined to occur between
the molecule and the ligand in the absence of the candidate
compound indicates that the candidate compound is a compound that
affects the binding of the molecule comprising an SH3 domain and
the ligand.
123. A kit comprising, in one or more containers: (a) a purified
first molecule comprising an SH3 domain; (b) a purified second
molecule that binds to the SH3 domain.
124. The kit of claim 123 wherein said second molecule comprises a
peptide having an amino acid sequence selected from the group
consisting of: SEQ ID NOs:107- 112, 114-126, 128-140, 142-159, 167,
168-172, 174, 176-195, 197-209, 211-221, 224-232, 234-237, 239-242,
250-252, 254, 256-259, 261, 262, 264-266, 269-272, 275, 280, 281,
286-289, 291, 294-453.
125. A purified peptide that binds to the SH3 domain of Src, said
peptide comprising the amino acid sequence
LX.sub.1X.sub.2RPLPX.sub.3.psi.PX.sub.- 4X.sub.5 (SEQ ID NO:454)
where .psi. represents aliphatic amino acid residues and X.sub.1,
X.sub.2, X.sub.3, X.sub.4, and X.sub.5 represent any amino acid;
except that if X.sub.3=P, .psi.=L, X.sub.4=P, and X.sub.5=P, then:
where X.sub.1=F, then X.sub.2 is not H or R; or where X.sub.1=S,
then X.sub.2 is not R, H, A, N, T, G, V, M, or W; or where
X.sub.1=C, then X.sub.2 is not S or G; or where X.sub.1=R, then
X.sub.2 is not T or F; or where X.sub.1=A, then X.sub.2 is not R,
Q, N, S, or L; or where X.sub.1=Q, then X.sub.2 is not M; or where
X.sub.1=L, then X.sub.2 is not R; or where X.sub.1=I, then X.sub.2
is not A; or where X.sub.1=P, then X.sub.2 is not P, W, or R; or
where X.sub.1=G, then X.sub.2 is not S or R; or where X.sub.1=T,
then X.sub.2 is not T.
126. A purified peptide that binds to the SH3 domain of Yes, said
peptide comprising the amino acid sequence
.psi.X.sub.1X.sub.2RPLPX.sub.3LPX.sub.- 4X.sub.5 (SEQ ID NO:455)
where .psi. represents aliphatic amino acid residues and X.sub.1,
X.sub.2, X.sub.3, X.sub.4, and X.sub.5 represent any amino acid;
except that if X.sub.3=P, X.sub.4=P, and X.sub.5=P, then: when
.psi.=L, where X.sub.1=F, then X.sub.2 is not H or R; or where
X.sub.1=S, then X.sub.2 is not R, H, A, N, T, G, V, M, or W; or
where X.sub.1=C, then X.sub.2 is not S or G; or where X.sub.1=R,
then X.sub.2 is not T or F; or where X.sub.1=A, then X.sub.2 is not
R, Q, N, S, or L; or where X.sub.1=Q, then X.sub.2 is not M; or
where X.sub.1=L, then X.sub.2 is not R; or where X.sub.1=I, then
X.sub.2 is not A; or where X.sub.1=P, then X.sub.2 is not P, W, or
R; or where X.sub.1=G, then X.sub.2 is not S or R; or where
X.sub.1=T, then X.sub.2 is not T; and when .psi.=P, where
X.sub.1=A, then X.sub.2 is not R; or where X.sub.1=S, then X.sub.2
is not R or Y; or where X.sub.1=M, then X.sub.2 is not S; or where
X.sub.1=V, then X.sub.2 is not G; or where X.sub.1=R, then X.sub.2
is not S; or where X.sub.1=I, then X.sub.2 is not R; and when
.psi.=A, where X.sub.1=A, then X.sub.2 is not K; and when .psi.=V,
where X.sub.1=A, then X.sub.2 is not C or Q; or where X.sub.1=P,
then X.sub.2 is not P; and when .psi.=I, where X.sub.1=G, then
X.sub.2 is not H; or where X.sub.1=T, then X.sub.2 is not S; or
where X.sub.1=R, then X.sub.2 is not S.
Description
[0001] This application is a continuation-in-part of co-pending
U.S. patent application Ser. No. 08/483,555 filed Jun. 7, 1995
which in turn is a continuation-in-part of U.S. patent application
Ser. No. 08/278,865 filed Jul. 22, 1994, the entire contents of
each of which are incorporated herein by reference.
1. FIELD OF THE INVENTION
[0002] The present invention relates to SH3 binding peptides having
a broad range of binding specificities. That is, certain members of
the SH3 binding peptides disclosed bind with approximately the same
facility with SH3 domains derived from different SH3
domain-containing proteins. Other members, in contrast, bind with a
much greater degree of affinity for specific SH3 domains. The SH3
binding peptides are obtained from random peptide libraries that
are also phage-displayed. Methods are described of obtaining the
phage clones that bind to the SH3 domain targets and of determining
their relevant nucleotide sequences and consequent primary amino
acid sequence of the binding peptides. The resulting SH3 binding
proteins are useful in a number of ways, including, but not limited
to, providing a method of modulating signal transduction pathways
at the cellular level, of modulating oncogenic protein activity or
of providing lead compounds for development of drugs with the
ability to modulate broad classes, as well as specific classes, of
proteins involved in signal transduction.
2. BACKGROUND OF THE INVENTION
[0003] 2.1. Src and the SH3 Domain
[0004] Among a number of proteins involved in eukaryotic cell
signaling, there is a common sequence motif called the SH3 domain.
It is 50-70 amino acids in length, moderately conserved in primary
structure, and can be present from one to several times in a large
number of proteins involved in signal transduction and in
cytoskeletal proteins.
[0005] The protein pp60c-src represents a family of at least nine
non-receptor protein tyrosine kinases (NR-PTKs). Members of this
family share an overall structural organization comprising a series
of catalytic and non-catalytic domains. In Src, a 14-amino-acid
myristylation signal resides at the extreme amino-terminus, and is
followed by a unique region that is not highly conserved among
family members. Following this region are two highly conserved
60-and 100-amino-acid regions, the Src homology (SH) domains 3 and
2, respectively. SH2 and SH3 domains have been shown to play an
important role in mediating protein-protein interactions in a
variety of signaling pathways. Koch, C. A., et al., in Science
(1991) 252:668-74. The carboxy-terminal half of Src contains the
PTK catalytic domain, as well as a negative regulatory tyrosine
(Y527) near the carboxy terminus. Phosphorylation of this residue
(e.g., by Csk) results in the inhibition of PTK activity. Cooper,
J. A., et al., in Science (1986) 231:1431-1434. Mutation of
Y527.fwdarw.F generates forms of Src with increased PTK and
oncogenic activity. Cartwright, C. A., et al., in Cell (1987)
49:83-91; Kmiecik, T. E., et al., in Cell (1987) 49:65-73; and
Piwicna-Worms, H., et al., in Cell (1987) 75-82.
[0006] The fact that some mutations which result in increased Src
PTK and transforming activity map to the Src SH2 (Seidel-Dugan, C.,
et al., in Mol. Cell. Biol. (1992) 12:1835-45; and Hirai, H. and
Varmus, H. E. in Mol. Cell. Biol. (1990) 10:1307-1318) and SH3
domains (Seidel-Dugan, C., et al., supra; Hirai, H. and Varmus,
H.E., supra; Superti-Furga, G., et al., in Embo. J. (1993)
12:2625-34; and Potts, W. M., et al., in Oncogene Res. (1988)
3:343-355) suggests a negative regulatory role for these domains.
That phosphotyrosine residues within specific sequence contexts
represent high affinity ligands for SH2 domains suggests a model in
which the SH2 domain participates in Y527-mediated inhibition of
PTK activity by binding phosphorylated Y527, thereby locking the
kinase domain in an inactive configuration. Matsuda, M., Mayer, B.
J., et al., in Science (1990) 248:1537-1539. This model is
supported by the observation that phosphopeptides corresponding to
the carboxy-terminal tail of Src bind active, but not inactive,
variants of Src. Roussel, R. R., et al., in Proc. Natl. Acad. Sci.
U S A (1991) 88:10696-700; and Liu, X., et al., in Oncogene (1993)
8:1119-1126.
[0007] The mechanism of SH3-mediated inhibition of Src PTK activity
remains unclear. There is evidence that pY527-mediated inhibition
of Src PTK activity involves the SH3 domain as well as the SH2
domain. Okada, M., Howell, et al., in J. Biol. Chem. (1993)
268:18070-5; Murphy, S. M., et al., in Mol. Cell. Biol. (1993)
13:5290-300; and Superti-Furga, G., et al., supra. Although these
effects are thought to be a consequence of SH3-mediated
protein-protein interactions, precisely how the Src SH3 domain
exerts its negative regulatory effect is unclear. Identification of
high affinity ligands for the Src SH3 domain could help resolve
these issues.
[0008] 2.2. Protein Tyrosine Kinases and The Immune Response
[0009] Src-related tyrosine kinases are expressed in a variety of
cell types including those of the immune system (lymphocytes, T
cells, B cells, and natural killer cells) and the central nervous
system (neural cells, neurons, oligodendrocytes, parts of the
cerebellum, and the like). Umemori, H. et al., in Brain Res. Mol.
Brain Res. (1992) Dec. 16(3-4):303-310. Their presence in these
cells and tissues and their interaction with specific cell surface
receptors and immunomodulatory proteins (such as T cell antigen
receptor, CD14, CD2, CD4, CD40 or CD45) suggest that these kinases
serve an important role in the signalling pathways of not only the
central nervous system but of the immune system, as well. See,
e.g., Ren, C. L. et al., in J. Exp. Med. (1994) 179(2):673-680
(signal transduction via CD40 involves activation of Lyn kinase);
Donovan, J. A. and Koretzky, G. A., in J. Am. Soc. Nephrol. (1993)
4(4):976-985 (CD45, the immune response, and regulation of Lck and
Fyn kinases); and Carmo, A. M. et al., in Eur. J. Immunol. (1993)
23(9):2196-2201 (physical association of the cytoplasmic domain of
CD2 with p56lck and p59fyn).
[0010] For instance, mice with disruptions in their Src-like genes,
Hck and Fgr, possess macrophages with impaired phagocytic activity
or exhibit a novel immunodeficiency characterized by an increased
susceptibility to infection with Listeria monocytogenes. Lowell, C.
A. et al., in Genes Dev. (1994) 8(4):387-398. Also, it has been
shown that bacterial lipopolysaccharide (LPS) activates
CD14-associated p56lyn, p68hck, and p59c-fgr, while inducing the
production of lymphokines, such as TNF-alpha, IL-1, IL-6, and IL-8.
Inhibition of the protein tyrosine kinases blocks production of
TNF-alpha and IL-1.
[0011] 2.3. SH3 Binding Peptides
[0012] As mentioned above, it has long been suspected that SH3
domains are sites of protein-protein interaction, but it has been
unclear what SH3 domains actually bind. Efforts to identify ligands
for SH3 domains have led to the characterization of a number of
SH3-binding proteins, including 3BP1 and 2 (Ren, R., Mayer, et al.,
in Science (1993) 259:1157-61), SOS (Olivier, J. P., et al., in
Cell (1993) 73:179-91; and Rozakis-Adcock, M., et al., in Nature
(1993) 363:83-5), p85 PI-3' Kinase (Xingquan, L., et al., in Mol.
Cell. Biol. (1993) 13:5225-5232), dynamin (Gout, I., et al., in
Cell (1993) 75:25-36), AFAP-110 (Flynn, D. C., et al., in Mol.
Cell. Biol. (1993) 13:7892-7900), and CD42 (Barfod, E. T., et al.,
in J. Biol. Chem. (1993) 268:26059-26062). These proteins tend to
possess short, proline-rich stretches of amino acids, some of which
have been directly implicated in SH3 binding. A variety of
consensus sequences have been proposed, although the similarity
among proline-rich regions of different SH3-binding proteins tends
to be fairly low. Also, attempts to build consensus sequences are
likely complicated by the incorporation of data from proteins that
bind different SH3 domains.
[0013] Thus, Cicchetti, P., et al., in Science (1992) 257:803-806,
published their work relating to the isolation and sequencing of
two naturally-occurring proteins that could be bound in vitro by
the SH3 domain of the abl oncogene product. These workers found
that SH3 domains bind short, proline-rich regions of such proteins.
Subsequently, this same group disclosed further results (Ren, R. et
al., supra) in which the SH3 binding sites of the SH3 binding
proteins were localized to "a nine- or ten-amino acid stretch rich
in proline residues." A consensus sequence incorporating the
features of the SH3 binding sites of four SH3 binding proteins was
proposed: XPXXPPPXP (SEQ ID NO:1), wherein X indicates a position
in the amino acid sequence which is not conserved among the four
SH3 binding proteins, P represents proline, and indicates a
hydrophobic amino acid residue, such as P or L.
[0014] The screening of complex random peptide libraries has been
used to identify peptide epitopes for monoclonal (Scott, J. K. and
Smith, G. P. in Science (1990) 249:386-390) and polyclonal (Kay, B.
K., et al., in Gene (1993) 128:59-65) antibodies, as well as
peptide ligands for a variety of proteins, including streptavidin
(Devlin, J. J., et al., in Science (1990) 249:404-406; and Lam, K.,
et al., in Nature (1991) 354:82-84), the endoplasmic reticulum
chaperone BiP (Blond-Elguindi, S., et al., in Cell (1993)
75:717-728), and CaM (Dedman, J. R., et al., in J. Biol. Chem.
(1993) 268:23025-23030).
[0015] Recently, Chen, J. K. et al., in J. Am. Chem. Soc. (1993)
115:12591-12592, described ligands for the SH3 domain of
phosphatidylinositol 3-kinase (PI-3' Kinase) which were isolated
from a biased combinatorial library. A "biased" library is to be
distinguished from a "random" library in that the amino acid
residue at certain positions of the synthetic peptide are fixed,
i.e., not allowed to vary in a random fashion. Indeed, as stated by
these research workers, screening of a "random" combinatorial
library failed to yield suitable ligands for a PI-3' Kinase SH3
domain probe. The binding affinities of these unsuitable ligands
was described as weak, >100 .mu.M, based on dissociation
constants measured by the Biosensor System (BIAcore).
[0016] More recently, Yu, et al. (Yu, H., et al., in Cell (1994)
76:933-945) used a "biased" synthetic peptide library of the form
XXXPPXPXX (SEQ ID NO:2), wherein X represents any amino acid other
than cysteine, to identify a series of peptides which bind the Src
and PI-3' Kinase SH3 domains. The bias was accomplished by fixing
the proline residues at the specific amino acid positions indicated
for the "random" peptide. As stated previously, without this bias,
the technique disclosed fails to identify any SH3 domain-binding
peptides.
[0017] A consensus sequence, based on 13 binding peptides was
suggested: RXLPPRPXX (SEQ ID NO:3), where X tends to be a basic
residue (like R, K or H). The binding affinities of several SH3
binding peptides were disclosed as ranging from 8.7 to 30 .mu.M. A
"composite" peptide, RKLPPRPRR (SEQ ID NO:4), was reported to have
a binding affinity of 7.6 .mu.M. This value compares favorably to
the binding affinity of the peptide, VPPPVPPRRR (SEQ ID NO:5), to
the N-terminal SH3 domain of Grb2. See, Kraulis, P.J. J. Appl.
Crystalloqr. (1991) 24:946. Recognizing the limitations of their
technique, Chen and co-workers, supra, stated that their results
"illustrate the utility of biased combinatorial libraries for
ligand discovery in systems where there is some general knowledge
of the ligand-binding characteristics of the receptor" (emphasis
added).
[0018] Yu and co-workers, supra, further described an SH3 binding
site consensus sequence, Xp.phi.PpXP (SEQ ID NO:6), wherein X
represents non-conserved residues, .phi. represents hydrophobic
residues, P is proline, and p represents residues that tend to be
proline. A consensus motif of RXLPPRPXX (SEQ ID NO:7), where X
represents any amino acid other than cysteine, was proposed for
ligands of PI-3' Kinase SH3 domain. A consensus motif of
RXLPPLPR.phi. (SEQ ID NO:8), where .phi. represents hydrophobic
residues, was proposed for ligands of Src SH3 domain. Still, the
dissociation constants reported for the 9-mer peptides ranged only
from about 8-70 .mu.M and selectivity between one type of SH3
domain and another was relatively poor, the KDS differing by only
about a factor of four.
[0019] Hence, there remains a need to develop techniques for the
identification of Src SH3 binding peptides which do not rely on
such "biased" combinatorial peptide libraries that are limited to a
partially predetermined set of amino acid sequences. Indeed, the
isolation of SH3 binding peptides from a "random" peptide library
has not been achieved successfully before now. Furthermore,
particular peptides having much greater binding affinities, whether
general or more selective binding for specific SH3 domains, remain
to be identified. Binding peptides specific for particular SH3
domains are useful, for example, in modulating the activity of a
particular SH3 domain-containing protein, while leaving others
bearing an SH3 domain unaffected. Still, the more promiscuous
general binding peptides are useful for the modulation of a broad
spectrum of SH3 domain-containing proteins.
[0020] The present invention relates to such SH3 binding peptides,
methods for their identification, and compositions comprising same.
In particular, peptides comprising particular sequences of amino
acid residues are disclosed which were isolated from random peptide
libraries. In the present invention, clones were isolated from a
phage-displayed random peptide library which exhibited strong
binding affinities for SH3 domain-containing protein targets. Some
of these protein targets, include Abl, Src, Grb2, PLC-.delta.,
PLC-.gamma., Ras GAP, Nck, and p85 PI-3' Kinase. From the
nucleotide sequence of the binding phage, the amino acid sequence
of the peptide inserts has been deduced. Synthetic peptides having
the desired amino acid sequences are shown to bind the SH3 domain
of the target proteins. In particular, synthetic peptides combining
a core consensus sequence and additional amino acid residues
flanking the core sequence are especially effective at binding to
particular target protein SH3 domains. The SH3 binding peptides
disclosed herein can be utilized in a number of ways, including the
potential modulation of oncogenic protein activity in vivo. These
peptides also serve as useful leads in the production of
peptidomimetic drugs that modulate a large class of proteins
involved in signal transduction pathways and oncogenesis.
3. SUMMARY OF THE INVENTION
[0021] Accordingly, three phage-displayed random peptide libraries
were screened for isolates that bind to bacterial fusion proteins
consisting of the Src homology region 3 (SH3) and glutathione
S-transferase (GST). DNA sequencing of the isolates showed that
they contained sequences that resemble the consensus motif, RPLPPLP
(SEQ ID NO:9), within their 8, 22, or 36 amino acid long random
regions. When peptides were synthesized corresponding to the pIII
inserts of the SH3-binding phage, they bound to the GST fusions of
the SH3 domains of Src and the Src-related proteins, such as Yes,
but not of Grb2, Crk, Abl, or PLC.gamma.1. The synthesized peptides
bind quite well to the Src SH3 domain and act as potent competitors
of natural Src SH3 interactions in cell lysates. For instance,
these peptides can compete with radiolabelled proteins from cell
lysates in binding to immobilized Src-GST, with an apparent
IC.sub.50 of 1-10 .mu.M. When a peptide, bearing the consensus
sequence RPLPPLP (SEQ ID NO:9) was injected into Xenopus laevis
oocytes, it accelerated the rate of progesterone-induced
maturation. These results demonstrate the utility of
phage-displayed random peptide libraries in identifying SH3-binding
peptide sequences and that such identified peptides exhibit both in
vivo and in vitro biological activity.
[0022] Thus, it is an object of the present invention to provide
peptides having at least nine and up to forty-five amino acid
residues, including an amino acid sequence of the formula,
R-2-L-P-5-6-P-8-9 (SEQ ID NO:10), positioned anywhere along the
peptide, in which each number represents an amino acid residue,
such that 2 represents any amino acid residue except cysteine, 5
and 6 each represents a hydrophobic amino acid residue, 8
represents any amino acid residue except cysteine, and 9 represents
a hydrophilic amino acid residue except cysteine, each letter being
the standard one-letter symbol for the corresponding amino acid,
said peptide exhibiting a binding affinity for the SH3 domain of
Src, provided that said peptide is not R-P-L-P-P-L-P-T-S (SEQ ID
NO:11). In a particular embodiment of the present invention, the
peptides also exhibit a binding affinity for the SH3 domain of
Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck and
Fgr.
[0023] The present invention also contemplates SH3 domain-binding
peptides that further comprise a C-terminal-flanking amino acid
sequence of the formula 10, 10-11, 10-11-12, 10-11-12-13 (SEQ ID
NO:12) or 10-11-12-13-14 (SEQ ID NO:13), in which each number
represents any amino acid residue except cysteine, such that 10 is
bound to 9 by a peptide bond. Furthermore, peptides are also
provided which further comprise an N-terminal-flanking amino acid
sequence of the formula 1', 2'-1', 3'-2'-1' or 4'-3'-2'-1' (SEQ ID
NO:14) in which each number represents any amino acid residue
except cysteine, such that 1' is bound to R by a peptide bond.
[0024] Thus, in a particular embodiment, a peptide is disclosed
having at least thirteen and up to forty-five amino acid residues,
including an amino acid sequence of the formula,
3'-2'-1'-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere
along the peptide, in which each number represents an amino acid
residue, such that 3', 2', 1', 2, 8, and 10 each represents any
amino acid residue except cysteine, 5 and 6 each represents a
hydrophobic amino acid residue, and 9 represents a hydrophilic
amino acid residue except cysteine, each letter being the standard
one-letter symbol for the corresponding amino acid, said peptide
exhibiting a binding affinity for the SH3 domain of Src.
[0025] The present invention also seeks to provide new consensus
sequences or motifs that reflect variations in SH3 domain binding
selectivities or specificities. The present invention also
contemplates conjugates of the SH3 binding peptides and a second
molecule or chemical moiety. This second molecule may be any
desired substance whose delivery to the region of the SH3 domain of
a particular protein (or cell containing the protein) is sought.
Possible target cells include, but are not limited to, neural
cells, immune cells (e.g., T cells, B cells, natural killer cells,
and the like), osteoclasts, platelets, epidermal cells, and the
like, which cells express Src, Src-related proteins, and
potentially, other SH3 domain-containing proteins. In this manner,
the modulation of the biological activity of proteins bearing an
SH3 domain can be accomplished.
[0026] Other methods and compositions consistent with the
objectives of the present invention are likewise disclosed. in
particular, a method is disclosed of modulating the activity of Src
or Src-related proteins comprising administering a composition
comprising an effective amount of a peptide of the present
invention and a carrier, preferably a pharmaceutically acceptable
carrier. In a specific embodiment, the contemplated method results
in the inhibition of the activity of Src or Src-related proteins.
Alternatively, the method is effective to activate Src or
Src-related proteins.
[0027] In yet another embodiment, a method is disclosed of
identifying a peptide having a region that binds to an SH3 domain
comprising: (a) providing an immobilized target protein comprising
an SH3 domain; (b) incubating the immobilized target protein with
an aliquot taken from a random peptide library; (c) washing unbound
library peptides from the immobilized target protein; (d)
recovering the peptide bound to the immobilized target protein; and
(e) determining the primary sequence of the SH3 domain-binding
peptide.
[0028] Moreover, a method is disclosed of imaging cells, tissues,
and organs in which Src or Src-related proteins are expressed,
which comprises administering an effective amount of a composition
comprising an SH3 domain-binding peptide conjugated to detectable
label or an imaging agent.
[0029] Other objectives of the present invention will become
apparent to one of ordinary skill in the art after consideration of
the above disclosure and the following detailed description of the
preferred embodiments.
[0030] The invention also provides assays for identifying a
compound that affects the binding between a first molecule
comprising an SH3 domain and a second molecule that binds to the
SH3 domain comprising incubating one or more candidate compounds
from which it is desired to select such a compound with the first
molecule and the second molecule under conditions conducive to
binding and detecting the one or more compounds that affect binding
of the first molecule to the second molecule.
[0031] Also provided are kits for performing such assays comprising
a first molecule comprising an SH3 domain and a second molecule
that binds to the SH3 domain.
4. BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 illustrates a scheme for the generation of a random
36 amino acid peptide library (TSAR-9; e.g., SEQ ID NO:16).
Oligonucleotides were synthesized (SEQ ID NOS:17-18), converted
into double-stranded DNA, cleaved with restriction enzymes (SEQ ID
NOS:19-20), and cloned into the M13 vector, m663. The random
peptide region encoded by the oligonucleotides is shown in the box
(SEQ ID NO:16) and is situated at the N-terminus of mature protein
III (SEQ ID NO:21). SEQ ID NO:22 includes the three amino acids
preceding the signal peptidase cleavage site.
[0033] FIG. 2 illustrates a scheme for the generation of a random
22 amino acid peptide library (TSAR-12; e.g., SEQ ID NO:23).
Oligonucleotides were synthesized (SEQ ID NOS:24-25), converted
into double-stranded DNA, cleaved with restriction enzymes (SEQ ID
NOS:26-27), and cloned into the M13 vector, m663. The random
peptide region encoded by the oligonucleotides is shown in the box
(SEQ ID NO:23) and is situated at the N-terminus of mature protein
III (SEQ ID NO:28). SEQ ID NO:29 includes the three amino side
preceding the signal peptidase cleavage site.
[0034] FIG. 3 illustrates a scheme for the generation-of a random 8
amino acid peptide library (R8C; SEQ ID NO:30). oligonucleotides
were synthesized (SEQ ID NOS:31-32), converted into double-stranded
DNA, cleaved with restriction enzymes (SEQ ID NOS:33-34), and
cloned into the M13 vector, m663. The random peptide region (SEQ ID
NO:30) is flanked by cysteine residues and is situated at the
N-terminus of mature protein III (SEQ ID NO:35).
[0035] FIG. 4 illustrates the possible origin of one class of
double-insert R8C recombinants (e.g., encoding SEQ ID NO:36).
Double-stranded oligonucleotides (e.g., SEQ ID NO:37) may have
ligated in a head-to-head fashion at the Xba I site prior to
cloning in the Xho I- Xba I cleaved M13 vector.
[0036] FIG. 5 shows a list of random peptide recombinants (SEQ ID
NOS:38-61 and 106) isolated by the method of the present invention
and the displayed peptide sequence. The amino acid sequences are
aligned to highlight the core sequences. The flanking sequences are
shown to the N-terminal and C-terminal ends of the core sequence.
SEQ ID NOS:38-61 are shown in order from top to bottom except that
SSCDHTLGLGWCGSRSTRQLPIPP TTTRPSR is SEQ ID NO:106 and RPLPPLP is
SEQ ID NO:9. T12.Src3.1 is a Class II ligand (See Section
6.14.5).
[0037] FIG. 6 graphically illustrates the relative binding
affinities of selected phage clones for various SH3 domains. The
results indicate that certain amino acid sequences provide generic
SH3 domain binding, while others can provide greater selectivity
for the SH3 domain of Src. Still other clones exhibit Src SH3
domain preferential binding.
[0038] FIG. 7 shows the binding of synthetic peptides (SEQ ID NOS:9
and 62-70) representing Src SH3-selected phage inserts to Src
SH3-GST fusion target (shaded columns) over background GST binding
(unshaded columns) relative to the core peptide RPLPPLP (SEQ ID
NO:9) and proline-rich peptide segments derived from naturally
occurring proteins. Bound biotinylated peptide was detected with
streptavidin-alkaline phosphatase ELISA. Each point was performed
in triplicate; average absorbance at 405 nm is presented. Error
bars represent SD. SEQ ID NOS:62-70 are shown in order from top to
bottom except that RPLPPLP is SEQ ID NO:9.
[0039] FIG. 8 illustrates the relative specificity of selected
peptides (SEQ ID NOS:9 and 62-70) for SH3 domains derived from
different proteins. In particular, the binding affinities of the
peptides for the SH3 domains of the following protein fusion
targets were tested: Src SH3-GST, Yes SH3-GST, Grb2-GST, Crk
SH3-GST, Abl SH3-GST, PLC.gamma.1 SH2SH3-GST. Bound biotinylated
peptide was detected with streptavidin-alkaline phosphatase. Each
point was performed in triplicate; values are average signal
(absorbance at 405 nm) above GST background, with error bars
representing standard deviation. Hatched bars indicate saturation
of the ELISA signal. SEQ ID NOS:62-70 are shown in order from top
to bottom except that RPLPPLP is SEQ ID NO:9.
[0040] FIG. 9 presents the results of competition experiments in
which selected peptides were found to inhibit the binding of
proteins from cell lysates to immobilized Src SH3-GST or Abl
SH3-GST protein fusion targets.
[0041] FIG. 10 presents a graph illustrating the increased rate of
progesterone-induced maturation of oocytes injected with an SH3
domain-binding peptide, VLKRPLPIPPVTR (SEQ ID NO:64), of the
present invention. Briefly, Stage VI oocyted were prepared and
injected as previously described (see, Kay, B. K., in Methods in
Cell Biol. (1991) 36:663-669). Oocytes were injected with 40 nL of
100 .mu.M test peptide or water. After injection, the oocytes were
placed in 2 .mu.g/mL progesterone (Sigma, St. Louis, Mo.) and
scored hourly for germinal vesicle breakdown (GVBD). LAPPKPPLPEGEV
is SEQ ID NO:70.
[0042] FIG. 11 shows the results of fluorescence experiments in
which certain peptides, Panel A=VRPLPIPPVTR (SEQ ID NO:64), Panel
B=GILAPPVPPRNTR (SEQ ID NO:63), Panel C=RSTPRPLPPLPTTR (SEQ ID
NO:67), of the invention were shown to localize within cellular
compartments thought to contain Src or Src-related proteins.
[0043] FIG. 12 illustrates a scheme for the generation of a biased
peptide library. Oligorncleotides were synthesized (SEQ ID
NOS:162-163), converted into double-stranded DNA (SEQ ID NO:454),
cleaved with restriction enzymes XhoI and XbaI (SEQ ID
NOs:455-456), and cloned into the mBAX vector (SEQ ID NOs:457-458),
described further below in the Examples section. The biased peptide
region (SEQ ID NO:459) is situated at the N-terminus of mature pIII
protein. CTAGACGTGTCAGT is a portion of SEQ ID NO:162. ACTGACACGT
is a portion of SEQ ID NO:454. TCGAGGCACAG is a portion of SEQ ID
NO:454.
[0044] FIG. 13 illustrates the peptide sequence encoded in the mBAX
vector situated at the N-terminus of mature pIII protein.
TCCTCGAGTATCGACATGCCTTAGACTGCTAGCACTATGTACAACATGCTT CATCGCAACGAGCCA
is SEQ ID NO:460. SSIDMP*TASTMYNM LHRNEP is SEQ ID NO:461.
GGTGGGAGGAAGTTGAGCCCGCCCGCCAACGA CATGCCGCCCGCCCTCCTGAAGAGGTCTAGA is
SEQ ID NO:462. GGRKLSPPANDMPPALLKRSR is SEQ ID NO:463.
[0045] FIG. 14 illustrates the relative binding of SH3-selected
phage clones to various SH3 domains. Two clones (A and B)
representing each consensus motif were assayed for binding to 1
.mu.g of each immobilized GST-SH3 fusion protein. Bound phage were
detected by anti-phage ELISA. Sequences of peptides displayed by
each clone are aligned with their respective consensus motifs.
Invariant proline residues are underlined. Solid bars, specific
binding; open bars, cross-reactive binding. Values are average
OD.sub.405.+-.SD (N=3).
5. DETIALED DESCRIPTION OF THE INVENTION
[0046] 5.1. General Considerations
[0047] The present invention relates to peptides that exhibit a
binding affinity for an SH3 domain, which domain has been found to
be present in a number of physiologically significant proteins. In
particular, peptides are disclosed which exhibit general binding
characteristics to the SH3 domains found in a group of proteins,
including but not limited to Abl, Src, Grb2, PLC-.delta.,
PLC-.gamma., Ras GAP, Nck, and p85 PI-3' Kinase. Preferred peptides
exhibit selective, if not specific, binding affinity for the SH3
domain of Src. As described herein, the peptides of the present
invention include a core sequence, preferably a consensus seqeunce,
and additional amino acid residues that flank the core sequence.
These peptides, including the methods for their identification, are
described in greater detail, below.
[0048] Thus, in a specific embodiment of the invention, peptides
are provided which have at least nine and up to about forty-five
amino acid residues, including an amino acid sequence resembling
the formula,
[0049] R-2-L-P-5-6-P-8-9 (SEQ ID NO:10),
[0050] positioned anywhere along the peptide. In the
above-mentioned formula, each number represents an amino acid
residue, such that 2 represents any amino acid residue except
cysteine, 5 and 6 each represents a hydrophobic amino acid residue,
8 represents any amino acid residue except cysteine, and 9
represents a hydrophilic amino acid residue except cysteine. Each
letter used in the formulas herein represent the standard
one-letter symbol for the corresponding amino acid. When the
peptide is a 9-mer, the peptide R-P-L-P-P-L-P-T-S (SEQ ID NO:ll) is
excluded. The peptides of particular interest are those that
exhibit a binding affinity for the SH3 domain of Src and
Src-related proteins, including Yes, Fyn, Lyn, Lck, Hck and Fgr.
Preferably, the peptides of the invention exhibit a binding
affinity for the SH3 domain of Src, which is at least three-fold,
more preferably at least four-fold, most preferably at least about
five-fold greater than that exhibited by the peptide RPLPPLP (SEQ
ID NO:9). In still other embodiments, the peptides exhibit a
binding affinity for the SH3 domain of Src which is at least
ten-fold greater than that exhibited by the peptide RPLPPLP (SEQ ID
NO:9).
[0051] In specific embodiments, peptides are disclosed in which the
various amino acid residues at the indicated positions may
independently have the following preferred identities: 2 is a P, R,
A, L, Q, E or S, more preferably P or R; 5 represents a P, M, I or
L, more preferably P or M; 6 is a P, L, I or V, more preferably P
or L; 8 is a T, R, P, I, N, E, V, S, A, G or L, more preferably T
or R; and 9 is a T, R, S, H or D, more preferably T or R. Despite
the preference for hydrophobic amino acid residues at 5 and 6, in
some cases it may be desirable to have hydrophilic amino acid
residues at these positions. Specifically, amino acid residue 5 may
be a T, R or S, and amino acid residue 6 may be a T or R. Likewise,
while a hydrophilic amino acid residue is preferred at position 9,
in some instances a hydrophobic residue, such as a P or A, may be
desirable.
[0052] The present invention also contemplates SH3 domain-binding
peptides with a minimum length of 10, 11, 12, 13, 14, 15 or more
amino acids. Such peptides contain additional amino acid residues
flanking the core sequence of R-2-L-P-5-6-P (SEQ ID NO:71) either
at the C-terminal end, the N-terminal end or both. Thus, for
example, such peptides include those that further comprise a
C-terminal-flanking amino acid sequence of the formula 10, 10-11,
10-11-12, 10-11-12-13 (SEQ ID NO:12) or 10-11-12-13-14 (SEQ ID
NO:13), in which each number represents any amino acid residue
except cysteine, such that the amino acid residue 10 is bound to
the amino acid residue 9 by a peptide bond. In that case, specific
embodiments include an amino acid residue 10 which is T, R, L, S,
D, P, A or N, preferably T or R, an amino acid residue 11 which is
R, P, A, Q, S or T, preferably R or P, an amino acid residue 12
which is P, S, R or T, preferably P or S, an amino acid residue 13
which is P, S, R, F, H or T, preferably P or S, and an amino acid
residue 14 which is S, R, G or T, preferably, S or R.
[0053] Furthermore, peptides are also provided which further
comprise an N-terminal-flanking amino acid sequence of the formula
1', 2'-1', 3'-2'-1' or 4'-3'-2'-1' (SEQ ID NO:14) in which each
number represents any amino acid residue except cysteine, such that
1' is bound to R by a peptide bond. In such a case, specific
embodiments are provided in which the amino acid residue 1' is T,
P, S, N, F, W, K, H, Q or G, preferably T or P, wherein the amino
acid residue 2' is S, T, G, P, R, Q, L, A or H, preferably S or T,
wherein the amino acid residue 3' is R, S, P, G, A, V, Y or L,
preferably S or T, and wherein the amino acid residue 4' is R, S,
V, T, G, L or F, preferably R or S.
[0054] In a particular embodiment, a peptide is disclosed having at
least thirteen and up to forty-five amino acid residues, including
an amino acid sequence of the formula,
3'-2'-1'-R-2-L-P-5-6-P-8-9-10 (SEQ ID NO:15), positioned anywhere
along the peptide, in which each number represents an amino acid
residue, such that 3', 2', 1', 2, 8, and 10 each represents any
amino acid residue except cysteine, 5 and 6 each represents a
hydrophobic amino acid residue, and 9 represents a hydrophilic
amino acid residue except cysteine, each letter being the standard
one-letter symbol for the corresponding amino acid, said peptide
exhibiting a binding affinity for the SH3 domain of Src. Preferred
13-mers include, but are not limited to, those having an amino acid
residue 5 which is a P or M, an amino acid residue 1' which is T,
P, S or N, an amino acid residue 2' which is S or T, an amino acid
residue 3' which is R or S, and an amino acid residue 10 which is T
or R. In all the SH3 domain-binding peptides described herein, the
prohibition against the use of the hydrophilic amino acid residue
cysteine (C) does not extend beyond the 7-mer "core" sequence and
the additional amino acid residues flanking the core up to a total
(core+flanking) of about 20 amino acids. That is, the occasional
use of a cysteine is not absolutely prohibited. What should be kept
in mind is that the potential for the formation of intramolecular
disulfide bonds, to form a cyclic structure, be minimized as much
as possible. Applicants have found that cyclized structures appear
to be disfavored, at least with potential binding peptides of less
than about 15 amino acid residues in length. The concern for the
formation of cyclized structures comprising the core sequence
diminishes with increasing size of the peptide. Presumably, a large
enough structure, though cyclic, may allow the critical core
sequence to adopt a more or less linear conformation.
[0055] In particular, specific peptides are disclosed which exhibit
binding affinities to SH3 domains. These include the peptides,
RSTPRPLPMLPTTR (SEQ ID NO. 62), RSTPRPLPPLPTTR (SEQ ID NO. 67),
GILAPPVPPRNTR (SEQ ID NO. 63), VLKRPLPIPPVTR (SEQ ID NO. 64),
GPHRRLPPTPATR (SEQ ID NO. 65), and ANPSPATRPLPTR (SEQ ID NO.
66).
[0056] Phage clones are also disclosed, along with the amino acid
sequences that are responsible for SH3 domain binding. These phage
clones are identified in FIG. 5.
[0057] In other embodiments of the present invention, SH3
domain-binding peptides are contemplated which have a total of 11,
13, 14, 18, 20, 22, 23, 25, 30, 36, 38 or 45 amino acid
residues.
[0058] The peptides of the present invention, having been disclosed
herein, may be prepared by any number of practicable methods,
including but not limited to solution-phase synthesis, solid-phase
synthesis, protein expression by a transformed host, cleavage from
a naturally-derived, synthetic or semi-synthetic polypeptide, or a
combination of these techniques.
[0059] The SH3 binding peptides exhibit a wide range of biological
activity which includes the enhancement (or inhibition, depending
on the particular peptide or the nature of the peptide's target
molecule, in this case a protein bearing an SH3 domain) of the
natural function or biological activity of the peptide's target
molecule. For example, the interaction of the binding peptide of
the present invention could result in the modulation of the
oncogenic activity of the target molecule bearing the SH3 domain.
If the target molecule has, in turn, a natural binding partner or
ligand, the peptides of the present invention may also exhibit
antagonistic or agonistic activity in relation to the biological
activity of the natural binding partner.
[0060] Thus, it is an object of the present invention to provide a
method of activating Src or Src-related protein tyrosine kinases by
administering an effective amount of the SH3 domain-binding
peptides generally described herein. The intensity of the immune
response can thus be stimulated for example, by the increased
production of certain lymphokines, such as TNF-alpha and
interleukin-1. As is generally known to those of ordinary skill in
the art, a more intense immune response may be in order in certain
conditions, such as in combating a particularly tenacious
infection, viral or otherwise, or a malignancy.
[0061] Furthermore, in a specific embodiment of the present
invention, a conjugate compound is contemplated which comprises the
peptide of the present invention and a second chemical moiety. The
second chemical moiety can be selected from a wide variety of
chemical compounds including the peptide itself. Typically,
however, the second chemical moiety is selected to be other than
the peptide of the present invention, including but not limited to
an amino acid, a peptide other than an SH3 binding peptide of the
present invention, a polypeptide or protein (i.e., the conjugate is
a fusion protein), a nucleic acid, a nucleoside, a glycosidic
residue (i.e., any sugar or carbohydrate), a label or
image-enhancing agent (including metals, isotopes, radioisotopes,
chromophores, fluorophores (such as FITC, TRITC, and the like), and
enzyme substrates), a drug (including synthetic, semisynthetic, and
naturally-occurring compounds), small molecules (e.g., biotin,
hormones, factors) and the like.
[0062] The peptide of the present invention can be conjugated to
the second chemical moiety either directly (e.g., through
appropriate functional groups, such as an amine or carboxylic acid
group to form, for example, an amine, imine, amide, ester, acyl or
other carbon-carbon bond) or indirectly through the intermediacy of
a linker group (e.g., an aliphatic or aromatic polyhydroxy,
polyamine, polycarboxylic acid, polyolefin or appropriate
combinations thereof). Moreover, the term "conjugate," as used
herein, is also meant to encompass non-covalent interactions,
including but not limited to ionic, affinity or other complexation
interactions. Preferably, such other non-covalent interactions
provide definable, most preferably, isolatable chemical conjugate
species.
[0063] As described further herein, the peptides of the present
invention have been shown to localize within certain cellular
compartments which contain Src or Src-related proteins.
Consequently, the above-described conjugate can be utilized as a
delivery system for introduction of a drug to cells, tissues or
organs that include SH3 domain-containing proteins.
[0064] It should also be pointed out that the present invention
seeks to provide a recombinant construct comprising a nucleic acid
or its complement that includes codons or nucleotide and sequences
encoding a peptide having a region that binds to an SH3 domain,
preferably the Src SH3 domain. The recombinant nucleic acid may be
a DNA or RNA polynucleotide.
[0065] In a specific embodiment, the present invention contemplates
a recombinant construct which is a transforming vector. Such
vectors include those well known to those of ordinary skill in the
art, which effect the transfer or expression of the nucleotide
sequence after introduction to a host, such as recombinant plasmid,
phage or yeast artificial chromosome. These vectors may be closed
circular loops or they may be linearized. The vectors contemplated
include those that exist extrachromosomally after host
transformation or transfection, as well as those that integrate
within or even displace portions of the host chromosome. The
vectors may be introduced to the cell with the help of transfection
aids or techniques well-known in the art. For example, these aids
or techniques may take the form of electroporation, use of calcium
chloride, calcium phosphate, DEAE dextran, liposomes or polar lipid
reagents known as LIPOFECTIN or LIPOFECTAMINE. In addition, the
present invention contemplates the direct introduction of the
desired nucleic acid to the host cell, for instance, by
injection.
[0066] Transformed host cells are also obtained by the methods of
the present invention which are capable of reproducing the
polynucleotide sequences of interest and/or expressing the
corresponding peptide products. A variety of hosts are
contemplated, including prokaryotic and eukaryotic hosts. In
particular, bacterial, viral, yeast, animal, and plant cells are
potentially transformable hosts. Thus, a method is disclosed to
obtain a transformed host cell that can produce, preferably
secrete, a peptide having a region that binds to an SH3 domain
comprising (a) providing an expression vector, preferably a
secretory expression vector, comprising a nucleotide sequence
encoding at least one copy of a peptide having a region that binds
to an SH3 domain; and (b) introducing the vector to a competent
host cell.
[0067] The peptides, thus produced, may then be introduced to
cells, tissues, organs, or administered to the subject for the
purpose of modulating the biochemical activity of the SH3
domain-containing proteins present therein. Accordingly, in
specific embodiments of the present invention, compositions are
provided which comprise an SH3 domain-binding peptide, including a
core sequence and flanking sequences, and a suitable carrier.
[0068] The compositions contemplated by the present invention may
also include other components, from those that facilitate the
introduction or administration of the compositions to those that
have their own innate activity, such as a prophylactic, a
diagnostic or a therapeutic action. Such innate activity may be
distinct from that of the peptides of the present invention or be
complementary thereto. In any event, the compositions of the
present invention include those that are suitable for
administration into mammals, including humans. Preferably, the
compositions (including necessarily the carrier) of the present
invention are sterile, though others may need only be cosmetically,
agriculturally or pharmaceutically acceptable. Still other
compositions may be adapted for veterinary use.
[0069] The compositions, including the drug delivery systems
described herein, are contemplated to be administered in a variety
of ways, such as parenterally, orally, enterally, topically or by
inhalation. The compositions may also be adminstered intranasally,
opthalmically or intravaginally. Furthermore, the compositions of
the invention can take several forms, such as solids, gels,
liquids, aerosols or patches.
[0070] In another embodiment of the present invention a method is
provided of identifying a peptide having a region that binds to an
SH3 domain comprising: (a) providing an immobilized target protein
comprising an SH3 domain; (b) incubating the immobilized target
protein with an aliquot taken from a phage-displayed random peptide
library, which library includes peptides having a random sequence
of .gtoreq.8 amino acid residues; (c) washing unbound phage from
the immobilized target protein; (d) recovering the phage bound to
the immobilized target protein; and (e) determining the relevant
nucleotide sequence of said binding phage nucleic acid and deducing
the primary sequence corresponding to the SH3 domain-binding
peptide. Preferably, the method further comprises amplifying the
titer of the recovered phage and repeating the steps of incubation,
washing and recovery to provide SH3 domain-binding peptide-enriched
phage.
[0071] Any other mode by which the peptide library, random or
otherwise, can be "displayed" can be utilized in the present
invention, however. Moreover, the present applicants believe that
longer random peptide sequences (e.g., >6 amino acid residues,
preferably >10, and most preferably, >12) provide not only
much greater diversity but also a richer degree of secondary
structure conducive to binding activity. If the random region of
the peptide is less than or equal to an 8-mer, it should preferably
not be cyclized.
[0072] 5.2. Preparation of Random Peptide Libraries
[0073] The preparation and characterization of the preferred
phage-displayed random peptide libraries have been described
elsewhere. See, for example, Kay, B. K. et al. in Gene (1992)
128:59-65, for a description of the preparation of the
phage-displayed random peptide library known as TSAR-9, more below.
In particular, by cloning degenerate oligonucleotides of fixed
length into bacteriophage vectors, recombinant libraries of random
peptides can be generated which are expressed at the amino-terminus
of the pIII protein on the surface of M13 viral particles. (There
are 3-5 copies of the pIII-fusion on the surface of each particle.)
Phage display offers several conveniences: first, the expressed
peptides are on the surface of the viral particles and accessible
for interactions; second, the recombinant viral particles are
stable (i.e., can be frozen, exposed to pH extremes); third, the
viruses can be amplified; and fourth, each viral particle contains
the DNA encoding the recombinant genome. Consequently, these
libraries can be screened by isolating viral particles that bind to
targets. The isolates can be grown up overnight, and the displayed
peptide sequence responsible for binding can be deduced by DNA
sequencing.
[0074] These libraries have approximately >108 different
recombinants, and nucleotide sequencing of the inserts suggests
that the expressed peptides are indeed random in amino acid
sequence. These libraries are referred to herein as TSAR libraries,
where TSAR stands for Totally Synthetic Affinity Reagents. The
preparation of the TSAR libraries are described further below.
[0075] 5.3. SH3 Binding Clones And Their Characteristics
[0076] Accordingly, peptides have been isolated from an
unconstrained random peptide library which exhibit a binding
affinity for SH3 domains. Furthermore, the binding affinities
exhibited by the disclosed peptides differ in their selectivities
with certain peptides showing comparable binding affinities for SH3
domains derived from different proteins, while others manifest
greater affinities for specific SH3 domains.
[0077] The amino acid sequence of various peptides isolated by the
present method are listed in FIG. 5. As can be seen from this list,
certain groups of SH3 domain binding peptides are isolated from
three separate random peptide libraries, each based on a different
type of random peptide insert, all displayed at the amino-terminus
of the pIII protein on the surface of M13 viral particles. Ten
clones were isolated from the R8C library, seven from the TSAR-12
library, and seven from the TSAR-9 library. The sequences are
presented to highlight the particular amino acid residues believed
to bind directly to the SH3 domain, as well as to point out the
remaining amino acid resiudes of the random insert and the viral
flanking sequences and complementary site amino acid residues
common to each group of clones. The frequency with which each
particular clone is found in each library is also indicated in FIG.
5. Thus, clones T12.SRC3.1 and T12.SRC3.2 are by far the most
abundant clones found among the three libraries.
[0078] Interestingly, all the binding peptides are found to have
the proline-rich amino acid residue motif, which is apparently
responsible for binding, the motif being located predominantly at
the C-terminal end of the insert, although each clone also contains
an insert at the N-terminal end. The significance of this
observation is not presently understood, although this finding may
indicate the possible importance of the C-terminal viral flanking
sequences in SH3 domain binding.
[0079] Indeed, a synthetic peptide bearing only the core consensus
sequence RPLPPLP (SEQ ID NO:9) was less effective in binding to
target SH3 domains than synthetic peptides that also included
additional amino acid residues flanking the core sequences. Thus,
13-mers and 14-mers having the sequences RSTPRPLPMLPTTR (SEQ ID
NO:62), RSTPRPLPPLPTTR (SEQ ID NO:67), GILAPPVPPRNTR (SEQ ID
NO:63), GPHRRLPPTPATR (SEQ ID NO:65), and VLKRPLPIPPVTR (SEQ ID
NO:64) have been prepared and shown to bind to SH3 domains, such as
those of Src and Yes, much more avidly than the 7-mer, RPLPPLP (SEQ
ID NO:9). The 13-mer ANPSPATRPLPTR (SEQ ID NO:66) has been shown to
have binding affinities comparable to the core consensus sequence.
In each case, the 13-mers comprise a 7-mer "core" sequence plus
additional amino acid residues flanking same, some of which
additional amino acid residues are contributed by the viral
flanking sequences.
[0080] Thus, in one embodiment of the present invention, a 7-mer
core includes a consensus motif of the formula RXLP.phi..phi.P (SEQ
ID NO:71), wherein R is arginine, L is leucine, P is proline, X
represents any amino acid except cysteine and .phi. represents a
hydrophobic amino acid residue. By "hydrophobic amino acid
residue," the applicants mean to include F, Y, W, V, A, I, L, P or
M, each letter representing the standard one-letter designation for
the corresponding amino acid residue.
[0081] Furthermore, a preferred 9-mer peptide comprising two
additional amino acids on the C-terminal end of the core sequence
is envisioned having a consensus motif of the formula
RXLP.phi..phi.PX.psi. (SEQ ID NO:10). In this preferred 9-mer
consensus motif, the symbol .psi. represents a hydrophilic amino
acid residue, except cysteine. By "hydrophilic amino acid residue,"
the applicants mean to include K, R, H, D, E, N, Q, T, S or C, and
the other symbols are as defined above. For the purposes of the
present invention, a glycine residue (G) may be considered either a
hydrophobic or a hydrophilic amino acid residue. The one-letter
symbols B and Z, which stand for N or D and Q or E, respectively,
are considered hydrophilic amino acid residues.
[0082] Particular 13-mer peptides of the present invention include
those listed, below. It is noted, however, that not all the
following 13-mer peptides correlate strictly to or comply with the
preferred 9-mer consensus motif, described above. Those peptides
that do not comply (indicated in italics, with the non-complying
amino acid residues underscored) can, thus, be described as
"resembling" those that do comply (indicated in normal type) with
the preferred 9-mer consensus motif: PGFRELPPLPPSR (SEQ ID
NO:72),
[0083] AQSRPLPIPPETR (SEQ ID NO:73), VLKRPLPIPPVTR (SEQ ID
NO:64),
[0084] PPNSPLPPLPTHL (SEQ ID NO:74), TGRGPLPPLPNDS (SEQ ID
NO:75),
[0085] YSTRPVPPITRPS (SEQ ID NO:76), SHKSRLPPLPTRP (SEQ ID
NO:77),
[0086] YRFRALPSPPSAS (SEQ ID NO:78), GPHRRLPPTPATR (SEQ ID
NO:65),
[0087] LAQRQLPPTPGRD (SEQ ID NO:79), ALQRRLPRTPPPA (SEQ ID
NO:80),
[0088] PATRPLPTRPSRT (SEQ ID NO:81), YSTRPLPSRPSRT (SEQ ID
NO:82),
[0089] XPGRILLLPSEPR (SEQ ID NO:83), SGGILAPPVPPRN (SEQ ID
NO:84),
[0090] RSTRPLPILPRTT (SEQ ID NO:85), STPRPLPMLPTTR (SEQ ID
NO:86),
[0091] STNRPLPMIPTTR (SEQ ID NO:87), RSTRPLPSLPITT (SEQ ID
NO:88),
[0092] STSRPLPSLPTTR (SEQ ID NO:89), RSTRSLPPLPPTT (SEQ ID
NO:90),
[0093] RSTRQLPIPPTTT (SEQ ID NO:91), STPRPLPLIPTTP (SEQ ID
NO:92),
[0094] RSTRPLPPTPLTT (SEQ ID NO:93), and RSTRPQPPPPITT (SEQ ID
NO:94). Accordingly, other peptides not specifically disclosed,
which either comply with or "resemble" the preferred 9-mer
consensus motif, can be readily envisioned by those of ordinary
skill in the art and are considered to be equivalent to those that
are specifically disclosed above. In particular, non-compliance at
positions 1 (S, G, and I, in place of R, are tolerated), 3 (V, A,
and Q, in place of L, are tolerated), 4 (L, in place of P, is
tolerated), 5 (hydrophilic amino acid residues, S, R, and T, are
tolerated in place of a hydrophobic amino acid residue), 6
(hydrophilic amino acid residues, R and T, are tolerated in place
of a hydrophobic amino acid residue), 7 (T, and S, in place of P,
are tolerated), and 9 (P and A are tolerated in place of a
hydrophilic amino acid residue) have been observed.
[0095] 5.3.1. Binding specificities
[0096] It has been discovered that certain of the binding peptides
disclosed have a greater relative binding affinity for one SH3
domain over another. Referring now to FIG. 8, the relative binding
affinities of the various peptides described above toward different
SH3 domain targets are graphically presented. As one can see, the
relative binding affinities of the respective peptides can differ
by orders of magnitude. Thus, as shown in FIG. 8, the peptide
GPHRRLPPTPATR (SEQ ID NO:65), having the relevant sequence of the
phage clone identified as T12.SRC3.3, is specific to Src family SH3
domains, including, but not limited to, Src, Yes, Lck, Hck, Fgr,
Fyn, and Lyn. This SH3 binding peptide has little affinity for SH3
domains derived from PLC.gamma. or Grb2. On the other hand, the
peptide GILAPPVPPRNTR (SEQ ID NO:63), corresponding to the relevant
sequence of the phage clone T12.SRC3.1, which is one of the most
abundant binding clones found by the present method, binds
generically to a broad range of SH3 domains, including Src,
PLC.gamma., and Grb2.
[0097] On an intermediate level, the present invention has also
uncovered a peptide, VLKRPLPIPPVTR (SEQ ID NO:64), corresponding to
the relevant sequence of the phage clone T12.SRC3.6, which is Src
preferential; that is, this peptide exhibits strong binding
affinities for members of the Src family, some binding affinities
for Grb2 proteins, but little binding affinities for PLC.gamma.
domains. The peptide ANPSPATRPLPTR (SEQ ID NO:66), corresponding to
the relevant sequence of the phage clone T12.SRC3.2, also exhibits
Src family specificity similar to GPHRRLPPTPATR (SEQ ID NO:65). The
peptides RSTPRPLPMLPTTR (R8C.YES3.5; SEQ ID NO:62) and
RSTPRPLPPLPTTR (representative consensus motif; SEQ ID NO:67) are
highly specific for SH3 domain of Src, Yes, and other Src-related
proteins.
[0098] 5.4. Further Discussion of Binding Experiments
[0099] At the outset it is apparent that the binding affinity of
certain peptides to the SH3 domain of Src and Src-related proteins
is governed by more than just the presence of the preferred core
consensus sequences, RPLPPLP (SEQ ID NO:9) or RPLPMLP (SEQ ID
NO:95; i.e., RPLP(P/M)LP, SEQ ID NO:96). Thus, while the synthetic
peptides RSTPRPLPMLPTTR (R8C.YES3.5; SEQ ID NO:62) and
RSTPRPLPPLPTTR (consensus; (SEQ ID NO:67) exhibit a strong specific
binding affinity for Src SH3, the other synthetic peptides tested
also exhibited an avid binding affinity to SH3 domains relative to
the 7-mer, RPLPPLP (SEQ ID NO:9). These other peptides,
GILAPPVPPRNTR (SEQ ID NO:63), VLKRPLPIPPVTR (SEQ ID NO:64),
GPHRRLPPTPATR (SEQ ID NO:65), and ANPSPATRPLPTR (SEQ ID NO:66),
sport core sequences and flanking sequences that do not closely
adhere to the preferred core consensus sequences. Thus, these
results suggest that binding affinity to SH3 domains is governed to
a large extent by the nature of the amino acid residues flanking
the core 7-mer sequence.
[0100] The binding characteristics of Src SH3-selected peptides was
determined using synthetic biotinylated peptides corresponding to
the sequences displayed by Src SH3-selected phage. These
biotinylated peptides were assayed for direct binding to
immobilized Src SH3-GST. Each of the five library-derived peptides
tested were found to bind to Src SH3-GST and Yes SH3-GST over
background (FIG. 8). Furthermore, a strong correlation was observed
between the similarity of a given peptide to the preferred core
consensus sequence RPLP(P/M)LP (SEQ ID NO:96) and the peptidets
affinity for Src SH3-GST. The core sequence of the clone T12.SRC3.1
(GILAPPVPPRNTR; SEQ ID NO:63) appears to provide more generic SH3
domain-binding characteristics.
[0101] Experiments comparing the relative binding of various phage
clones to SH3 domains taken from a variety of proteins demonstrated
the preference of these clones for Src and Src-related SH3 domains
over SH3 domains taken from other proteins.
[0102] It was further found that while the 7-mer having the
consensus sequence RPLPPLP (SEQ ID NO:9) bound to Src SH3-GST only
weakly, peptides comprising the consensus sequence flanked by
residues encoded by one of the Src SH3-selected clones
(R8C.YES3.5), RSTP (SEQ ID NO:97) at the N-terminal end and TTR at
the C-terminal end, bound significantly better than any of the
peptides tested (FIG. 7). Thus, as stated previously, sequences
that flank the RPLP(P/M)LP (SEQ ID NO:96) core appear to be
important contributors to SH3 binding. It is further surmised that
a peptide having or resembling the sequence RSTPAPPVPPRTTR (SEQ ID
NO:98) should exhibit strong but generic binding to a variety of
SH3 domains.
[0103] Similarly, it is observed that most of the Src SH3-binding
motifs are located near the carboxy-terminus of the random
peptides, adjacent to sequences which are fixed in every clone
(FIG. 5). The exceptional clones tend to possess sequences that
resemble motifs that include fixed flanking sequences. This
clustering contrasts with previous results, in which binding motifs
are distributed throughout the random peptide. Kay, B. K., et al.,
in Gene (1993) 128:59-65.
[0104] The binding of the library-derived Src SH3-binding peptides
was compared to that of peptides corresponding to proline-rich
regions of natural proteins. Peptides corresponding to SH3-binding
regions in human PI-3' Kinase (KISPPTPKPRPPRPLPV; SEQ ID NO:69) and
human SOS1.20 (GTVEPVPPPVPPRRRPESA; SEQ ID NO:68), as well as a
proline-rich region of the cytoskeletal protein vinculin
(LAPPKPPLPEGEV; SEQ ID NO:70), bound Src SH3 much less well than
the library-derived peptides (FIG. 7).
[0105] As mentioned above, the relative specificity of binding was
explored. Thus, the relative binding of Src SH3-selected peptides
to equal amounts of GST fusions to SH3 domains from different
proteins was determined (FIG. 8). While all of the library-derived
peptides bound the Src and Yes SH3 domains almost equally well,
none of the peptides (with the exception of peptide T12.SRC3.1, the
most divergent peptide tested) bound the SH3 domains of Grb2, Crk,
Abl or PLC.gamma.1 appreciably. Thus, the library-derived peptides,
in contrast with a peptide derived from SOS1, exhibit SH3 binding
that is relatively specific for Src-family members.
[0106] Next, it was determined whether the binding to the Src SH3
domain was qualitatively like the interactions of the SH3 domain
and natural proteins found in cell lysates. Thus, radiolabeled
proteins were prepared from NIH 3T3 cell lysates and
chromatographed over Src SH3-GST immobilized on glutathione linked
Sepharose. SDS-PAGE shows that a number of proteins can be affinity
purified in this manner. The synthesized peptides bind quite well
to the Src SH3 domain, as they can compete the binding of
radiolabeled proteins from cell lysates to immobilized Src-GST,
with an IC.sub.50 of 1-10 mM (FIG. 9). In conclusion, the peptides
can efficiently block the interaction of cellular proteins with Src
SH3 in vitro.
[0107] Moreover, Xenopus laevis oocytes injected with mRNA encoding
constitutively active Src undergo progesterone-induced maturation
at an accelerated rate relative to oocytes injected with water or
c-Src MRNA. Unger, T. F. and Steele, R. E. in Mol. Cell. Biol.
(1992) 12:5485-5498. To explore the ability of the library-derived
Src SH3-binding peptides to exert a biochemical effect in vivo, the
influence of the peptides on the maturation of Xenopus laevis
oocytes was examined. Hence, stage VI oocytes were injected with
peptide, exposed to progesterone, and scored for germinal vesicle
breakdown. FIG. 10 shows that the rate of maturation was
accelerated by approximately one hour when oocytes were injected
with the SH3-binding peptide consisting of RPLPPLP (SEQ ID NO:9)
flanked by residues from clone T12.SRC3.6 (VLKRPLPIPPVTR; SEQ ID
NO:64), but not with water or a peptide corresponding to a
proline-rich segment of vinculin (LAPPKPPLPEGEV; SEQ ID NO:70) as
controls. The magnitude of this effect is roughly equivalent to
that seen with injection of mRNA encoding constituitively active
Src. See, e.g., FIG. 3B in Unger, T. F. and Steele, R. E., supra.
This result suggests that the library-derived Src SH3-binding
peptide is effectively relieving an inhibitory effect of the Src
SH3 domain upon Src PTK activity. This model is consistent with a
number of studies which have demonstrated an inhibitory effect of
the Src SH3 domain upon Src kinase and transforming activity. See,
e.g., Okada, M., et al., supra; Murphy, S. M., et al., supra; and
Superti-Furga, G., et al., supra.
[0108] 5.5. Diagnostic And Therapeutic Agents Based on SH3 Binding
Peptides and Additional Methods of Their Use
[0109] As already indicated above, the present invention also seeks
to provide diagnostic, prophylactic, and therapeutic agents based
on the SH3 binding peptides described herein.
[0110] In one embodiment, diagnostic agents are provided,
preferably in the form of kits, comprising an SH3 domain-binding
peptide and a detectable label conjugated to said peptide directly,
indirectly or by complexation, said peptide comprising: (i) a core
sequence motif of the formula RXLP.phi..phi.P (SEQ ID NO:71),
wherein X represents any amino acid except cysteine and .phi.
represents a hydrophobic amino acid residue, including F, Y, W, V,
A, I, L, P, M or G, each letter representing the standard
one-letter designation for the corresponding amino acid residue;
and (ii) two or more additional amino acid residues flanking said
core sequence at its C-terminal end, N-terminal end or both.
[0111] The diagnostic agents of the present invention can be used
to detect the presence of SH3 domains of a generic or specific type
in cells, tissues or organs either in vitro or in vivo. For in vivo
applications, the diagnostic agent is preferably mixed with a
pharmaceutically acceptable carrier for administration, either
enterally, parenterally or by some other route dictated by the
needs of the particular application.
[0112] In a particular embodiment, for example, an assay based on a
fusion product is contemplated which comprises a Src SH3
domain-binding peptide of the invention and a substrate for
deregulated or "activated" Src. For instance, a muscle biopsy,
taken from a subject suspected of being infected by the Rous
sarcoma virus, can be treated with an effective amount of the
fusion product. By subsequent analysis of the degree of conversion
of the substrate, one can potentially detect infection by the Rous
sarcoma virus in the subject, particularly mammals, especially
chickens. The presence of the retrovirus, which causes the
expression of deregulated or "activated" Src, may thus be indicated
by unusually high levels of Src as revealed by large amounts of the
converted substrate. See, for example, Paxton, W. G. et al., in
Biochem. Biophys. Res. Commun. (1994) 200(1):260-267 (detection of
phosphorylated tyrosine and serine residues of angiotensin II AT1
receptor, a substrate of Src family tyrosine kinases); another
suitable substrate may be the protein p68 (Fumagalli, S. et al., in
Nature (1994) 368(6474):871-874; Taylor, S. J. and Shalloway, D.,
in Ibid. at 867-871.
[0113] Alternatively, the enzyme can be isolated by selective
binding to a form of the SH3 domain-binding peptides of the present
invention (e.g., biotin-peptide conjugate). After isolation of the
protein-peptide conjugate complex (e.g., on a column comprising
streptavidin), the activity of the enzyme can then be assayed by
conventional methods to determine its level of protein kinase
activity which can be taken as an indication of the presence of the
deregulated or "activated" form of the enzyme. An assay for Src
kinase has been described by Klinz and Maness, in Neuroprotocols (a
companion to Neuroscience) (1992) 1(3):224-231.
[0114] Moreover, the diagnostic agents of the invention can also
serve as imaging agents of cells, tissues or organs, especially
those that contain proteins with an SH3 domain. For example, neural
cells (e.g., neurons, other areas of the brain), osteoclasts,
osteoblasts, platelets, immune cells, and other dividing cells are
known to express or contain proteins with SH3 domains. Thus, an
image can be taken of portions of the body to serve as a baseline
for subsequent images to detect physiologic or biochemical changes
in the subject's body. For instance, changes in the condition of
cellular levels of Src or a transformation of the cellular Src to
an "activated" form may be detected using the diagnostic or imaging
agents of the present invention.
[0115] Accordingly, it has been demonstrated that an SH3-binding
peptide tagged with a fluorescence emitter can provide an image of
the cytoskeleton. The images are presented in FIG. 11. As can be
seen from FIG. 11, panels A, B, and C show the fluorescence image
that is obtained on treating NIH 3T3 fibroblasts with SH3
domain-binding peptides modified to include a fluorescent tag. In
sharp contrast, panel D shows only a dark image that is produced
when the cells are treated with a proline-rich segment of vinculin
as a control.
[0116] In another embodiment, an SH3 domain-binding
peptide-horseradish immunoperoxidase complex or related
immunohistochemical agent could be used to detect and quantitate
specific receptor molecules in tissues, serum or body fluids. In
particular, the present invention provides useful diagnostic
reagents for use in immunoassays, Southern or Northern
hybridization, and in situ assays. Accordingly, the diagnostic
agents described herein may be suitable for use in vitro or in
vivo.
[0117] In addition, the diagnostic or imaging agent of the present
invention is not limited by the nature of the detectable label.
Hence, the diagnostic agent may contain one or more such labels
including, but not limited to, radioisotope, fluorescent tags,
paramagnetic substances, heavy metals, or other image-enhancing
agents. Those of ordinary skill in the art would be familiar with
the range of label and methods to incorporate or conjugate them
into the SH3 domain-binding peptide to form diagnostic agents.
[0118] In yet a further embodiment, pharmaceutical compositions are
provided comprising an SH3 domain-binding peptide and a
pharmaceutically acceptable carrier. In a specific embodiment of
the invention, the pharmaceutical composition is useful for the
modulation of the activity of SH3 domain-containing proteins. By
"modulation" is meant either inhibition or enhancement of the
activity of the protein target. Accordingly, a pharmaceutical
composition is disclosed comprising an SH3 domaih-binding peptide
and a pharmaceutically acceptable carrier, said peptide comprising:
(i) a 9-mer sequence motif of the formula RXLP.phi..phi.PX.psi.
(SEQ ID NO:10), wherein X represents any amino acid except
cysteine, .phi. represents a hydrophobic amino acid residue, and
wherein .psi. is a hydrophilic amino acid residue except cysteine,
each letter representing the standard one-letter designation for
the corresponding amino acid residue; and, optionally, (ii)
additional amino acid residues flanking the 9-mer sequence at its
C-terminal end, N-terminal end or both, up to a total of 45 amino
acid residues, including said 9-mer sequence. Preferably, the
peptide comprises at least one, more preferably at least two, and
most preferably at least three additional amino acids flanking the
9-mer sequence.
[0119] As stated above, the therapeutic or diagnostic agents of the
invention may also contain appropriate pharmaceutically acceptable
carriers, diluents and adjuvants. Such pharmaceutical carriers can
be sterile liquids, such as water and oils including those of
petroleum, animal, vegetable or synthetic origin, such as peanut
oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, magnesium carbonate,
magnesium stearate, sodium stearate, glycerol monostearate, talc,
sodium chloride, dried skim milk, glycerol, propylene, glycol,
water, ethanol and the like. These compositions can take the form
of solutions, suspensions, tablets, pills, capsules, powders,
sustained-release formulations and the like. Suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin.
[0120] Such compositions will contain an effective therapeutic
amount of the active compound together with a suitable amount of
carrier so as to provide the form for proper administration to the
subject. While intravenous injection is a very effective form of
administration, other modes can be employed, including but not
limited to intramuscular, intraperitoneal, and subcutaneous
infection, and oral, nasal, enteral, and parenteral
administration.
[0121] The therapeutic agents and diagnostic agents of the instant
invention are used for the treatment and/or diagnosis of animals,
and more preferably, mammals including humans, as well as dogs,
cats, horses, cows, pigs, guinea pigs, mice and rats. Accordingly,
other methods contemplated in the present invention, include, but
are not limited to, a method of modulating, i.e., inhibiting or
enhancing, bone resorption in a mammal (see, e.g., Hall, T. J., in
Biochem. Biophys. Res. Commun. (1994) 199(3):1237-44), a method of
disrupting protein tyrosine kinase-mediated signal transduction
pathways or a method of regulating the processing, trafficking or
translation of RNA in a cell by introducing or administering an
effective amount of an SH3 domain-binding peptide of the present
invention (see, e.g., Taylor, S. J. and Shalloway, D., supra).
[0122] The diagnostic or therapeutic agents of the present
invention can be modified by attachment to soluble macromolecules
such as proteins, polysaccharides, or synthetic polymers. For
example, the peptide could be coupled to styrene-maleic acid
copolymers (see, e.g., Matsumura and Maeda, Cancer Res. (1986)
46:6387), methacrylamide copolymers (Kopececk and Duncan, J.
Controlled Release (1987) 6:315), or polyethylene glycol (PEG)
(e.g., Hershfield and Buckley, N. Engl. J. Med. (1987) 316:589; Ho
et al., Drug Metab. Discos. (1986) 14:349; Chua et al., Ann.
Intern. Med. (1988) 109:114). The agents, if desired, are further
targeted by attachment to an antibody, especially a monoclonal
antibody. Such antibodies include but are not limited to chimeric,
single chain, Fab fragments, and Fab expression libraries. In one
embodiment the agent is coupled to the macromolecule via a
degradable linkage so that it will be released in vivo in its
active form.
[0123] In another embodiment, the therapeutic or diagnostic agent
may be delivered in a vesicle, in particular a liposome. See,
Langer, Science (1990) 249:1527-1533; Treat et al., in Liposomes in
the Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, N.Y. (1989) pp. 353-365; Lopez-Berestein,
ibid., pp. 317-327.
[0124] In yet another embodiment, the therapeutic or in vivo
diagnostic agent can be delivered in a controlled release system.
In one embodiment, a pump may be used (see Langer, supra; Sefton,
CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery
(1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). In
another embodiment, polymeric materials may be used (see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Pres., Boca Raton, Fla., 1974; Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.) Wiley,
N.Y. 1984; Raner and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.
(1983) 23:61; see, also, Levy et al., Science (1985) 228:190;
During et al., Ann. Neurol. (1989) 25:351; Howard et al., J.
Neurosurg. (1989) 71:105). In a preferred embodiment, a controlled
release system may be placed next to the therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, (1984)
2:115-138). It will be recognized by one of ordinary skill in the
art that a particular advantage of the invention is that a peptide
will not be subject to the problems of denaturation and aggregation
associated with proteins held in the warm, most environment of a
body in a controlled release system.
[0125] Other controlled release systems are discussed in the review
by Langer, in Science (1990) 249:1527-1533.
[0126] 5.6 Identification of Compounds that Affect Binding of SH3
Domain-containing Proteins and their Ligands
[0127] A common problem in the development of new drugs is that of
identifying a single, or a small number, of compounds that possess
a desirable characteristic from among a background of a large
number of compounds that lack that desired characteristic. This
problem arises both in the testing of compounds that are natural
products from plant, animal, or microbial sources and in the
testing of man-made compounds. Typically, hundreds, or even
thousands, of compounds are randomly screened by the use of in
vitro assays such as those that monitor the compound's effect on
some enzymatic activity or its ability to bind to a reference
substance such as a receptor or other protein.
[0128] The compounds which pass this original screening test are
known as "lead" compounds. These lead compounds are then put
through further testing, including, eventually, in vivo testing in
animals and humans, from which the promise shown by the lead
compounds in the original in vitro tests is either confirmed or
refuted. See Remington's Pharmaceutical Sciences, 1990, A. R.
Gennaro, ed., Chapter 8, pages 60-62, Mack Publishing Co., Easton,
PA; Ecker and Crooke, 1995, Bio/Technology 13:351-360.
[0129] There is, of course, a continual need for new compounds to
be tested in the in vitro assays that make up the first testing
step described above. There is also a continual need for new assays
by which the pharmacological activities of these compounds may be
tested. It is an object of the present invention to provide such
new assays to determine whether a candidate compound is capable of
affecting the binding between a protein or polypeptide containing
an SH3 domain and a ligand of the SH3 domain. A compound capable of
affecting this binding would be useful as a means of modulating the
pharmacological activity of proteins or polypeptides containing the
SH3 domain. The present invention provides suitable ligands for SH3
domains for use in such assays. Such assays can be performed where
the SH3 domains include, but are not limited to, SH3 domains from
Cortactin, Nck, Abl, PLC.gamma., Src, p53bp2, Crk, Yes, and
Grb2.
[0130] The present invention provides methods of identifying a
compound that affects the binding of a molecule comprising an SH3
domain and a ligand of the SH3 domain. The effect on binding can be
an increase or decrease in total amount of binding or in affinity
of bidning. Preferably, the effect is an inhibition (reduction in
or loss of binding).
[0131] Accordingly, the invention provides a method of identifying
an inhibitor of the binding between a first molecule comprising an
SH3 domain and a second molecule that binds to the SH3 domain
comprising incubating one or more compounds from which it is
desired to select such an inhibitor with the first molecule and the
second molecule under conditions conducive to binding and detecting
the one or more compounds that inhibit binding of the first
molecule to the second molecule.
[0132] In a particular embodiment of the above-described method,
the second molecule is obtained by:
[0133] (i) screening a peptide library with the SH3 domain to
obtain peptides that bind the SH3 domain;
[0134] (ii) determining a consensus sequence for the peptides
obtained in step (i);
[0135] (iii) producing a peptide comprising the consensus
sequence;
[0136] wherein the second molecule comprises the peptide comprising
the consensus sequence.
[0137] In another embodiment, the second molecule is obtained
by:
[0138] (i) screening a peptide library with the SH3 domain to
obtain peptides that bind the SH3 domain;
[0139] (ii) determining a consensus sequence for the peptides
obtained in step (i);
[0140] (iii) searching a database to identify amino acid sequences
that resemble the consensus sequence of step (ii);
[0141] (iv) producing a peptide comprising an amino acid sequence
identified in step (iii);
[0142] wherein the second molecule comprises the peptide comprising
an amino acid sequence identified in step (iii).
[0143] Second molecules that bind SH3 domains can be obtained by,
e.g., the use of diversity libraries, such as random or
combinatorial peptide or nonpeptide libraries which can be screened
for molecules that specifically bind to SH3 domains. Many libraries
are known in the art that can be used, e.g., chemically synthesized
libraries, recombinant (e.g., phase display libraries), and in
vitro translation-based libraries.
[0144] Examples of chemically synthesized libraries are described
in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991,
Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski,
1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal
Chemistry 37(9):1233-1251; ohlmeyer et al., 1993, Proc. Natl. Acad.
Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci.
USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618;
Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT
Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc.
Natl. Acad. Sci. USA 89:5381-5383.
[0145] Examples of phage display libraries are described in Scott
and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science,
249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol.
227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et
al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318
dated Aug. 18, 1994.
[0146] In vitro translation-based libraries include but are not
limited to those described in PCT Publication No. WO 91/05058 dated
Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci.
USA 91:9022-9026.
[0147] By way of examples of nonpeptide libraries, a benzodiazepine
library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA
91:4708-4712) can be adapted for use. Peptoid libraries (Simon et
al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be
used. Another example of a library that can be used, in which the
amide functionalities in peptides have been permethylated to
generate a chemically transformed combinatorial library, is
described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA
91:11138-11142).
[0148] Screening the libraries can be accomplished by any of a
variety of commonly known methods. See, e.g., the following
references, which disclose screening of peptide libraries: Parmley
and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith,
1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques
13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA
89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al.,
1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;
Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.
5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346,
all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673;
and PCT Publication No. WO 94/18318.
[0149] In a specific embodiment, screening can be carried out by
contacting the library members with an SH3 domain immobilized on a
solid phase and harvesting those library members that bind to the
SH3 domain. Examples of such screening methods, termed "panning"
techniques are described by way of example in Parmley and Smith,
1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques
13:422-427; PCT Publication No. WO 94/18318; and in references
cited hereinabove.
[0150] In another embodiment, the two-hybrid system for selecting
interacting proteins in yeast (Fields and Song, 1989, Nature
340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA
88:9578-9582) can be used to identify molecules that specifically
bind to SH3 domains.
[0151] A typical assay of the present invention consists of at
least the following components: (1) a molecule (e.g., protein or
polypeptide) comprising an SH3 domain; (2) a ligand of the SH3
domain; (3) a candidate compound, suspected of having the capacity
to affect the binding between the protein containing the SH3 domain
and the ligand. The assay components may further comprise (4) a
means of detecting the binding of the protein comprising the SH3
domain and the ligand. Such means can be e.g., a detectable label
affixed to the protein, the ligand, or the candidate compound.
[0152] In another specific embodiment, the invention provides a
method of identifying a compound that affects the binding of a
molecule comprising an SH3 domain and a ligand of the SH3 domain
comprising:
[0153] (a) contacting the SH3 domain and the ligand under
conditions conducive to binding in the presence of a candidate
compound and measuring the amount of binding between the SH3 domain
and the ligand;
[0154] (b) comparing the amount of binding in step (a) with the
amount of binding known or determined to occur between the molecule
and the ligand in the absence of the candidate compound, where a
difference in the amount of binding between step (a) and the amount
of binding known or determined to occur between the molecule and
the ligand in the absence of the candidate compound indicates that
the candidate compound is a compound that affects the binding of
the molecule comprising an SH3 domain and the ligand.
[0155] A kit is provided that comprises, in one or more containers,
one or more components of the assay of the invention, e.g., a first
molecule comprising an SH3 domain and a second molecule that binds
to the SH3 domain.
[0156] In one embodiment, the assay comprises allowing the protein
or polypeptide containing an SH3 domain to contact the ligand of
the SH3 domain in the presence and in the absence of the candidate
compound under conditions such that binding of the ligand to the
protein containing an SH3 domain will occur unless that binding is
disrupted or prevented by the candidate compound. By detecting the
amount of binding of the ligand to the protein containing an SH3
domain in the presence of the candidate compound and comparing that
amount of binding to the amount of binding of the ligand to the
protein or polypeptide containing an SH3 domain in the absence of
the candidate compound, it is possible to determine whether the
candidate compound affects the binding and thus is a useful lead
compound for the modulation of the activity of proteins containing
the SH3 domain. The effect of the candidate compound may be to
either increase or decrease the binding.
[0157] One version of an assay suitable for use in the present
invention comprises binding the protein containing an SH3 domain to
a solid support such as the wells of a microtiter plate. The wells
contain a suitable buffer and other substances to ensure that
conditions in the wells permit the binding of the protein or
polypeptide containing an SH3 domain to its ligand. The ligand and
a candidate compound are then added to the wells. The ligand is
preferably labeled, e.g., it might be biotinylated or labeled with
a radioactive moiety, or it might be linked to an enzyme, e.g.,
alkaline phosphatase. After a suitable period of incubation, the
wells are washed to remove any unbound ligand and compound. If the
candidate compound does not interfere with the binding of the
protein or polypeptide containing an SH3 domain to the labeled
ligand, the labeled ligand will bind to the protein or polypeptide
containing an SH3 domain in the well. This binding can then be
detected. If the candidate compound interferes with the binding of
the protein or polypeptide containing an SH3 domain and the labeled
ligand, label will not be present in the wells, or will be present
to a lesser degree than is the case when compared to control wells
that contain the protein or polypeptide containing an SH3 domain
and the labeled ligand but to which no candidate compound is added.
Of course, it is possible that the presence of the candidate
compound will increase the binding between the protein or
polypeptide containing an SH3 domain and the labeled ligand.
Alternatively, the ligand can be affixed to solid substrate during
the assay.
[0158] The present invention provides ligands capable of binding
SH3 domains that are suitable for incorporation into assays such as
those described above. Ligands provided by the present invention
include those SH3 domain-binding amino acid sequences disclosed in
Tables 1-13 below and proteins or polypeptides containing those
amino acid sequences. Also provided are nucleic acids encoding the
SH3 domain-binding amino acid sequences disclosed in Tables 1-13
below.
6. EXAMPLES
[0159] 6.1. Preparation of the TSAR-9 Library
[0160] 6.1.1. synthesis and Assembly of Oligonucleotides
[0161] FIG. 1 shows the formula of the oligonucleotides and the
assembly scheme used in construction of the TSAR-9 library. The
oligonucleotides were synthesized with an applied Biosystems 380a
synthesizer (Foster City, Calif.), and the full-length
oligonucleotides were purified by HPLC.
[0162] Five micrograms of each of the pair of oligonucleotides were
mixed together in buffer (10 mM Tris-HCl, pH 8.3, 15 mM KCl, 0.001%
gelatin, 1.5 mM magnesium chloride), with 0.1% Triton X-100, 2 mM
dNTP's, and 20 units of Tag DNA polymerase. The assembly reaction
mixtures were incubated at 72.degree. C. for 30 seconds and then
30.degree. C. for 30 seconds; this cycle was repeated 60 times. It
should be noted that the assembly reaction is not PCR, since a
denaturation step was not used. Fill-in reactions were carried out
in a thermal cycling, device (Ericomp, LaJolla, Calif.) with the
following protocol: 30 seconds at 72.degree. C., 30 seconds at
30.degree. C., repeated for 60 cycles. The lower temperature allows
for annealing of the six base complementary region between the two
sets of the oligonucleotide pairs. The reaction products were
phenol/chloroform extracted and ethanol precipitated. Greater than
90% of the nucleotides were found to have been converted to double
stranded synthetic oligonucleotides.
[0163] After resuspension in 300 AL of buffer containing 10 mM
Tris-HCI, pH 7.5, 1 mM EDTA (TE buffer), the ends of the
oligonucleotide fragments were cleaved with Xba I and Xho I (New
England BioLabs, Beverly, Mass.) according to the supplier's
recommendations. The fragments were purified by 4% agarose gel
electrophoresis. The band of correct size was removed and
electroeluted, concentrated by ethanol precipitation and
resuspended in 100 .mu.L TE buffer. Approximately 5% of the
assembled oligonucleotides can be expected to have internal Xho I
or Xba I sites; however, only the full-length molecules were used
in the ligation step of the assembly scheme. The concentration of
the synthetic oligonucleotide fragments was estimated by comparing
the intensity on an ethidium bromide stained gel run along with
appropriate quantitated markers. All DNA manipulations not
described in detail were performed according to Maniatis,
supra.
[0164] To demonstrate that the assembled enzyme digested
oligonucleotides could be ligated, the synthesized DNA fragments
were examined for their ability to self-ligate. The digested
fragments were incubated overnight at 18.degree. C. in ligation
buffer with T4 DNA ligase. When the ligation products were examined
by agarose gel electrophoresis, a concatamer of bands was visible
upon ethidium bromide staining. As many as five different unit
length concatamer bands (i.e., dimer, trimer, tetramer, pentamer,
hexamer) were evident, suggesting that the synthesized DNA
fragments were efficient substrates for ligation.
[0165] 6.1.2. Construction of Vectors
[0166] The construction of the M13 derived phage vectors useful for
expressing a TSAR library has been recently described (Fowlkes, D.
et al. BioTech. (1992) 13:422-427). To express the TSAR-9 library,
an M13 derived vector, m663, was constructed as described in
Fowlkes. The m663 vector contains the pIII gene having a
c-myc-epitope, i.e., as a stuffer fragment, introduced at the
mature N-terminal end, flanked by Xho I and Xba I restriction sites
(see also, FIG. I of Fowlkes).
[0167] 6.1.3. Expression of the TSAR-9 Library
[0168] The synthesized oligonucleotides were then ligated to Xho I
and Xba I double-digested m663 RF DNA containing, the pIII gene
(Fowlkes) by incubation with ligase overnight at 12.degree. C. More
particularly, 50 ng of vector DNA and 5 ng of the digested
synthesized DNA and was mixed together in 50 .mu.L ligation buffer
(50 mM Tris, pH 8.0, 10 mM MgCl.sub.2, 20 mM DTT, 0.1 mM ATP) with
T4 DNA ligase. After overnight ligation at 12.degree. C., the DNA
was concentrated by ethanol precipitation and washed with 70%
ethanol. The ligated DNA was then introduced into E. coli
(DH5.alpha.F'; GIBCO BRL, Gaithersburg, Md.) by
electroporation.
[0169] A small aliquot of the electroporated cells was plated and
the number of plaques counted to determine that 10.sup.8
recombinants were generated. The library of E. coli cells
containing recombinant vectors was plated at a high density
(.about.400,000 per 150 mM petri plate) for a single amplification
of the recombinant phage. After 8 hr, the recombinant bacteriophage
were recovered by washing each plate for 18 hr with SMG buffer (100
mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl.sub.2, 0.05% gelatin)
and after the addition of glycerol to 50% were frozen at
-80.degree. C. The TSAR-9 library thus formed had a working titer
of -2.times.10.sup.11 pfu/ml.
[0170] 6.2. Preparation of the TSAR-12 Library
[0171] FIG. 2 shows the formula for the synthetic oligonucleotides
and the assembly scheme used in the construction of the TSAR-12
library. As shown in FIG. 2, the TSAR-12 library was prepared
substantially the same as the TSAR-9 library described in Section
6.1 above with the following exceptions: (1) each of the variant
non-predicted oligonucleotide sequences, i.e., NNB, was 30
nucleotides in length, rather than 54 nucleotides; (2) the
restriction sites included at the 5' termini of the variant,
non-predicted sequences were Sal I and Spe 1, rather than Xho I and
Xba I; and (3) the invariant sequence at the 3' termini to aid
annealing of the two strands was GCGGTG and CGCCAC rather than
CCAGGT and GGTCCA (5' to 3').
[0172] After synthesis including numerous rounds of annealing and
chain extension in the presence of dNTP's and Taq DNA polymerase,
and purification as described above in Section 6.1.1, the synthetic
double stranded, oligonucleotide fragments were digested with Sal I
and Spe I restriction enzymes and ligated with T4 DNA ligase to the
nucleotide sequence encoding the M13 pIII gene contained in the
m663 vector to yield a library of TSAR-expression vectors as
escribed in Sections 6.1.2 and 6.1.3. The ligated DNA was hen
introduced into E. coli (DH5.alpha.F'; GIBCO BRL, aithersburg, MD
by electroporation. The library of E. coli cells were plated at
high density (.about.400,000 per 150 mm petri late) for
amplification of the recombinant phage. After bout 8 hr, the
recombinant bacteriophage were recovered by ashing, for 18 hr with
SMG buffer and after the addition of lycerol to 50% were frozen at
-80.degree. C.
[0173] The TSAR-12 library thus formed had a working titer of
.about.2.times.10.sup.11 pfu/mL.
[0174] 6.3. Characterization of the TSAR-9 and -12 Libraries
[0175] The inserted synthetic oligonucleotides for each of the TSAR
libraries, described in Sections 6.1 and 6.2 above, ad a potential
coding complexity of 20.sup.36 (.about.10.sup.47) and 20.sup.20,
respectively, and since .about.10.sup.14 molecules were used in
each transformation experiment, each member of these TSAR libraries
should be unique. After plate amplification the library solution or
stock has 10.sup.4 copies of each member/mL.
[0176] It was observed that very few (<10%) of the inserted
oligonucleotide sequences characterized so far in both of the
libraries have exhibited deletions or insertions. This is likely a
reflection of the accuracy assembling the oligonucleotides under
the conditions used and the fact that certain types of mutations
(i.e., frame-shifts) would not be tolerated as pIII an essential
protein for phage propagation.
[0177] In order to determine whether any coding bias existed in the
variant non-predicted peptides expressed by these libraries,
perhaps due to biases imposed in vitro during synthesis of the
oligonucleotides or in vivo during expression by the reproducing
phage, inserts were sequenced as set forth below.
[0178] 6.3.1. Characterization of TSAR-9 Library
[0179] Inserted synthetic oligonucleotide fragments of 23 randomly
chosen isolates were examined from the TSAR-9 library. Individual
plaques were used to inoculate I ml of 2XYT broth containing E.
coli (DH5.alpha.F') cells and the cultures were allowed to grow
overnight at 37.degree. C. with aeration. DNA was isolated from the
culture supernatants according to Maniatis, supra. Twenty-three
individual isolates were sequenced according to the method of
Sanger (Proc. Natl. Acad. Sci. USA (1979) 74:5463-5467) using as a
primer the oligonucleotide 5'-AGCGTAACGATCTCCCG (SEQ ID NO. 99),
which is 89 nucleotides downstream of the pIII gene cloning site of
the m663 vector used to express the TSARS.
[0180] Nucleotide sequences and their encoded amino acid sequences
were analyzed with the MacVector computer program (IBI, New Haven,
Conn.). The Microsoft EXCEL program was used to evaluate amino acid
frequencies. Such analyses showed that the nucleotide codons coding
for and hence most amino acids, occurred at the expected frequency
in the TSAR-9 library of expressed proteins. The notable exceptions
were glutamine and tryptophan, which were over- and
under-represented, respectively.
[0181] It is of interest to note the paucity of TAG stop codons in
the inserts, i.e., only 2 of .about.200 isolates characterized
contained a TAG stop codon. About half [1-(47/48).sup.36] of the
phage inserts were expected to have at least one TAG codon in view
of the assembly scheme used. However, most of the TAG-bearing phage
appear to have been lost from the library, even though the
bacterial host was supE. This may be a consequence of suppression
being less than 100% effective.
[0182] The amino acids encoded by the inserted double stranded
synthesized oligonucleotide sequences, excluding the fixed
PG-encoding centers, were concatenated into a single sequence and
the usage frequency determined for each amino acid using the
Microsoft EXCEL program. These frequencies were compared to that
expected from the assembly scheme of the oligonucleotides, and the
divergence from expected values represented by the size of the bars
above and below the baseline. Chi square analysis was used to
determine the significance of the deviations. The majority of amino
acids were found to occur at the expected frequency, with the
notable exceptions that glutamine and tryptophan were somewhat
over- and under-represented, respectively. Thus, except for the
invariant Pro-Gly, any position could have any amino acid; hence,
the sequences are unpredicted or random.
[0183] 6.3.2. Characterization of TSAR-12 Library
[0184] Approximately 10 randomly chosen inserted oligonucleotides
from the TSAR-12 library were examined by DNA sequencing as
described above in Section 6.3.1. The isolates were chosen at
random from the TSAR-12 library and prepared for sequencing, as
were the TSAR-9 isolates. Analysis showed that except for the
invariant Gly any position could have any amino acid; hence, the
sequences are unpredicted or random.
[0185] 6.4. Preparation of R8C Library
[0186] Referring now to FIG. 3, two oligonucleotides were
synthesized on an Applied Biosystems Model 380a machine with the
sequence
5'-TGACGTCTCGAGTTGTNNKNNKNNKNNKNNKNNKNNKNNKTGTGGATCTAGAAGGATC-3'
(SEQ ID NO:31) and 5'-GATCCTTCTAGATCC-3' (SEQ ID NO:32), where N is
an equimolar ratio of deoxynucleotides A, C, G, and T, and K is an
equimolar ratio of G and T. Fifty pmol of each oligonucleotide was
incubated at 42.degree. C. for 5 min, then 37.degree. C. for 15
min, in 50 .mu.L of Sequenase.TM. buffer (U.S. Biochemicals,
Cleveland, Ohio) with 0.1 .mu.g/.mu.L acetylated BSA, and 10 mM
DTT. After annealing, 10 units of Sequenase.TM. (U.S. Biochemicals)
and 0.2 mM of each dNTP were added and incubated at 37.degree. C.
for 15 min. The sample was then heated at 65.degree. C. for 2 hr,
digested with 100 units of both Xho I and Xba I (New England
BioLabs, Beverly, Mass.), phenol extracted, ethanol precipitated,
and resolved on a 15% non-denaturing polyacrylamide gel. The
assembled, digested fragment was gel purified prior to ligation.
The. vector, m663 (Fowlkes, D. et al. Biotech. (1992) 13:422-427),
was prepared by digestion with Xho I and Xba I, calf alkaline
phosphatase (Boehringer Mannheim, Indianapolis, Ind.) treatment,
phenol extracted, and purified by agarose gel electrophoresis. To
ligate, 20 .mu.g vector was combined with 0.2 .mu.g insert in 3 mL
with T4 DNA ligase (Boehringer Mannheim), according to the
manufacturer. S After removal of the protein and buffer by phenol
extraction and ethanol precipitation, the ligated DNA was
electroporated into XL1-Blue E. coli (Stratagene, San Diego,
Calif.) and plated for eight hours at 37.degree. C. To recover the
recombinant phage, the top agar was collected with a spatula, mixed
with an equal volume of 100 mM NaCl, 10 mM MgCl.sub.2, and 50 mM
Tris-HCI (pH7.5), and disrupted by two passes through an 18-gauge
syringe needle. The bacterial cells were removed by centrifugation,
and phage particles were collected by polyethylene glycol
precipitation and stored at -70.degree. C. in 25% glycerol. The
library had 10.sup.8 total recombinants and a working titer of
6.times.10.sup.13pfu/mL.
[0187] Members of the library were checked for inserts by the
polymerase chain reaction (Saiki, et al. Science (1988)
239:487-491). Individual plaques on a petri plate were touched with
a sterile toothpick and the tip was stirred into 2xYT with F.sup.+
E. coli bacteria and incubated overnight at 37.degree. C. with
aeration. Five microliters of the phage supernatant were then
transferred to new tubes containing buffer (67 mM Tris-HCl, pH
8.8/10 mM .beta.- mercaptoethanol/16.6 mM ammonium sulfate/6.7 mM
EDTA/50 .mu.g bovine serum albumin per mL), 0.1 mM deoxynucleotide
triphosphates, and 1.25 units of Taq DNA polymerase (Boehringer
Mannheim, Indianapolis, Ind.) with 100 pmoles of oligonucleotide
primers. The primers flanked the cloning site in gene III of m663
(5'-TTCACCTCGAAAGCAAGCTG-3' (SEQ ID NO:100) and
[0188] 5'-CCTCATAGTTAGCGTAACG-3' (SEQ ID NO:101)). The assembly
reactions were incubated at 94.degree. C. for 1 min, 56.degree. C.
for 2 min, and 72.degree. C. for 3 min; this cycle was repeated 24
times. The reaction products were then resolved by electrophoresis
on a NuSieve 2.0% agarose gel (FMC, Rockland, Me.). Gels revealed
that for 20 plaques tested, all were recombinant and had single
inserts of the expected size.
[0189] Based on the sample size of the library, it was anticipated
that 100% of the recombinants had single inserts. However, all of
the SH3-binding phage isolated from the R8C library had
double-inserts. Such phage are presumed rare (i.e., <5%) within
the library, yet because the SH3-binding peptide appears to need to
be linear they were selected for by our screening methods. Most
likely they were formed during the generation of the library; one
scenario is that the inserts ligated together to form head-to-head
dimers and that they were subsequently cloned into m663 DNA by
ligation with the vector's Xho I sticky end and by illegitimate
ligation with the vector's Xba I site (see, FIG. 4).
[0190] 6.5. Preparation Of Target-Coated Microtiter Wells
[0191] 6.5.1. Preparation Of GST-SH3 Fusion Proteins
[0192] The preparation of Src-GST fusion protein was first
described by Smith and Johnson, in Gene (1988) 67:31, the
disclosure of which is incorporated by reference herein. Briefly,
pGEX-derived (Pharmacia, Piscataway, N.J.) constructs expressing
GST fusion proteins containing the SH3 domains of Src, Grb2, Crk,
Abl, or PLC.gamma. were obtained from Dr. Channing Der (University
of North Carolina at Chapel Hill); a construct expressing the SH3
domain of Yes was obtained from Dr. Marius Sudol (Rockefeller
University). The use of the pGEX bacterial expression vector for
the production of GST-SH3 fusion proteins is well-known to those in
the art. See, e.g., Cicchetti, P. et al., in Science (1992)
257:803-806. Briefly, the coding region for a particular SH3 domain
can be fused in-frame at the Bam HI site of pGEX-2T. Thus, fusion
proteins were prepared as per the manufacturer's instructions, and
quantified by Coomassie Blue staining of SDS-polyacrylamide gels.
Microtiter wells were coated with 5-20 .mu.g GST-SH3 fusion protein
in 100 mM NaHCO.sub.3, pH 8.5, blocked with 100 mM NaHCO.sub.3 (pH
8.5) 1% BSA, and washed. All washes consisted of five applications
of 1XPBS, 0.1% Tween 20, 0.1% BSA (Buffer A). Where appropriate,
the amount of protein bound to each well was quantified with an
anti-GST antibody-based ELISA (Pharmacia, Piscataway, N.J.), and
with a GST-binding phage, isolated during the course of this
work.
[0193] 6.5.2. Coating of Microtiter Wells
[0194] Bacterially expressed Src SH3 glutathione-S-transferase
(Src-GST) fusion protein was purified from bacterial lysates using
glutathione agarose 4B (Pharmacia), according to the manufacturer's
instructions. Bourd Src-GST fusion protein was eluted from the
glutathione agarose with 10 mM glutathione in PBS. Microtiter wells
were then coated with Src-GST fusion protein (1-10 .mu.g/well, in
50 mM NaHCO.sub.3, pH 8.5) overnight at 4.degree. C. To block
non-specific binding of phage, 100 .mu.L 1% BSA in 100 mM
NaHCO.sub.3, pH 8.5, was added to each well and allowed to incubate
at room temperature for 1 hour. The wells were then washed five
times with 200 .mu.L PBS, 0.1% Tween 20, 0.1% BSA (Buffer A).
[0195] 6.6. Biopanning And Subsequent Characterization of
Phage-Displayed Random Peptide Libraries With Src-GST Fusion
Protein As Target Molecule
[0196] 6.6.1. Isolation of Src SH3-Binding Phage
[0197] Library screens were performed as previously described. Kay,
B. K., et al., in Gene (1993) 128:59-65. Briefly, 1.times.10.sup.11
pfu TSAR 9, TSAR 12, or R8C phage in Buffer A were incubated in a
Src SH3-GST-coated well for 2 hours. The wells were washed, and
bound phage were eluted with 100 .mu.L 50 mM glycine.HCl (pH 2.2),
transferred to a new well, and neutralized with 100 mL 200 mM
NaHPO.sub.4 (pH 7.0). Recovered phage were used to infect
1.times.10.sup.9 DH5.alpha.F' E. coli cells in 20 mL 2.times.YT;
the infected cells were grown overnight, resulting in a 1000- to
10,000-fold amplification of phage titer. Amplified phage were
panned twice more, as above, excepting the amplification step.
Binding phage recovered after the third round of panning were
plated at a low density on a lawn of DH5.alpha.F' E. coli cells to
yield isolated plaques for clonal analysis. Isolated plaques were
used to produce small cultures from which phage stocks and DNA were
recovered for phage binding experiments and dideoxy sequencing
(Sanger, F., et al., in Proc. Nati. Acad. Sci. USA (1977)
74:5463-5467), respectively. Clones were confirmed as binding the
SH3 domain by applying equal titers of phage to wells containing
Src SH3-GST or GST alone, and titering the number of eluted
particles from each well, or detecting bound phage with an
anti-phage antibody-based ELISA (Pharmacia).
[0198] Indeed, the ability of isolated phage clones to bind to
several SH3 domains derived from a variety of different proteins
can be investigated by the manner described above. GST-SH3 fusion
proteins containing SH3 domains from a variety of different
proteins are bound to microliter wells. An aliquot of the
aforementioned phage stocks (50 .mu.L) is introduced into wells
containing the different GST-SH3 fusion proteins. After room
temperature incubation for 1-2 hours, the liquid contents of the
microtiter plates are removed, and the wells are washed 5 times
with 200 AL Buffer A. Bound phage are eluted with 100 AL 50 mM
glycine (pH 2.2), transferred to a new well, and neutralized with
100 .mu.L 200 mM NaHPO.sub.4 (pH 7.0). The phage are diluted
10.sup.3- to 10.sup.-6-fold, and aliquots are plated onto lawns of
DH5.alpha.F' E. coli cells to establish the number of plaque
forming units in the output sample. From these experiments, the
relative specificity of different Src SH3 binding clones for SH3
domains derived from other proteins is determined.
[0199] 6.6.2. Phage ELISA and Nucleotide Sequencing
[0200] To evaluate the binding of isolates to various targets
proteins, enzyme-linked-immuno-assays (ELISA) were also performed.
Bacterial cultures were infected with phage isolates and cultured
overnight in 2XYT at 37.degree. C. The cells were spun down and 25
mL of supernatant was added to microtiter plate wells coated with
50 AL of protein (1 mg/mL in 100 mM NaHCO.sub.3, pH 8.4; overnight
at 4.degree. C. or for a few hours at room temperature) and blocked
(1 mg/mL BSA in 100 mM NaHCO.sub.3, pH 8.4; for about one hour).
The phage are incubated in the well with 25 .mu.L of PBS-0.1% Tween
20 at RT for 2 hr. The wells are then washed multiple times over 30
minutes. To each well is added 50 .mu.L of polyclonal anti-phage
antibody conjugated to horseradish peroxidase. The antibody is
diluted 1:3000 in BSA-PBS-Tween 20; it was obtained from Pharmacia
(Piscataway, N.J.; catalog number 27-9402-01). After minutes, the
wells are washed again with BSA-PBS-Tween 20 for .sup.-20 minutes.
Finally, 100 AL of ABTS reagent (Pharmacia, with H.sub.2O.sub.2)
are added to each well for the development of color. Plates are
read with a plate reader (Molecular Devices, Menlo Park, Calif.) at
405 nm wavelength.
[0201] The nucleotide sequence of the relevant segments of the Src
SH3 binding clones (or phage clones that bind to SH3 domains of
other proteins) were sequenced using standard methods. Sanger, F.,
et al., in Proc. Natl. Acad. Sci. USA (1977) 74:5463-5467. The
oligo primer 5'-AGCGTAACGATCTAAA-3' (SEQ ID NO:102) was used, which
is 89 nucleotides downstream of the gene III cloning site of M13
m666. The nucleotide sequences were analyzed with the MacVector
computer program (IBI, New Haven, Conn., USA). From this nucleotide
sequence information the primary sequence of each Src SH3 binding
peptide was deduced. The corresponding synthetic peptides were then
prepared by techniques well known in the art with or without
flanking sequences. Indeed, these synthetic peptides have been
shown to bind to SH3 domain targets, with those possessing the
phage flanking amino acid residues exhibiting greater binding
affinity.
[0202] 6.7 In Vitro Peptide Binding Assays
[0203] Peptides were obtained from Research Genetics (Birmingham,
Ala.), Chiron Mimotopes (Victoria, Australia), or synthesized by
conventional techniques by Dr. J. Mark Carter of Cytogen
Corporation (Princeton, N.J.). Peptide purity was assessed by HPLC
and/or mass spectrometry. Biotinylated peptides were synthesized
with either a KSGSG (SEQ ID NO:103) or a GSGS (SEQ ID NO:104)
peptide linker (a spacer) between the biotin and the N-terminus of
the peptide. Binding experiments were performed as above, excepting
the use of 10 AM peptide instead of phage. Bound biotinylated
peptide was detected with streptavidin conjugated to alkaline
phosphatase (Sigma Chemical Co., St. Louis, Mo.). After one hour
incubation period at room temperature, the wells were washed, and a
solution of 3 mM p-nitrophenyl-phosphate (US Biochemicals,
Cleveland, Ohio) in 50 mM NaCO.sub.3 (pH 9.8), and 50 mM MgCl.sub.2
was added and color allowed to develop. Signals were read with an
ELISA plate reader (Molecular Devices, Menlo Park, Calif.) at 405
nm wavelength. Binding experiments were performed in triplicate.
The results are presented in FIGS. 7 and 8.
[0204] 6.8. Peptide Competition of GST-SH3 Affinity Precipitations
of Cell Lysates
[0205] Labeled proteins are-prepared by incubating a culture of
HeLa cells overnight with .gtoreq.100 .mu.Ci/mL
.sup.35S-methionine. The cells are then washed and lysed with mild
detergent. This mixture of radioactive proteins is incubated with
Src-GST fusion protein that has been immobilized on
glutathione-linked Sepharose beads (Pharmacia, Piscataway, N.J.).
After several hours of tumbling, the beads are pelleted gently by
low-speed centrifugation, and the supernatant is discarded. The
beads are then resuspended into a slurry in PBS-0.1% Tween 20,
pelleted, and washed several additional times. Finally, a 2% SDS
solution is added to the sample, which is then boiled at
100.degree. C. for 3 minutes. Afterward, the sample is centrifuged,
and the supernatant loaded on a 10% polyacrylamide SDS gel for
electrophoresis. After the proteins have been resolved, the gel is
fixed, dried down, and exposed to X-ray film for autoradiography or
phosphor plates for scanning by a Molecular Dynamics
PhosphorImager.
[0206] The ability of Src SH3 to bind certain .sup.35S-labeled
proteins is examined for competability with exogenous peptides.
Synthetic peptides corresponding to phage-displayed inserts and
motifs are added at the time that the lysate is incubated with the
Src-GST fusion protein immobilized on glutathione-linked sepharose
beads. The SH3 inding peptides block binding of all or some of the
labeled roteins while negative control peptides (unrelated peptide
sequences) do not. The amount of competition is quantified and
correlated with the amount of added SH3-domain binding
peptides.
[0207] Alternatively, NIH 3T3 cells were grown in Dulbecco's
Modified Eagle Medium (DME)+10% fetal calf serum (FCS)+80 .mu.Ci/mL
Tran.sup.35Slabel (ICN), washed with PBS, lysed in RIPA buffer, and
pelleted. Supernatant from 1.5.times.10.sup.6 cells was recleared
with 100 .mu.g glutathione-agarose-immobilized GST. The supernatant
was then incubated with 10 .mu.g glutathione-agarose-immobilized
GST-SH3 fusion protein with or without added test peptide in a
final volume of 250 .mu.L. Pelleted beads were washed with 1 mL
each of RIPA, RIPA+1% deoxycholate+0.1% SDS, and PBS, resuspended
in 50 .mu.L SDS-PAGE sample buffer, boiled, and subjected to
SDS-PAGE (7.5%). Labeled proteins were detected by phosphorimaging
(Molecular Dynamics). The results are presented in FIG. 9.
[0208] 6.9. Peptide Competition of GST-SH3 Affinity Precipitations
of PI-3' Kinase From Cell Lysates
[0209] It is possible to follow the precipitation of PI-3' Kinase
by Src from cell lysates in the presence or absence of SH3-binding
peptides. HeLa cells are lysed with detergent and the protein
mixtures are incubated for several hours with the Src-GST fusion
protein immobilized on glutathione-linked Sepharose beads. After
several hours of tumbling, the beads are pelleted gently by
low-speed centrifugation and the supernatant is discarded. The
beads are then resuspended into a slurry in PBS-0.1% Tween 20,
pelleted, and washed several additional times. Finally, an SDS
solution is added to the sample, which is then boiled at
100.degree. C. for 3 minutes. Subsequently, the sample is
centrifuged, and the supernatant is loaded on a 10% polyacrylamide
SDS gel for electrophoresis. After the proteins have been resolved,
the gel is blotted to nitrocellulose or nylon (i.e., western blot).
The filter is then probed with a PI-3' Kinase antibody (monoclonal
and polyclonal antibodies are available from Upstate Biotechnology
Incorporated, Lake Placid, N.Y.) and an enzyme-linked secondary
antibody. The amount of PI-3' Kinase is then quantitated.
[0210] The ability of Src SH3 to bind PI-3' Kinase is examined for
competability with exogenous peptides. Synthetic peptides
corresponding to phage-displayed inserts and motifs are added at
the time that the lysate is incubated with the Src-GST fusion
protein that has been immobilized on glutathione-linked sepharose
beads. Ten-fold and one hundred-fold molar excess of peptides are
used relative to SH3 proteins. The SH3 binding peptides block
binding of the PI-3' Kinase as detected on western blots while
negative control peptides (unrelated peptide sequences) do not. The
amount of competition is quantified and correlated with the amount
of added SH3-domain binding peptides.
[0211] 6.10. In Vivo Association Of SH3-Binding Peptides With
SH3-Domains Of Proteins
[0212] To demonstrate association of the SH3-binding peptides with
SH3-domains of proteins inside cells, the SH3-binding peptides are
tagged and localized in cells. For example, Bar-Sagi et al., in
Cell (1993) 74:83-91, have shown that SH3-binding proteins localize
to the cytoskeleton when expressed in cells. Thus, the SH3
domain-binding peptides of the present invention can serve as
cellular targetting signals (e.g., to the cytoskeleton).
Accordingly, the peptides are tagged with biotin and, subsequently,
injected into cells. Alternatively, one can transfect into cells a
recombinant plasmid that expresses a fusion protein comprising of
the SH3-binding peptide and the green fluorescent protein (GFP,
Chalfie et al., in Science (1994) 263:802-805). The location of the
biotinylated peptide or the GFP fusion protein is then assayed with
FITC-labeled streptavidin in paraformaldehyde-fixed cells or by
direct fluorescence in living cells, respectively. Localization of
the SH3-binding peptides to the cytoskeleton demonstrates that the
SH3-binding peptides can bind SH3-domain proteins in vivo. In
addition, focal adhesions, which are rich in Src, are also sites of
potential subcellular localization of SH3-binding peptides.
[0213] Thus, NIH 3T3 fibroblasts were cultured in vitro on glass
coverslips coated with fibronectin. After two days of growth at
37.degree. C., the cells were fixed for one hour at room
temperature in the presence of 2% paraformaldehyde (pH 7.5). The
coverslips were washed with PBS-0.1% Tween 20 several times to
remove the fixative. Next, the coverslips were dipped into acetone
(chilled at -20.degree. C.) for approximately 20 seconds and
allowed to air-dry. The coverslips were washed again with PBS-0.1%
Tween 20, containing BSA (1 mg/mL), and incubated for 2 hours at
room temperature with different biotinylated peptides in PBS-0.1%
Tween 20. The coverslips were washed and then incubated with 1
mg/mL streptavidin-Cy3 (Jackson Immunoresearch Co., West Grove,
Pa.) for 1 hour at room temperature. Finally, the coverslips were
washed in PBS-0.1% Tween 20, mounted in a glycerol solution on a
glass slide, and viewed with a Nikon Optiphot epifluorescence
microscope and a 60.times. oil immersion lens.
[0214] The results are presented in FIG. 11, in which panel A
displays cells stained with the conjugate
biotin-spacer-VLKRPLPIPPVTR (SEQ ID NO:64); panel B exhibits cells
stained with the conjugate, biotin-spacer-GILAPPVPPRNTR (SEQ ID
NO:63); panel C shows cells stained with the long consensus
peptide, biotin-spacer-RSTPRPLPPLPTTR (SEQ ID NO:67); and panel D
shows cells stained with the proline-rich vinculin peptide
conjugate, biotin-spacer-LAPPKPPLPEGEV (SEQ ID NO:70). The "spacer"
sequence is KSGSG (SEQ ID NO:103). As shown in FIG. 11, the panels
in which SH3 domain-binding peptides were used present a bright
display of fluorescence activity that is in sharp contrast to the
relatively "dark" features of panel D (non-SH3 domain binding
vinculin segment). These results demonstrate further the ability of
the SH3 domain-binding peptides of the present invention to
localize to protein targets (e.g., Src and Src-related proteins)
within cells and provide an image thereof.
[0215] 6.11. In Vivo Modulation of Src In Oocytes With SH3-Binding
Peptides
[0216] When Xenopus laevis oocytes are injected with mRNA encoding
deregulated Src, there are dramatic cytological and biochemical
changes in the oocyte (Unger, T.F. and Steele, R. E., in Mol. Cell.
Biol. (1992) 12:5485-5498). The applicants have obtained plasmids
for generating wild type and deregulated Src mRNA, which are
available from Dr. Robert Steele (University of California at
Irvine). Synthetic SH3-binding peptides are injected into oocytes
that have been previously injected with Src mRNA. The state of the
cytoskeleton is inspected visually by observing the arrangement of
cortical pigment granules under a dissecting microscope. The state
of phosphorylation of several proteins is examined by western
blotting with an anti-phosphotryosine monoclonal antibody (4G10;
Upstate Biotechnology Incorporated), as described in Unger and
Steele, above.
[0217] 6.12. Progesterone-induced X. laevis oocyte Maturation
[0218] Segments of adult ovary were removed surgically and
incubated in 0.1% collagenase type D (Boehringer Mannheim,
Indianapolis, Ind.) in Ca.sup.2+-free OR2 (82.5 mM NaCl, 2.5 mM
KCl, 1.0 mM MgCl.sub.2, 1.0 mM Na.sub.2HPO.sub.4, 5.0 mM HEPES, and
3.8 mM NaOH, pH 7.6). Oocytes were then washed 3-5 times with OR2
containing 1.0 mM CaCl.sub.2 and allowed to recover in OR2
overnight at 18.degree. C. Stage VI oocytes were injected with 40
nL of 100 mM peptide or water. After injection, the oocytes were
placed in OR2 with 2 mg/mL progesterone (Sigma, St Louis, Mo.) and
incubated at 20.degree. C. Oocytes were scored at hourly time
points for germinal vesicle breakdown (GVBD).
[0219] FIG. 10 presents the results of this experiment. As shown by
the graph, oocytes injected with the SH3 domain-binding peptide
VLKRPLPIPPVTR (SEQ ID NO:64) exhibit a faster rate of
progesterone-induced germinal vesicle breakdown relative to oocytes
that had been injected with water or with the proline-rich vinculin
peptide, LAPPKPPLPEGEV (SEQ ID NO:70). These results parallel those
of Unger and Steele, supra, wherein oocytes injected with
deregulated or active Src RNA matured at a faster rate than oocytes
injected with water or wild-type Src mRNA (See FIG. 3B of the Unger
and Steele article).
[0220] The present results obtained with Src SH3 domain-binding
peptides suggest that these peptides modulate the biochemical
activity of "cellular" Src; in particular, it is proposed that at
least some of the Src SH3 domain-binding peptides of the present
invention upregulate the biochemical activity of "cellular" Src,
which may be downregulated or inhibited in its normal state. Hence,
the administration of the SH3 domain-binding peptides of the
present invention can constitute a novel method of modulating the
activity of Src or Src-related proteins. Specifically, certain of
these peptides are able to activate Src-family proteins.
[0221] 6.13. In Vivo Antagonism Of Src In src Transformed Cells
With SH3-Binding Peptides
[0222] The coding regions for SH3-binding peptides are cloned into
vectors that direct their expression in animal cells. A bipartite
gene is constructed, encoding a protein with c-myc epitope and
SH3-binding peptide, which is transcribed from a strong
constitutive promoter (e.g., SV40, CMV, HSV TK, calmodulin). The
vector is introduced into either normal or Src-transformed cells
via transfection (e.g., electroporation, calcium phosphate,
liposomes, DEAE dextran). Transfected cells express the bipartite
gene transiently in culture. To create stable transformed cell
lines, the vector carries a selectable marker (e.g., neomycin
resistance) or transfection is performed in the presence of excess
plasmid carrying a selectable marker (e.g., neomycin resistance)
and cells selected for the marker. Transfected cells are stained by
immunofluorescence to detect expression of the bipartite protein.
The hybridoma 9E10 secretes a monoclonal antibody that is highly
specific for the c-myc epitope (EQKLISEEDLN [SEQ ID NO:105]; see,
Evan, G.A. et al., in Mol. Cell. Biol. (1985) 5:3610-3616). This
antibody is sed in immunofluorescence experiments to demonstrate
that he bipartite protein is expressed inside the cells, and in
some cases, localized to subcellular structures enriched in SH3
domain bearing proteins.
[0223] There are several controls used in these experiments. First,
cells are transfected with vectors that do not have he SH3-binding
peptide coding region. Second, normal (non-ransformed) cells are
transfected. Third, cells transformed by oncogenes other than Src
are used in the transfection experiments. Fourth, cells are stained
with other monoclonal antibodies that do not recognize the c-myc
epitope.
[0224] Transfected cells are examined for any changes in cell
shape, behavior, and metabolism as a consequence of expressing the
SH3 binding peptides. Cell shape is examined by phase contrast
microscope at several times after transfection; in particular, the
flatness of the cells, their adhesion to the substrate, and the
degree of cell ruffling are monitored. Cell division rates, cell
migration, and contact inhibition are also observed over time.
Finally, the amount of phosphorylated tyrosine in transfected cells
is quantitated by phosphoaminoacid analysis and with an anti-
phosphotryosine monoclonal antibody (4G10; Upstate Biotechnology
Incorporated) in western blotting experiments.
[0225] 6.14 Distinct Ligand Preferences of Various 8H3 Domains
[0226] 6.14.1 Preparation of PXXP (SEQ ID NO: 161) Biased Peptide
Libraries
[0227] Using procedures similar to those described in Sections 6.1
and 6.4 and also described in Sparks, A. B., et al., in Methods in
Enzymology, (1995) 255:498-509, oligonucleotide inserts were
constructed according to the schematic provided in FIG. 12. The two
synthetic oligonucleotides
(5'-ctgtgcctcgagk(nnk).sub.6cca(nnk).sub.2cca(nnk).sub.6tctagacgtgtcagt-3-
' (SEQ ID NO:162) and 5'-actgacacgtctaga-3'(SEQ ID NO:163), where
k=g+t and n=g+a+t+c) were annealed and filled in with Sequenase
(Amersham, Arlington Heights, Ill.). The inserts were then digested
with Xho I and Xba I and were ligated into gene III of the mBAX
vector.
[0228] The mBAX vector was created by generating cloning sites in
gene III of the M13mpl8 vector (Messing, J., 1991, "Cloning in M13
phage or how to use biology at its best," Gene 100, 3-12) in the
manner of Fowlkes et al., 1992, Biotechniques 13, 422-427. The mBAX
vector displays a peptide sequence at the N-terminus of the mature
pIII protein that encodes the epitope for the mouse monoclonal
antibody 7E11 (see FIG. 13); it includes the stop codon TAG in the
coding region, which is suppressed in E. coli carrying suppressor
tRNA gene mutations known as supE or supF. There are no other stop
codons in the mBAX genome. The mBAX vector also carries a segment
of the alpha fragment of .beta.-galactosidase; bacterial cells
expressing the omega fragment of .beta.-galactosidase that are
infected by a bacteriophage that expresses the alpha fragment
convert the clear XGal substrate into an insoluble blue
precipitate. Thus, plaques of such bacteriophage on such cells
appear blue.
[0229] Recombinant mBAX molecules can be distinguished easily from
non-recombinant molecules due to the TAG codon in the XhoI-XbaI
segment in gene III of mBAX. When recombinants are generated by
replacing the Xho I-Xba I fragment with oligonucleotides encoding
random peptides, the recombinants can be grown in bacteria with
(e.g., DH5.alpha.F') or without (e.g., JS5) suppressor tRNA mutant
genes. On the other hand, the non-recombinant mBAX molecules fail
to produce plaques on bacterial lawns where the bacteria (e.g.,
JS5) lack such suppressor genes. This is because in JS5, the TAG
codon serves as a stop codon to yield a truncated pIII molecule
during translation; since pIII is an essential protein component of
viable M13 viral particles, no plaques will form.
[0230] The ligated DNA was electroporated into JS5 E. coli and
recombinant phage were propagated on two hundred 100 mm 2xYT +0.8%
agar plates as described in Sambrook, J., Frisch, E, F., &
Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory, Plainview, N.Y.) (Sambrook et al.). To
minimize the recovery of sibling clones during affinity
purification of binding phage, six distinct library fractions were
prepared by dividing the plates into six roughly equal groups. Each
fraction was treated separately in all subsequent manipulations.
Phage particles were harvested from each fraction by diffusion into
100 ml PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na.sub.2HPO.sub.4, 1.4
mM KH.sub.2PO.sub.4), concentrated by polyethylene glycol
precipitation as in Sambrook et al. (1989, supra), and resuspended
in 10 ml PBS+10% glycerol. Each fraction contained approximately
5.times.10.sup.7 unique recombinants, for a total library
complexity of approximately 3.times.10.sup.8. The resulting
phage-displayed library contained peptides of the form
X.sub.6PXXPX.sub.6 (SEQ ID NO:164), where X represents any amino
acid.
[0231] 6.14.2 Affinity purification of SH3-binding phage
[0232] Library screens were performed as described in Sparks, A.
B., et al., in Methods in Enzymology, (1995) 255:498-509. Briefly,
wells of an ELISA microtiter plate were coated with 10 .mu.g
GST-SH3 fusion protein in 100 mM NaHCO.sub.3 (pH 8.5) for 3 hours
and blocked with Superblock (Pierce, Rockford, Ill.) for 1 hour.
Approximately 5.times.10.sup.11 infectious particles from each
library fraction were diluted in 200 .mu.l PBS+0.1% Tween 20 and
incubated in a GST-SH3-coated well for 3 hours. The wells were
washed five times with PBS+0.1% Tween 20, and bound phage were
eluted with 50 mM glycine-HCl (pH 2.2). Recovered phage were
propagated in 10 ml 2xYT media and 100 .mu.l of a saturated
DH5.alpha.F' E. coli culture and affinity purified twice more as
above. Affinity purified phage were plated onto 2xYT+0.8% agar
plates to yield isolated plaques from which clonal phage stocks and
DNA were produced. Phage binding was confirmed by incubating equal
amounts of a clonal phage stock in wells coated with 1 .mu.g
GST-SH3 or GST. The wells were washed five times with PBS+0.1%
Tween 20, and bound phage were detected by anti-phage ELISA
according to the manufacturer's instructions (Pharmacia,
Piscataway, N.J.). Clones with strong SH3-binding activity were
selected for further analysis. The sequences of peptides displayed
by these clones were determined by DNA sequencing of phage
inserts.
[0233] 6.14.3 Preparation of GST-SH3 fusion proteins
[0234] Constructs encoding GST fusions to the Grb2 N-terminal (Grb2
N, aa 1-58), Nck N-terminal (Nck N, aa 1-68), Nck middle (Nck M, aa
101-166), Nck C-terminal (Nck C, aa 191-257), p53bp2 (aa 454-530),
or Src (aa 87-143) SH3 domains were generated by PCR cloning of the
appropriate cDNAs into pGEX-2T (Pharmacia, Piscataway, NJ; a
general reference for the PGEX vectors is Smith, D. B., &
Johnson, K. S. (1988) Gene 67, 31-40). The integrity of the
constructs was confirmed by DNA sequencing. pGEX-derived constructs
expressing GST fusions to the SH3 domains of Yes, Cortactin, Crk,
Abl, and PLC.gamma. were kindly provided by M. Sudol (Rockefeller
University), J. T. Parsons (University of Virginia at
Charlottsville), M. Matsuda (Tokyo, Japan), A. M. Pendergast (Duke
University), and S. Earp (University of North Carolina at Chapel
Hill), respectively. Alternatively, the GST-SH3 fusion proteins for
Yes, Cortactin, Crk, Abl, and PLC.gamma. could have been prepared
as above for Grb2 N, Nck N, Nck M, Nck C, p53bp2, and Src, using
published sequence information for these proteins. See, e.g., Suen
et al., (1993) Mol. Cell. Biol. 13, 5500-5512 (Grb2); Lehmann et
al., (1990) Nucleic Acids Res. 18, 1048 (Nck); Iwabuchi et al.,
(1994) Proc. Natl. Acad. Sci. USA 91, 6098-6102 (p53bp2); Takeya et
al., (1983) Cell 32, 881-890 (Src); Sudol et al., (1988) Nucleic
Acids Res. 16, 9876 (Yes); Wu et al., (1991) Mol. Cell. Biol. 11,
5113-5124 (Cortactin); Matsuda et al., (1992) Mol. Cell. Biol. 12,
3482-3489 (Crk); Shtivelman et al., (1986) Cell 47, 277-284 (Abl);
Burgess et al., (1990) Mol. Cell. Biol. 10, 4770-4777 (PLC.gamma.).
GST-SH3 fusion proteins were prepared as described in Smith, D. B.,
& Johnson, K. S. (1988) Gene 67, 31-40. The integrity and
purity of the fusion proteins were confirmed by SDS-PAGE. Protein
concentrations were determined using a the BioRad protein assay
(BioRad, Hercules, Calif.).
[0235] 6.14.4 SH3 Domain Binding Peptides and Consensus
Sequences
[0236] The use of second generation or biased peptide libraries,
which fix all or part of the PXXP (SEQ ID NO:161) consensus motif
for SH3 domain binding peptides and randomize flanking residues,
has defined additional sequence residues exhibiting selective SH3
domain binding.
[0237] Tables 1-5, below, list some of the relevant amino acid
sequences obtained when the biased peptide library described in
Section 6.14.1 was screened with GST-SH3 fusion proteins. The
underscored amino acid residues in Tables 1-5 indicate the fixed
positions. Also, indicated for each set of new binders is a
"consensus" sequence, which seeks to include the additional
features gleaned from the new binding peptides. The symbol ".phi."
in the consensus sequences of Tables 1-5 represents a hydrophobic
residue. The symbol x in the consensus sequences of Tables 1-5
represents any amino acid. For the Nck SH3 domain binding clones, a
GST-SH3 fusion protein containing the middle SH3 domain of Nck was
used.
1TABLE 1 CORTACTIN SH3-BINDING PEPTIDES SEQ. ID NO.
PXXP.CORT.M1/2/3.PP SSLLGPPVPPKPQTLFSFSR 107 PXXP.CORT.M4.PP
SRLGEFSKPPIPQKPTWMSR 108 PXXP.CORT.N2.PP SRTERPPLPQRPWLSYSSR 109
PXXP.CORT.N3.PP.INC SREPDWLCPNCPLLLRSDSR 110 PXXP.CORT.01/2/3.PP
SSSSHNSRPPLPEKPSWLSR 111 PXXP.CORT.04.PP SRLTPQSKPPLPPKPSAVSR 112
CONSENSUS KPP.phi.PxKPxW 113 R
[0238]
2TABLE 2 NCK SH3-BINDING PEPTIDES SEQ. ID NO. PXXP.NCK.Q1/4.PP
SSLGVGWKPLPPMRTASLSR 114 PXXP.NCK.Q2/3.PP.INC SSVGFADRPRPPLRVESLSR
115 PXXP.NCK.R1.PP.INC SSAGILRPPEKPXRSFSLSR 116 PXXP.NCK.R2.PP
SSPYTGDVPIPPLRGASLSR 117 PXXP.NCK.R3.PP SSLMGSWPPVPPLRSDSLSR 118
PXXP.NCK.R4.PP SSIGEDTPPSPPTRRASLSR 119 PXXP.NCK.S1/4.PP
SRSLSEVSPKPPIRSVSLSR 120 PXXP.NCK.S2.PP.INC SSVSEGYSPPLPPRSTSLSR
121 PXXP.NCK.S3.PP SSSFTLAAPTPPTRSLSLSR 122 PXXP.NCK.T1.PP
SSPPYELPPRPPNRTVSLSR 123 PXXP.NCK.T2.PP SRVVDGLAPPPPVRLSSLSR 124
PXXP.NCK.T3.PP.INC SSLGYSGAPVPPHRxSSLSR 125 PXXP.NCK.T4.PP
SSISDYSRPPPPVRTLSLSR 126 CONSENSUS .phi.xxxxxPxPP.phi.RSxSL 127
T
[0239]
3TABLE 3 ABL SH3 BINDING PEPTIDES SEQ. ID NO. PXXP.ABL.G1/2.PP
SRPPRWSPPPVPLPTSLDSR 128 PXXP.ABL.G3/4.PP SSPPDYAAPAIPSSLWVDSR 129
PXXP.ABL.H1/3/4.PP SSPPHWAPPAPPAMSPPISR 130 PXXP.ABL.H2.PP.INC
SSDRCWECPPWPAGGQRGSR 131 PXXP.ABL.I1/2/3.PP SSPPKFSPPPPPYWQLHASR
132 PXXP.ABL.I4.PP SSPPSFAPPAAPPRHSFGSR 133 PXXP.ABL.J1.PP
SSAPKKPAPPVPMMAHVMSR 134 PXXP.ABL.J2.PP.INC SSPTYPPPPPPDTAKGASR 135
PXXP.ABL.J3.PP.INC SSPPXXXPPPIPNSPQVLSR 136 PXXP.ABL.J4.PP
SSPPTWTPPKPPGWGVVFSR 137 PXXP.ABL.L1.PP SSAPTWSPPALPNVAKYKSR 138
PXXP.ABL.L2/3.PP SSIKGPRFPVPPVPLNGVSR 139 PXXP.ABL.L4.PP
SSPPAWSPPHRPVAFGSTSR 140 CONSENSUS PPxWxPPP.phi.P 141
[0240]
4TABLE 4 PLC.gamma. SH3-BINDING PEPTIDES SEQ. ID NO.
PXXP.PLC.gamma..P1.PP SSMKVHNFPPLPSYETSR 142 PXXP.PLC.gamma..P2.PP
SRVPPLVAPRPPSTLNSLSR 143 PXXP.PLC.gamma..PE.PP.INC
SSLYWQHGPDPPVGAPQLSR 144 PXXP.PLC.gamma..P4.PP SSHPLNSWPGGPFRHNLSSR
145
[0241]
5TABLE 5 SRC SH3-BINDING PEPTIDES SEQ. ID NO. PXXP.SRC.A1.PP
SSRALRVRPLPPVPGTSLSR 146 PXXP.SRC.A2.PP SSFRALPLPPTPDNPFAGSR 147
PXXP.SRC.A3.PP SRDAPGSLPFRPLPPVPTSR 148 PXXP.SRC.A4.PP
SSISQRALPPLPLHSDPASR 149 PXXP.SRC.B1.PP SSPAYRPLPRLPDLSVIYSR 150
PXXP.SRC.B2/3/PP SSFINRRLPALPPDNSLLSR 151 PXXP.SRC.B4.PP
SRLTGRPLPALPPPFSDFSR 152 PXXP.SRC.C1.PP SRMKDRVLPPIPTVESAVSR 153
PXXP.SRC.C2.PP.INC SSLYSAIAPDPPPRNSSSSR 154 PXXP.SRC.C3.PP
SSLASRPLPLLPNSAPGQSR 155 PXXP.SRC.D1.PP SSLTSRPLPDIPVRPSKSSR 156
PXXP.SRC.D2.PP.INC SSLKWRALPPLPETDTPYSR 157 PXXP.SRC.D3.PP
SSNTNRLPPPTPDGLDVRSR 158 PXXP.SRC.D4.PP SSLQSRPLPLPPQSSYPISR 159
CONSENSUS RPLPPLP 9
[0242] In addition to the consensus sequence shown in Table 5, the
amino acid sequences of the inserts from the Src SH3 domain-binding
phage isolated from the PXXP (SEQ ID NO:161) biased peptide library
described in Section 6.14.1 also give rise to the consensus
sequence LXXRPLPX.psi.P (SEQ ID NO:165), as shown in Table 6,
below. In the consensus sequence LXXRPLPX.psi.P (SEQ ID NO:165),
.psi. represents aliphatic amino acid residues (A, V, L, I, P); X
represents any amino acid.
6TABLE 6 Src SH3 Binding Peptides LASRPLPLLPNSAPGQ (a portion of
SEQ ID NO:155) LTGRPLPALPPPFSDF (a portion of SEQ ID NO:152)
PAYRPLPRLPDLSVIY (a portion of SEQ ID NO:150) RALRVRPLPPVPGTSL (a
portion of SEQ ID NO:146) DAPGSLPFRPLPPVPT (a portion of SEQ ID
NO:148) LKWRALPPLPETDTPY (a portion of SEQ ID NO:157)
ISQRALPPLPLMSDPA (a portion of SEQ ID NO:149) LTSRPLPDIPVRPSKS (a
portion of SEQ ID NO:156) NTNRPLPPTPDGLDVR (a portion of SEQ ID
NO:158) MKDRVLPPIPTVESAV (a portion of SEQ ID NO:153)
LQSRPLPLPPQSSYPI (a portion of SEQ ID NO:159) FINRRLPALPPDNSLL (a
portion of SEQ ID NO:151) FRALPLPPTPDNPFAG (a portion of SEQ ID
NO:147) LYSAIAPDPPPRNSSS.diamond-solid. (a portion of SEQ ID
NO:154) LXXRPLPX.psi.P=CONSENSUS (SEQ. ID NO: 165)
[0243] In Table 6, .psi. represents aliphatic amino acid residues
(A, V, L, I, P); X represents any amino acid; .diamond-solid.
putative class II peptide (see Section 6.14.5). Invariant proline
residues are underlined.
[0244] Another consensus sequence that can be derived from the
amino acid sequences of the inserts from the Src SH3 domain-binding
phage is:
[0245] LX.sub.1X.sub.2RPLPX.sub.3.psi.PX.sub.4.sub.5 (SEQ ID
NO:454)
[0246] where .psi. represents aliphatic amino acid residues (A, V,
L, I, P) and X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5
represent any amino acid; except that if
[0247] X.sub.3=P, .psi.=L, X.sub.4=P, and X.sub.5=P, then:
[0248] where X.sub.1=F, then X.sub.2 is not H or R; or
[0249] where X.sub.1=S, then X.sub.2 is not R, H, A, N, T, G, V, M,
or W; or
[0250] where X.sub.1=C, then X.sub.2 is not S or G; or
[0251] where X.sub.1=R, then X.sub.2 is not T or F; or
[0252] where X.sub.1=A, then X.sub.2 is not R, Q, N, S, or L;
or
[0253] where X.sub.1=Q, then X.sub.2 is not M; or
[0254] where X.sub.1=L, then X.sub.2 is not R; or
[0255] where X.sub.1=I, then X.sub.2 is not A; or
[0256] where X.sub.1=P, then X.sub.2 is not P, W, or R; or
[0257] where X.sub.1=G, then X.sub.2 is not S or R; or
[0258] where X.sub.1=T, then X.sub.2 is not T.
[0259] In addition to the consensus sequence shown in Table 1, the
amino acid sequences of the inserts from the cortactin SH3
domain-binding phage isolated from the PXXP (SEQ ID NO:161) biased
peptide library described in Section 6.14.1 also give rise to the
consensus sequence +PP.psi.PXKPXWL (SEQ ID NO:166), as shown in
Table 7, below.
7TABLE 7 Cortactin SH3 Binding Peptides LTPQSKPPLPPKPSAV (a portion
of SEQ ID NO:112) SSHNSRPPLPEKPSWL (a portion of SEQ ID NO:111)
PVKPPLPAKPWWLPPL (SEQ ID NO:167) TERPPLPQRPDWLSYS (a portion of SEQ
ID NO:109) LGEFSKPPIPQKPTWM (a portion of SEQ ID NO:108)
YPQFRPPVPPKPSLMQ (SEQ ID NO:168) VTRPPLPPKPGHMADF (SEQ ID NO:169)
VSLGLKPPVPPKPMQL (SEQ ID NO:170) LLGPPVPPKPQTLFSF (a portion of SEQ
ID NO:107) YKPEVPARPIWLSEL (SEQ ID NO:171) GAGAARPLVPKKPLFL (SEQ ID
NO:172) +PP.psi.PXKPXWL =CONSENSUS (SEQ ID NO:166)
[0260] In Table 7, +represents basic amino acid residues (R, K);
.psi.represents aliphatic amino acid residues (A, V, L, I, P); X
represents any amino acid. Invariant proline residues are
underlined.
[0261] In addition to the consensus sequence shown in Table 3, the
amino acid sequences of the inserts from the Abl SH3 domain-binding
phage isolated from the PXXP (SEQ ID NO:161) biased peptide library
described in Section 6.14.1 also give rise to the consensus
sequence PPX.theta.XPPP.psi.P (SEQ ID NO:173), as shown in Table 8,
below.
8TABLE 8 Ab1 SH3 Binding Peptides PPWWAPPPIPNSPQVL (SEQ ID NO:174)
PPKFSPPPPPYWQLHA (a portion of SEQ ID NO:132) PPHWAPPAPPAMSPPI (a
portion of SEQ ID NO:130) PPTWTPPKPPGWGVVF (a portion of SEQ ID
NO:137) PPSFAPPAAPPRHSFG (a portion of SEQ ID NO:133)
PTYPPPPPPDTAKGA.dagger-dbl. (a portion of SEQ ID NO:135)
GPRWSPPPVPLPTSLD (a portion of SEQ ID NO:128) APTWSPPALPNVAKYK (a
portion of SEQ ID NO:138) PPDYAAPAIPSSLWVD (a portion of SEQ ID
NO:129) IKGPRFPVPPVPLNGV (a portion of SEQ ID NO:139)
PPAWSPPHRPVAFGST (a portion of SEQ ID NO:140) APKKPAPPVPMMAHVM (a
portion of SEQ ID NO:134) PPX.theta.XPPP.psi.P = (SEQ ID NO:173)
CONSENSUS
[0262] In Table 8, .theta. represents aromatic amino acid residues;
.psi. represents aliphatic amino acid residues (A, V, L, I, P); X
represents any amino acid. Invariant proline residues are
underlined.
[0263] .dagger-dbl.This clone contained a three nucleotide deletion
in the random peptide coding sequence.
[0264] The amino acid sequences of the inserts from the PLC.gamma.
SH3 domain-binding phage isolated from the PXXP (SEQ ID NO:161)
biased peptide library described in Section 6.14.1 give rise to the
consensus sequence PPVPPRPXXTL (SEQ ID NO:175), as shown in Table
9, below.
9TABLE 9 PLC.gamma. SH3 Binding PeptideB MPPPVPPRPPGTLQVA (SEQ ID
NO:176) LSYSPPPVPPRPDSTL (SEQ ID NO:177) VLAPPVPPRPGNTFFT (SEQ ID
NO:178) YRPPVAPRPPSSLSVD (SEQ ID NO:179) LQCPDCPRVPPRPIPI (SEQ ID
NO:180) VPPLVAPRPPSTLNSL (a portion of SEQ ID NO:143)
LTPPPFPKRPRWTLPE (SEQ ID NO:181) YWPHRPPLAPPQTTLG (SEQ ID NO:182)
PPVPPRPXXTL=CONSENSUS (SEQ ID NO:175)
[0265] In Table 9, the symbol X represents any amino acid.
Invariant proline residues are underlined.
[0266] The PXXP (SEQ ID NO:161) biased peptide library described in
Section 6.14.1 was also used to obtain phage clones that
specifically bound the SH3 domain from the p53bp2 rotein. The amino
acid sequences of the peptides expressed by the p53bp2 SH3
domain-binding phage are shown in Table 10 below.
10TABLE 10 p53bp2 SH3 Binding Peptides YDASSAPQRPPLPVRKSRP (SEQ ID
NO:183) EYVNASPERPPIPGRKSRP (SEQ ID NO:184) WNGIAIPGRPEIPPRASRP
(SEQ ID NO:185) SMIFIYPERPSPPPRFSRP (SEQ ID NO:186)
GVEEWNPERPQIPLRLSRP (SEQ ID NO:187) WVVDSRPDIPLRRSLP (SEQ ID
NO:188) VVPLGRPEIPLRKSLP (SEQ ID NO:189) GGTVGRPPIPERKSVD (SEQ ID
NO:190) YSHAGRPEVPPRQSKP (SEQ ID NO:191) FSAAARPDIPSRASTP (SEQ ID
NO:192) LYIPKRPEVPPRRHEA (SEQ ID NO:193) NNISARPPLPSRQNPP (SEQ ID
NO:194) MAGTPRPAVPQRMNPP (SEQ ID NO:195) RPX.psi.P.psi.R +
SXP=CONSENSUS (SEQ ID NO:196)
[0267] In Table 10, + represents basic amino acid residues (R, K);
.psi. represents aliphatic amino acid residues (A, V, L, I, P); X
represents any amino acid. Invariant proline or flanking residues
are underlined.
[0268] The PXXP (SEQ ID NO:161) biased peptide library described in
Section 6.14.1 was also used to obtain phage clones that
specifically bound the SH3 domain from the N terminal portion of
the Crk protein. The amino acid sequences of the peptides expressed
by the Crk N terminal SH3 domain-binding phage are shown in Table
11 below.
11TABLE 11 Crk N SH3 Binding Peptides GQPAGDPDPPPLPAKF (SEQ ID NO:
197) FEQTGVPLLPPKSFKY (SEQ ID NO:198) IFGDPPPPIPMKGRSL (SEQ ID
NO:199) SNQGSIPVLPIKRVQY (SEQ ID NO:200) NYVNALPPGPPLPAKN (SEQ ID
NO:201) SSDPERPVLPPKLWSV (SEQ ID NO:202) HFGPSKPPLPIKTRIT (SEQ ID
NO:203) DWKVPEPPVPKLPLKQ (SEQ ID NO:204) ATSEGLPILPSKVGSY (SEQ ID
NO:205) NANVSAPRAPAFPVKT (SEQ ID NO:206) EMVLGPPVPPKRGTVV (SEQ ID
NO:207) AGSRHPPTLPPKESGG (SEQ ID NO:208) SVAADPPRLPAKSRPQ (SEQ ID
NO:209) .psi.P.psi.LP.psi.K=CONSENSUS (SEQ ID NO:210)
[0269] In Table 11, .psi. represents aliphatic amino acid residues
(A, V, L, I, P). Invariant proline residues are underlined.
[0270] The present invention provides a purified peptide that binds
to the SH3 domain of Crk, the purified peptide comprising the amino
acid sequence .psi.P.psi.LP.psi.K (SEQ ID NO:210), where .psi.
represents aliphatic amino acid residues (A, V, L, I, P), with the
proviso that the peptide does not comprise the amino acid sequence
WNERQPAPALPPKPPKPT (SEQ ID NO:456).
[0271] The PXXP (SEQ ID NO:161) biased peptide library described in
Section 6.14.1 was also used to obtain phage clones that
specifically bound the SH3 domain from the Yes protein. The amino
acid sequences of the peptides expressed by the Yes SH3
domain-binding phage are shown in Table 12 below.
12TABLE 12 Yes SH3 Binding Peptides ITMRPLPALPGHGQIH (SEQ ID
NO:211) LPRRPLPDLPMAAGKG (SEQ ID NO:212) LGSRPLPPTPRQWPEV (SEQ ID
NO:213) STIRPLPAIPRDTLLT (SEQ ID NO:214) RSGRPLPPIPEVGHNV (SEQ ID
NO:215) IGSRPLPWTPDDLGSA (SEQ ID NO:216) LAQRELPGLPAGAGVS (SEQ ID
NO:217) IPGRALPELPPQRALP (SEQ ID NO:218) FVGRELPPTPRTVIPW (SEQ ID
NO:219) DPRSALPALPLTPLQT (SEQ ID NO:220) SPHDVLPALPDSHSKS (SEQ ID
NO:221) .psi.XXRPLPXLP =CONSENSUS (SEQ ID NO:222)
[0272] In Table 12, .psi. represents aliphatic amino acid residues
(A, V, L, I, P); X represents any amino acid. Invariant proline
residues are underlined.
[0273] Another consensus sequence that can be derived from the
amino acid sequences of the inserts from the Yes SH3 domain-binding
phage is:
[0274] .psi.X.sub.1X.sub.2RPLPX.sub.3LPX.sub.4X.sub.5 (SEQ ID
NO:455)
[0275] where .psi. represents aliphatic amino acid residues (A, V,
L, I, P) and X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5
represent any amino acid; except that if
[0276] X.sub.3=P, X.sub.4=P, and X.sub.5=P, then:
[0277] when .psi.=L,
[0278] where X.sub.1=F, then X.sub.2 is not H or R; or
[0279] where X.sub.1=S, then X.sub.2 is not R, H, A, N, T, G, V, M,
or W; or
[0280] where X.sub.1=C, then X.sub.2 is not S or G; or
[0281] where X.sub.1=R, then X.sub.2 is not T or F; or
[0282] where X.sub.1=A, then X.sub.2 is not R, Q, N, S, or L;
or
[0283] where X.sub.1=Q, then X.sub.2 is not M; or
[0284] where X.sub.1=L, then X.sub.2 is not R; or
[0285] where X.sub.1=I, then X.sub.2 is not A; or
[0286] where X.sub.1=P, then X.sub.2 is not P, W, or R; or
[0287] where X.sub.1=G, then X.sub.2 is not S or R; or
[0288] where X.sub.1=T, then X.sub.2 is not T; and
[0289] when .psi.=P,
[0290] where X.sub.1=A, then X.sub.2 is not R; or
[0291] where X.sub.1=S, then X.sub.2 is not R or Y; or
[0292] where X.sub.1=M, then X.sub.2 is not S; or
[0293] where X.sub.1=V, then X.sub.2 is not G; or
[0294] where X.sub.1=R, then X.sub.2 is not S; or
[0295] where X.sub.1=I, then X.sub.2 is not R; and
[0296] when .psi.=A,
[0297] where X.sub.1=A, then X.sub.2 is not K; and
[0298] when .psi.=V,
[0299] where X.sub.1=A, then X.sub.2 is not C or Q; or
[0300] where X.sub.1=P, then X.sub.2 is not P; and
[0301] when .psi.=I,
[0302] where X.sub.1=G, then X.sub.2 is not H; or
[0303] where X.sub.1=T, then X.sub.2 is not S; or
[0304] where X.sub.1=R, then X.sub.2 is not S.
[0305] The present invention also provides a purified peptide that
binds to the SH3 domain of Yes, the purified peptide comprising the
amino acid sequence .psi.X.sub.1X.sub.2RPLPX.sub.3LPX.sub.4X.sub.5
(SEQ ID NO:455), where .psi. represents aliphatic amino acid
residues (A, V, L, I, P) and X.sub.1, X.sub.2, X.sub.3, X.sub.4,
and X.sub.5 represent any amino acid, with the proviso that the
peptide does not comprise the amino acid sequence AGDRPLPPLPYNPKS
(SEQ ID NO:457).
[0306] The PXXP (SEQ ID NO:161) biased peptide library described in
Section 6.14.1 was also used to obtain phage clones that
specifically bound the SH3 domain from the N terminal portion of
the Grb2 protein. The amino acid sequences of the peptides
expressed by the Grb2 N terminal SH3 domain-binding phage are shown
in Table 13 below. These sequences can be arranged into three
groups of sequences that have different, but related, consensus
sequences. An overall consensus sequence, +.theta.DXPLPXLP (SEQ ID
NO:223), can be derived for the three groups.
13TABLE 13 Grb2 N SH3 Binding Peptides KWDSLLPALPPAFTVE (SEQ ID
NO:224) RWDQVLPELPTSKGQI (SEQ ID NO:225) RFDFPLPTHPNLQKAH (SEQ ID
NO:226) RLDSPLPALPPTVMQN (SEQ ID NO:227) RWGAPLPPLPEYSWST (SEQ ID
NO:228) YWDMPLPRLPGEEPSL (SEQ ID NO:229) RFDYNLPDVPLSLGTA (SEQ ID
NO:230) TKKPNAPLPPLPAYMG (SEQ ID NO:231) KWDLDLPPEPMSLGNY (SEQ ID
NO:232) .theta.+DXPLPXLP =CONSENSUS (SEQ ID NO:223)
YYQRPLPPLPLSHFES (SEQ ID NO:234) YYRKPLPNLPRGQTDD (SEQ ID NO:235)
YFDKPLPESPGALMSL (SEQ ID NO:236) YFSRALPGLPERQEAH (SEQ ID NO:237)
Y.theta.X+PLPXLP =CONSENSUS (SEQ ID NO:238) SLWDPLPPIPQSKTSV (SEQ
ID NO:239) SYYDPLPKLPDPGDLG (SEQ ID NO:240) KLYYPLPPVPFKDTKH (SEQ
ID NO:241) DPYDALPETPSMKASQ (SEQ ID NO:242) .theta.DPLPXLP
=CONSENSUS (SEQ ID NO:243) .theta.+DXPLPXLP =OVERALL CONSENSUS (SEQ
ID NO:223)
[0307] In Table 13, + represents basic amino acid residues (R, K);
.theta. represents aromatic amino acid residues; X represents any
amino acid. Invariant proline residues are underlined.
[0308] 6.14.5 SH3 Ligand Binding Orientation
[0309] Peptide ligands bound to SH3 domains have been shown to
assume a left-handed polyproline type II (PPII) helix conformation
(Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A. W.,
& Schreiber, S. L. (1994) Cell 76, 933-45). SH3 ligands are
pseudo-symmetrical and may therefore bind in one of two opposite
orientations (Feng, S., Chen, J. K., Yu, H., Simon, J. A., &
Schreiber, S. L. (1994) Science 266, 1241-7) (Feng et al.). Feng et
al., supra, have demonstrated that peptides that bind in one or the
other orientation share different consensus motifs. Specifically,
ligands that bind in the Class I or Class II orientation conform to
the consensus +pYPpYP (SEQ ID NO:244) or YPpYPp+ (SEQ ID NO:245)
respectively, where uppercase positions represent conserved
residues that contact the SH3 domain and confer specificity, and
lowercase positions represent scaffolding residues that tend to be
proline.
[0310] According to this model, we predict that the peptides
selected by the Src, Yes, Abl, and Grb2 N SH3 domains bind in the
Class I orientation, whereas the peptides selected by the
Cortactin, p53bp2, PLC.gamma., and Crk N SH3 domains bind in the
Class II orientation (see Table 14). Interestingly, most of the SH3
ligand consensus motifs identified in this work contain additional
conserved residues flanking the SH3-binding core defined by Feng et
al., supra. Furthermore, these conserved residues are situated N-
and C-terminal of the SH3-binding core in Class I and Class II
motifs, respectively, and are therefore predicted to interact with
equivalent regions of their target SH3 domains (see Table 14).
14TABLE 14 Alignment of SH3 ligand consensus motifs SEQ ID NO:
Class I +p.psi.Pp.psi.P 244 Src LXXRPLPX.psi.P 165 Yes
.psi.XXRPLPXLP 222 Abl PPX.theta.XPPP.psi.P 173 Grb2 N
+.theta.DXPLPXLP 223 Y.theta.XRPLPXLP 246 .theta.DPLPXLP 243 Class
II .psi.Pp.psi.Pp+ 245 Cortactin +PP.psi.PXKPXWL 166 p53bp2
RPX.psi.P.psi.R+SXP 196 PLC.gamma. XPPVPPRPXXTL 247 Crk N
.psi.P.psi.LP.psi.K 210
[0311] In Table 14, each SH3 ligand consensus motif was assigned to
class I or II based on its agreement with the class I or II
consensus motif. Highly (>90%) conserved positions in each SH3
ligand consensus motif are listed in boldface and were interpreted
as SH3 contact residues. +represents basic amino acid residues (K,
R); .psi.represents aliphatic amino acid residues (A, V, L, I, P);
.theta.represents aromatic amino acid residues; X represents any
amino acid; lower case p represents residues that tend to be
proline.
[0312] The Src SH3 domain is capable of binding both Class I and
Class II peptides Feng et al., supra. Although Class I peptides
predominate in the population of Src SH3 ligands selected from the
PXXP (SEQ ID NO:161) library, one clone conforms well to the Class
II consensus (see Table 6). Previously, Sparks, A. B., Quilliam, L.
A., Thorn, J. M., Der, C. J., & Kay, B. K. (1994) J. Biol.
Chem. 269, 23853-6 and Yu, H., Chen, J. K., Feng, S., Dalgarno, D.
C., Brauer, 35 A. W., & Schreiber, S. L. (1994) Cell 76, 933-45
had isolated Class II Src SH3 ligands sharing the consensus
PP.psi.PPR (SEQ ID NO:248). Similarly, whereas the Grb2 N SH3
domain has been shown to bind peptides from SOS with the Class II
consensus sequence PP.psi.PPR (SEQ ID NO:248) (Rozakis-Adcock, M.,
Fernley, R., Wade, J., Pawson, T., & Bowtell, D. (1993) Nature
363, 83-5), we have isolated Grb2 N SH3 ligands that conform to the
Class I consensus (see Table 14). Thus, both the Src and the Grb2 N
SH3 domains apparently have the capacity to bind both Class I and
Class II peptide ligands.
[0313] 6.14.6 SH3 Ligand Binding Characteristics
[0314] To explore further the capacity of SH3 domains to
discriminate between different SH3 ligands, we investigated the
binding of phage expressing various peptide ligands to a panel of
SH3 domains. Equal titers of clonal phage stocks were incubated in
microtiter wells coated with different GST-SH3 fusion proteins. The
wells were washed several times, and bound phage were detected with
an anti-phage antibody (see FIG. 14). Positive ELISA signals were
equivalent to those obtained with previously characterized Src
SH3-binding clones (Sparks, A. B., Quilliam, L. A., Thorn, J. M.,
Der, C. J., & Kay, B. K. (1994) J. Biol. Chem. 269, 23853-6)
and are indicative of SH3:peptide affinities in the 5 to 75 .mu.M
range (Yu, H., Chen, J. K., Feng, S., Dalgarno, D. C., Brauer, A.
W., & Schreiber, S. L. (1994) Cell 76, 933-945; Rickles, R. J.,
Botfield, M. C., Weng, Z., Taylor, J. A., Green, 0. M., Brugge, J.
S., & Zoller, M. J. (1994) EMBO J. 13, 5598-604). Whereas the
Src, Yes, Crk, and Grb2 N SH3 domains cross-reacted with a few
phage clones selected with other SH3 domains, the Abl, Cortactin,
p53bp2, and PLC.gamma. SH3 domains displayed considerable
specificity. Significantly, only 33 of 220 potential instances of
cross-reactivity were observed, suggesting that SH3 selectivity is
the rule rather than the exception.
[0315] Each instance of cross-reactivity may be explained by
similarities between the sequences of the peptides and the ligand
preferences of the cross-reactive SH3 domains. For example, Crk SH3
cross-reacted with three phage clones selected with other SH3
domains; each of these clones coincidentally expressed peptides
conforming to the Crk SH3 preferred ligand consensus motif.
Similarly, the cross-reactivity observed between the Src, Yes, and
Grb2 SH3 domains and clones selected by other SH3 domains within
this group may be a consequence of the fact that these SH3 domains
prefer the same proline-rich core. Finally, the Src and Yes SH3
domains cross-reacted with the PLC.gamma. SH3 ligand MPPPVPPRPPGTL
(a portion of SEQ ID NO:176), which contains the Class II Src
SH3-binding sequence PPVPPR (SEQ ID NO:249). Taken together, these
data demonstrate the capacity of SH3 domains to discern subtle
differences in the primary structure of potential ligands.
[0316] 6.15 Use of Consensus sequences to Determine the Amino Acid
Sequences Responsible for Binding in Proteins that are Known to
Bind SH3 Domains
[0317] There are many proteins that are known to bind SH3 domains
but for which the specific sequences of those proteins that are
responsible for binding to SH3 domains are not known. The consensus
sequences shown above in Tables 1-13 can be used to search
databases (e.g., GenBank) containing the amino acid sequences of
those proteins in order to determine which sequences are
responsible for the binding of those proteins to SH3 domains. This
was done for a number of known SH3 domain binding proteins and
sequences resembling the consensus sequences of Tables 1-13 were
identified. The results are shown in Table 15. For comparison, also
shown in Table 15 are the amino acid sequences that had previously
been demonstrated to be responsible for SH3 domain binding for a
number of proteins.
15 TABLE 15 SEQ ID Refer- NO: ence Src SH3 Class I LXXRPLPX.psi.P
165 Hs AFAP-110 (62-73) PPQMPLPEIPQQ 250 1 (76-87) PPDNGPPPLPTS 251
1 Hs CDC42 GAP (250-261) TAPKPMPPRPPL 252 2 Hs hnRNP K (302-313)*
SRARNLPLPPPP 253 3 Mm p62 (328-339) TVTRGVPPPPTV 254 3 Hs P13K p85
(90-101)* RPPRPLPVAPGS 255 9 Hs Shc p52 (296-307) VRKQMLPPPPCP 256
3 Src SH3 Class II PP.psi.PPR 248 Hs Dynamin (810-820) GGAPPVPSRPG
257 6 (827-837) GPPPQVPSRPN 258 6 (838-848) RAPPGVPSRSG 259 6 Hs
hnRNP K (308-318)* PLPPPPPPRGG 260 3 Mm p62 (294-304) APPPPPVPRGR
261 3 Hs Paxillin (42-52) AVPPPVPPPPS 262 10 Hs PI3K p85 (302-312)*
QPAPALPPKPP 263 9 Hs Shb (50-60) GGPPPGPGRRG 264 11 (103-113)
TKSPPQPPRPD 265 11 Yes SH3 .psi.XXRPLPXLP 222 Hs Yap65 (240-251)
PVKQPPPLAPQS 266 4 Ab1 SH3 PPX.theta.XPPP.psi.P 173 Mm 3BP-1
(265-276)* RAPTMPPPLPPV 267 12 Mm 3BP-2 (200-211)* YPPAYPPPPVPV 268
12 Dm Ena (350-361) PGPGYGPPPVPP 269 5 PLC.gamma. SH3 PPVPPRPXXTL
175 Hs Dynamin (812-823) APPVPSRPGASP 270 6 (829-840) PPQVPSRPNRNR
271 6 Hs c-Cb1 (493-504) LPPVPPRLDLLP 272 7 Crk N SH3
P.psi.LP.psi.K 210 Hs Ab1 (524-533)* QAPELPTKTR 273 13 (568-577)*
VSPLLPRKER 274 13 (758-767) EKPALPRKRA 275 13 Hs C3G (282-291)*
PPPALPPKKR 276 14 (452-461)* TPPALPEKKR 277 14 (539-548)*
KPPPLPEKKN 278 14 (607-616)* PPPALPPKQR 279 14 Grb2 N SH3 Class I
+.theta.DXPLPXLP 233 Y.theta.X+PLPXLP 238 .theta.DPLPXLP 243 Hs
c-Cb1 (560-571) PQRRPLPCTPGD 280 8 (589-600) WLPRPIPKVPVS 281 8
Grb2 N SH3 Class II PPP.psi.PPR 282 Hs Ab1 (523-533)* LQAPELPTKTR
283 13 (567-577)* AVSPLLPRKER 284 13 (609-619)* KTAPTPPKRSS 285 13
Hs c-Cb1 (491-501) ASLPPVPPRLD 286 8 Hs Dynamin (810-820)
GGAPPVPSRPG 287 6 (827-837) GPPPQVPSRPN 288 6 (838-848) RAPPGVPSRSG
289 6 Hs S0S1 (1148-1158)* PVPPPVPPRRR 290 15 (1177-1187)
DSPPAIPPRQP 291 15 (1209-1219)* ESPPLLPPREP 292 15 (1287-1297)*
IAGPPVPPRQS 293 15 Rn Synapsin I (592-602) NLPEPAPPRPS 294 16
(670-680) PPGPAGPIRQA 295 16
[0318] In Table 15, + represents basic amino acid residues (R, K);
.psi. represents aliphatic amino acid residues (A, V, L, I, P);
.theta. represents aromatic amino acid residues; X represents any
amino acid. * represents amino acid sequences previously
demonstrated to bind their respective SH3 domains. Residues within
the sequences that agree with the most highly conserved residues of
the consensus motifs are shown in bold. Each entry shows an
abbreviation of the name of the SH3 domain binding protein and the
species from which it was derived. The amino acid positions in the
mature proteins of the sequences shown are indicated in
parentheses. For more details, see the reference listed for each
protein.
[0319] Reference 1 is Flynn, D. C., Leu, T. H., Reynolds, A. B.,
& Parsons, J. T. (1993) Mol Cell Biol 13, 7892-7900.
[0320] Reference 2 is Barfod, E. T., Zheng, Y., Kuang, W, J., Hart,
M. J., Evans, T., Cerione, R. A., & Ashkenazi, A. (1993) J Biol
Chem 268, 26059-62.
[0321] Reference 3 is Weng, Z., Thomas, S. M., Rickles, R. J.,
Taylor, J. A., Brauer, A. W., Seidel-Dugan, C., Michael, W. M.,
Dreyfuss, G., & Brugge, J. S. (1994) Mol Cell Biol 14,
4509-21.
[0322] Reference 4 is Sudol, M. (1994) Oncogene 9, 2145-52.
[0323] Reference 5 is Gertler, F. B., Comer, A. R., Juang, J. L.,
Ahern, S. M., Clark, M. J., Liebl, E. C., & Hoffmann, F. M.
(1995) Genes Dev 9, 521-33.
[0324] Reference 6 is Gout, I., Dhand, R., Hiles, I. D., Fry, M.
J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J.,
Booker, G, W. & et al. (1993) Cell 75, 25-36.
[0325] Reference 7 is Rivero-Lezcano, O. M., Sameshima, J. H., 30
Marcilla, A., & Robbins, K. C. (1994) J Biol Chem 269,
17363-6.
[0326] Reference 8 is Odai, H., Sasaki, K., Iwamatsu, A., Hanazono,
Y., Tanaka, T., Mitani, K., Yazaki, Y. & Hirai, H. (1995) J
Biol Chem 270, 10800-5.
[0327] Reference 9 is Kapeller, R., Prasad, K. V., Janssen, O.,
Hou, W., Schaffhausen, B. S., Rudd, C. E., & Cantley, L. C.
(1994) J Biol Chem 269, 1927-33.
[0328] Reference 10 is Weng, Z., Taylor, J. A., Turner, C. E.,
Brugge, J. S., & Seidel-Dugan, C. (1993) J Biol Chem 268,
14956-63.
[0329] Reference 11 is Karlsson, T., Songyang, Z., Landgren, E.,
Lavergne, C., Di-Fiore, P. P., Anafi, M., Pawson, T., Cantley, L.
C., Claesson-Welsh, L., & Welsh, M. (1995) Oncogene 10,
1475-83.
[0330] Reference 12 is Ren, R., Mayer, B. J., Cicchetti, P., &
Baltimore, D. (1993) Science 259, 1157-61.
[0331] Reference 13 is Ren, R., Ye, Z. S., & Baltimore, D.
(1994) Genes Dev 8, 783-95.
[0332] Reference 14 is Knudsen, B. S., Feller, S. M., &
Hanafusa, H. (1994) J Biol Chem 269, 32781-7.
[0333] Reference 15 is Rozakis-Adcock, M., Fernley, R., Wade, J.,
Pawson, T., & Bowtell, D. (1993) Nature 363, 83-5.
[0334] Reference 16 is McPherson, P, S., Czernik, A, J., Chilcote,
T, J., Onofri, F., Benfenati, F., Greengard, P., Schlessinger, J.,
& De-Camilli, P. (1994) Proc Natl Acad Sci USA 91, 6486-90.
[0335] The sequences shown in Table 15 are useful in that they can
be used as ligands in the assays for the identification of
compounds that affect binding of SH3 domain-containing proteins and
their ligands that is described above in Section 5.6.
[0336] 6.16 Use of Consensus Sequences to Identify Amino Acid
Sequences Resembling SH3 Domain-binding Sequences in Proteins that
are Not Known to Bind SH3 Domains
[0337] The consensus sequences shown above in Tables 1-13 can be
used to search databases (e.g., GenBank) containing the amino acid
sequences of proteins that are not known to bind to SH3 domains. In
this way, a large number of proteins not previously suspected of
containing amino acid sequences that bind SH3 domains have been
shown to contain such sequences. The portions of the amino acid
sequences of these proteins that resemble one or more of the
consensus motifs of Tables 1-13 are shown below in Table 16. The
SH3 domain-binding sequences of the proteins shown in Table 16 can
be used as ligands in the assays for the identification of
compounds that affect binding of SH3 domain-containing proteins and
their ligands that are described above in Section 5.6.
16TABLE 16 LOCUS ACCESSION #'S DESCRIPTION SEQUENCE SRC SRC ABL COR
P53 PLC GRB CRK ABL_DROME P00522 TYROSINE-PROTEIN KINASE DRO 132
146 LLQSRPLPHIPAGST (296) 1 DASH/AB 1380 1395 QIQQKPAVPHKPPLND
(297) 2 2 ABP1_YEAST P15891 ACTIN BINDING PROTEIN SAC 514 528
SSAAPPPPPRRATPE (298) 1 2 ACES_HUMAN P22303 ACETYLCHOLINESTERASE
HOM 73 87 MGPRRFLPPEPKQPW (299) 2 PRECURSOR ACM4_HUMAN P08173
MUSCARINIC HOM 276 290 PPPALPPPPRPVADK (300) 3 2 ACETYLCHOLINE
RECEPT ACRO_HUMAN P10323 ACROSIN PRECURSOR (EC HOM 329 343
QPPPRPLPPRPPAAQ (301) 1 2 1 3 4 21 10 AGIE_RAT Q00900 DNA-BINDING
PROTEIN AGIE- RAT 642 656 PNLRRGLPQVPYFSL (302) 2 BP1 (A ANDR_HUMAN
P10275 ANDROGEN RECEPTOR HOM 368 385 ALAGPPPPPPPHPHARI (303)
AOFB_HUMAN P27338 AMINE OXIDASE (FLAVIN- HOM 480 494
TFLERHLPSVPGLLR (304) 2 CONTAINING) AP2_HUMAN P05549 TRANSCRIPTION
FACTOR HOM 52 68 DFQPPYFPPPPYQPIYPQ (305) 2 AP-2 ATF3_HUMAN P18847
CYCLIC-AMP-DEPENDENT HOM 57 71 CFCHRPLPVPPGSLV (306) 1 TRANSCRIPT
B1AR_HUMAN P08588 BETA-1 ADRENERGIC HOM 282 296 APAPPPGPPRPAAAA
(307) 0 0 RECEPTOR B3AR_HUMAN P13945 BETA-3 ADRENERGIC HOM 361 375
CRCGRRLPPEPCAAA (308) 2 RECEPTOR BCL2_CHICK Q00709 APOPTOSIS
REGULATOR GAL 33 47 GEDRPPVPPAPAPAA (309) BCL-2 BNI1_YEAST P41832
BNI1 PROTEIN (SYNTHETIC SAC 1242 1256 PPPPPPPVPAKLFGE (310) 4 0
LETHAL CADM_MOUSE P33146 MUSCLE-CADHERIN (M- MUS 645 659
PQPHRVLPTSPSDIA (311) 3 CADHERIN) CALR_PIG P25117 CALCITONIN
RECEPTOR SUS 14 28 IFLNRPVLPDSAD (312) 1 PRECURSOR CBL_HUMAN P22681
PROTO-ONCOGENE C-CBL HOM 490 504 QASSLPPVPPRLDLLP (313) 1 1 536 555
PPTLRDLPPPPPPDRPYSVG 2 2 2 (314) 559 573 RPQRRPLPCTPGDCP (315) 2
CCB3_RABIT Q02343 BRAIN CALCIUM CHANNEL ORY 19 33 SDQGRNLPGTPVPAS
(316) 3 BII-1 PR 2100 2114 RHSRRQLPPVPPKPRPLL (317) 1 1 1 0
CCB4_RABIT Q02344 BRAIN CALCIUM CHANNEL ORY 19 33 SDQGRNLPGTPVPAS
(318) 3 BII-2 PRO CG2A_BOVIN P30274 G2/MITOTIC-SPECIFIC BOS 56 70
NDEYVPVPPWKANNK (319) 5 CYCLIN A CIC1_RAT P35524 CHLORIDE CHANNEL
RAT 724 741 QTPTPPPPPPPPLPPQFP (320) PROTEIN SKELE CIK5_HUMAN
P22460 POTASSIUM CHANNEL HOM 60 74 DSGVRPLPPLPDPGV (321) 0 PROTIEN
KV1 5 71 85 DPGVRPLPPLPEELP (322) 0 CINC_RAT P15389 SODIUM CHANNEL
PROTEIN, RAT 1723 1739 LNTGPPYCDPNLPNSNG (323) 3 CANDIAO CP12_RABIT
P00187 CYTOCHROME P450 IA2 (EC ORY 238 252 FPILRYLPNRPLQRF (324) 3
1 14 14 CP75_SOLME P37120 CYTOCHROME P450 LXXVA SOL 30 44
SWRRRKLPPGPEGWP (325) 2 (EC 1 14 CPC7_RAT P05179 CYTOCHROME P450
IIC7 (EC RAT 23 37 SSRRRKLPPGPTPLP (326) 2 1 14 CPC8_HUMAN P10631
CYTOCHROME P450 IIC8 (EC HOM 23 37 SCRRRKLPPGPTPLP (327) 2 1 14
CPCK_MACFA P33262 CYTOCHROME P450 IIC20 (EC MAC 23 37
SSGRRKLPPGPTPLP (328) 2 1 14 CPCM_RAT P19225 CYTOCHROME P450 IIC22
(EC RAT 23 37 HHVRRKLPPGPTPLP (329) 2 1 14 CPT7_MOUSE P27786
CYTOCHROME P450 XVIIA1 MUS 25 39 AKFPRSLPFLPLVGS (330) 2 (P450-C
CR2_MOUSE P19070 COMPLEMENT RECEPTOR MUS 22 38 NARKPYYSLPIVPGTVL
(331) 3 TYPE 2 PREG CTK1_YEAST Q03957 CTD KINASE ALPHA SUBUNIT SAC
30 44 QSLARPPPPKRIRTD (332) 3 1 3 (EC 2 CXA3_BOVIN P41987 GAP
JUNCTION ALPHA-3 BOS 287 301 ASPARALPGPPHPRR (333) 2 3 3 PROTEIN
CYA3_RAT P21932 ADENYLATE CYCLASE, RAT 829 843 TDSRLPLVPSKYSMT
(334) 4 1 OLFACTIVE TY CYGR_HUMAN Q02846 RETINAL GUANYLYL HOM 15 31
GLCGPAWWAPSLPRLPR (335) 3 CYCLASE PRECUR CYLI_HUMAN P35663 CYLICIN
(FRAGMENT) HOM 571 587 LCWCKMPPPPPKPRYAP (336) 2 3 2 CYRG_MOUSE
P34902 CYTOKINE RECEPTOR MUS 283 298 WLERMPPIPPIKNLED (337) 5 2
COMMON GAMMA DCD_HUMAN P20711 AROMATIC-L-AMINO-ACID HOM 31 47
PDVEPGYLRPLIPAAAP (338) 3 DECARBOXY DMD_HUMAN P11532 DYSTROPHIN HOM
700 714 QEELPPPPPQKKRQI (339) 1 DPOD_BOVIN P28339 DNA POLYMERASE
DELTA BOS 104 118 VAPARPLPGAPPPSQ (340) 1 CATALYTIC DRA_HUMAN
P40879 DRA PROTEIN (DOWN- HOM 319 335 GDMNPGFQPPITPDVET (341) 3
REGULATED IN DY15_DROME P13496 150 KD DYNEIN-ASSOCIATED DRO 1250
1264 ARSARRLPSWPPTLD (342) 3 POLYPE DYN1_HUMAN Q05193 DYNAMIN-1 HOM
809 823 LGGAPPVPSRPGASP (343) 1 1 E75C_DROME P13055
ECDYSONE-INDUCIBLE DRO 398 413 VMRPPPPPPPPKVKHA (344) 3 3 1 PROTEIN
E75- 587 601 MRHGRGLPSTPCHTS (345) 3 EGR2_HUMAN P11161 EARLY GROWTH
RESPONSE HOM 113 127 HLYSPPPPPPPYSGC (346) PROTEIN ELK1_MOUSE
P41969 PROTEIN ELK-1 (FRAGMENT) MUS 164 178 PQPQPPIPPRPASVL (347) 1
1 ENL_HUMAN Q03111 ENL PROTEIN HOM 272 286 PPPPPPPPPRASSKR (348) 1
2 452 467 LPSREPPPPQKPPPN (349) 2 EP15_HUMAN P42566 EPIDERMAL
GROWTH HOM 763 778 KSEDEPPALPPKIGTP (350) 3 0 FACTOR RECEPTOR
ERB3_HUMAN P21860 ERBB-3 RECEPTOR PROTEIN- HOM 1204 1218
RRHSPPHPPRPSSLE (351) 4 2 1 TYROSIN EZRI_HUMAN P15311 P23714 EZRLIN
(P81) (CYTOVILLIN) HOM 465 479 VMTAPPPPPPPVYEP (352) (VILLI
FAK_HUMAN Q05397 FOCAL ADHESION KINASE HOM 183 197 KEGERALPSIPKLAN
(353) 2 (EC 2 7 1 FASL_MOUSE P41047 FAS ANTIGEN LIGAND MUS 41 55
DQRRPPPPPPPVSPL (354) 3 FGR_FSVGR P00544 TYROSINE-PROTEINKINASE FEL
9 23 VCRPRPLPPLPPTAM (355) 0 TRANSFO FOR4_MOUSE Q05859 FORMIN 4
(LIMB DEFORMITY MUS 655 669 PPLIPPPPPLPPGLG (356) PROTEIN 681 700
CPVSPPPPPPPPPPTPVPPS (357) 699 718 PSDGPPPPPPPPPPLPNVLA (358) 721
740 NSGGPPPPPPPPPPPPGLAP (359) FOSB_MOUSE P13346 FOSB PROTEIN MUS
253 269 GWLLPPPPPPPLPFQSS (360) FOSB_CHICK P11939 P55-C-FOS
PROTO-ONCOGENE GAL 239 254 LMTEAPPAVPPKEPSG (361) 3 0 PROTEIN
FSH_DROME P13709 P13710 FEMALE STERILE HOMEOTIC DRO 4 20
SEPPPRYEPPVEPVNGI (362) 2 PROTEIN G33_RATE P05432 GENE 33
POLYPEPTIDE RAT 146 160 DRSSRPLPPLPISED (363) 0 281 295
IPPRVPIPPRPAKPD (364) 3 3 1 3 GLI3_HUMAN P10071 GLI3 PROTEIN HOM
789 804 MFPRLNPILPPKAPAV (365) 4 3 1 986 1000 AAPPRLLPPLPTCYG (366)
1 GTPA_BOVIN P09851 GTPASE-ACTIVATIGN BOS 127 141 GGGFPPLPPPPPQLP
(367) PROTEIN (GAP) HME1_MOUSE P09065 HOMEOBOX PROTEIN MUS 72 91
LPHPPPPPPPPPPPPPQHLA ENGRAILED-1 (M (368) HMOC_DROME P22810
HOMEOTIC PROTEIN DRO 453 467 SAPQRPMPPNRPSPP (369) 4 1 2
ORTHODENTICLE HS27_HUMAN P04792 HEAT SHOCK 27 KD PROTEIN HOM 48 64
GSSWPGYVRPLPPAAIE (370) 4 (HSP 2 HXA4_CHICK P17277 HOMEOBOX PROTEIN
HOX- GAL 42 59 HPHAPPPPPPPPPPHLHA (371) A4 (CHOX-1 127 141
GASPPPPPPAKGHPG (372) 3 5 HXAA.sub.-- P31260 HOMEOBOX PROTEIN HOX-
HOM 223 237 PQQQPPPPPQPPQPA (373) HUMAN A10 (HOX-1 HXB2_HUMAN
P14652 P17485 P10913 HOMEOBOX PROTEIN HOX-B2 HOM 75 91
GPALPPPPPPPLPAAPP (374) (HOX-2H HXB3_HUMAN P14651 P17484 HOMEOBOX
PROTEIN HOX-B3 HOM 280 296 HSMTPSYESPSPPAFGK (375) 4 (HOX-2G
HXB4_HUMAN P17483 HOMEOBOX PROTEIN HOX-B4 HOM 69 91
RDPGPPPPPPPPPPPPPPPGLSP (HOX-2 (376) HXC4_HUMAN P09017 HOMEOBOX
PROTEIN HOX-C4 HOM 50 64 QELYPPPPPRPSYPE (377) 1 (HOX-3 IBP1_BOVIN
P24591 INSULIN-LIKE GROWTH BOX 83 97 GLSCRALPGEPRPLH (378) 3 FACTOR
BIND IDE_HUMAN P14735 INSULIN-DEGRADING HOM 995 1009
TEFKRGLPLFPLVKP (379) 3 ENZYME (EC 3 IEFS_HUMAN P31948
TRANSFERMATION- HOM 195 211 EIATPPPPPPPKKETKP (380) 3 2 SENSITIVE
PROTEI IHBB_RAT P17491 INHIBIN BETA B CHAIN RAT 35 49
SPAAPPPPPPPGAPG (381) PRECURSOR IRS1_HUMAN P35568 INSULIN RECEPTOR
HOM 1197 1211 PEPQPPPPPPPHQPL (382) SUBSTRATE-1 (I ISP3_SCHPO
P40899 SEXUAL DIFFERENTIATION SCH 39 55 QHQQPTYWYPPPPPRHH (383) 2 3
2 PROCESS JUND_CHICK P27921 TRANSCRIPTION FACTOR GAL 203 218
PRLPPPPPPPLKDEPQ (384) 4 4 2 JUN-D KICH_HUMAN P35790 CHOLINE KINASE
(EC 2 1 1 32) HOM 53 67 ALALPPPPPLPLPLP (385) KIJ5_YEAST P40494
PROBABLE SAC 744 759 KDKSRPPRPPPKPLHL (386) 2
SERINE/THREONINE-PROTE KIR1_HUMAN Q04771 SERINE/THREONINE-PROTEIN
HOM 450 464 VDQQRPNIPNRWFSD (387) 3 1 KINASE KIR4_HUMAN P36897
SERINE/THREONINE-PROTEIN HOM 447 461 EQKLRPNIPNRWQSC (388) 1 KINASE
KRAF_CAEEL Q07292 RAF HOMOLOG CAE 458 473 LDAQRPRPPQKPHHED (389) 2
SERINE/THREONINE-P MAPA_RAT P34926 MICROTUBULE-ASSOCIATED RAT 1812
1826 VPKDRPLPPAPLSPA (390) 0 PROTEIN 2421 2437 GELSPSFLNPPLPPSTD
(391) 2 MAPB_MOUSE P14873 MICROTUBULE-ASSOCIATED MUS 520 535
DLTGQVPTPPVKQVKL (392) 5 PROTEIN MIS_HUMAN P03971 MUELLERIAN
INHIBITING HOM 266 280 LDTVPFPPPRPSAEL (393) 2 FACTOR 387 401
AAELRSLPGLPPATA (394) 2 MPK1_XENLA Q05116 DUAL SPECIFICITY MITOGEN-
XEN 286 300 ELAPRPRPPGRPISS (395) 3 0 3 ACTIVA MPK2_HUMAN P36507
DUAL SPECIFICITY MITOGEN- HOM 293 307 SISPRPRPPGRPVSG (396) 3 0 3
ACTIVA MYBB_CHICK Q03237 MYB-RELATED PROTEIN B GAL 512 526
YGPIRPLPQTPHLEE (397) 2 MYSA_CAEEL P12844 MYOSINE HEAVY CHAIN A CAE
561 577 LGKHPNFQKPKPPKGKQ (398) 4 (MHC A) MYSB_CAEEL P02566 MYOSINE
HEAVY CHAIN B CAE 559 575 LGKHPNFEKPKPPKGKQ (399) 4 (MHC B)
MYSC_CAEEL P12844 MYOSINE HEAVY CHAIN C CAE 562 578
LGKHPNFEKPKPPKGKQ (400) 4 (MHC C) MYSD_CAEEL P02567 MYOSINE HEAVY
CHAIN D CAE 556 572 LGKHPNFEKPKPPKGKQ (401) 4 (MHC D) NCF1_HUMAN
P14598 NEUTROPHIL CYTOSOL HOM 359 373 SKPQPAVPPRPSADL (402) 2 1
FACTOR 1 (N NEU_RAT P06494 NEU ONCOGENE PRECURSOR RAT 560 574
VSDKRCLPCHPECQP (403) 3 (EC 2 7 NG3_DROME P40140 NEW-GLUE PROTEIN 3
DRO 33 47 LRLPPPLPPRPRQPL (404) 0 0 PRECURSOR ( NME4_MOUSE Q03391
GLUTAMATE (NMDA) MUS 901 915 PPAKPPPPPQPLPSP (405) RECEPTOR SUBU
OIF_HUMAN PS0774 OSTEOINDUCTIVE FACTOR HOM 177 192 NQLLKLPVLPPKLTLF
(406) 3 PRECURSOR P11B_HUMAN P42338 PHOSPHATIDYLINOSITOL 3- HOM 309
323 SNLPLPLPPKKTRII (407) 4 KINASE ( P2B1_HUMAN P16298
SERINE/THREONINE PROTEIN HOM 7 25 ARAAPPPPPPPPPPPGADR 3 PHOSPH
(408) P53_CHICK P10360 CELLULAR TUMOR ANTIGEN GAL 45 62
EPSDPPPPPPPPPLPLAA (409) P53 P85A_HUMAN P27986 PHOSPHATIDYLINOSITOL
3- HOM 89 103 PRPPRPLPVAPGSSK (410) 1 KINASE P85B_BOVIN P23726
PHOSPHATIDYLINOSITOL 3- BOS 90 105 PRGPRPLPPARPRDGP (411) 2 3 0 3
KINASE 290 305 EQEVAPPALPPKPPKT (412) 2 0 PFTA_RAT Q04631 PROTEIN
RAT 18 34 QPEQPPPPPPPPPAQQP (413) FARNESYLTRANSFERASE AL PRGR_HUMAN
P06401 PROGESTERONE RECEPTOR HOM 419 433 LGPPPPLPPRATPSR (414) 0 1
(PR) (FOR PRO_DROME P29617 PROTEIN PROSPERO. DRO 1076 1090
YHPQPPPPPPPMMPV (415) PRPB_HUMAN P02814 PROLINE-RICH PEPTIDE P-B
HOM 17 33 QPFGPGFVPPPPPPPYG (416) 2 PTN1_HUMAN P18031
PROTEIN-TYROSINE HOM 302 316 PPEHIPPPPRPPKRI (417) 3 3 2 2
PHOSPHATASE 1 PTN3_HUMAN P26045 PROTEIN-TYROSINE HOM 860 874
CLTERNLPIYPLDIV (418) 3 PHOSPHATASE P PTN4_HUMAN P29074
PROTEIN-TYROSINE HOM 457 472 PGDGKPPALPPKQSKK (419) 3 PHOSPHATASE
ME PTP1_DROME P35992 PROTEIN-TYROSINE DRO 1430 1446
FTTWPDFGVPNPPQTLV (420) 4 PHOSPHATASE 10 PTPK_MOUSE P35822
PROTEIN-TYROSINE MUS 60 76 SAQEPHYLPPEMPQGSY (421) 2 PHOSPHATASE KA
RADI_HUMAN P35241 RADIXIN HOM 466 481 VMSAPPPPPPPPVIPP (422)
RB_HUMAN P06400 RETINOBLASTOMA- HOM 19 33 EPPAPPPPPPPEEDP (423)
ASSOCIATED PROTE ROG_HUMAN P38159 HETEROGENEOUS NUCLEAR HOM 92 106
GRRGPPPPPPSRGPP (424) 4 1 2 RIBONUCLE ROK_HUMAN Q07244
HETEROGENEOUS NUCLEAR HOM 267 281 GRGGRPMPPSRRDYD (425) 3 1
RIBONUCLE HOM 301 321 GSRARNLPLPPPPPPRGGDL 3 1 1 (426) ROL_HUMAN
P14866 HETEROGENEOUS NUCLEAR HOM 326 346 SRYGPQYGHPPPPPPPPEYGP 3
RIBONUCLE (427) RRG1_HUMAN P13631 RETINOIC ACID RECEPTOR HOM 76 90
SSPSPPPPPRVYKPC (428) 2 2 GAMMA-1 RRG2_HUMAN P22932 RETINOIC ACID
RECEPTOR HOM 65 79 SSPSPPPPPRVYKPC (429) 2 2 GAMMA-2 RRXB_HUMAN
P28702 RETINOIC ACID RECEPTOR HOM 95 109 GSGAPPPPPMPPPPL (430)
RXR-BETA RRXC_HUMAN P28703 RETINOIC ACID RECEPTOR 115 129
GSGAPPPPPMPPPPL (431) RXR-BETA RYNR_HUMAN P21817 RYANODINE
RECEPTOR, HOM 4516 4531 PKKQAPPSPPPKKEEA (432) 4 SKELETAL MU
SHC_HUMAN P29353 SHC TRANSFORMING HOM 297 311 RKQMPPPPPCPGREL (433)
PROTEINS 46 8 SLP1_DROME P32030 FORK HEAD DOMAIN DRO 242 258
GAPAPSYGYPAVPFAAA (434) 3 TRANSCRIPTION SOS_DROME P36675 SON OF
SEVENLESS PROTEIN DRO 1339 1353 RAVPPPLPPRRKERT (435) 0 1 1377 1391
ELSPPPIPPRLNHST (436) 0 1 ST20_YEAST Q03497
SERINE/THREONINE-PROTEIN SAC 533 547 EQPLPPIPPTKSKTS (437) KINASE
SUF_DROME P25991 SUPPRESSOR OR FORKED DRO 229 243 KGLNRNLPAVPPTLT
(438) 2 PROTEIN SXLF_DROME P19339 SEX-LETHAL PROTEIN, DRO 308 322
PANVPPPPPQPPAHM (439) FEMALE-SPEC TACT_HUMAN P40200 T-CELL SURFACE
PROTEIN HOM 538 553 PPPFKPPPPPIKYTCI (440) 1 4 TACTILE TGFB_HUMAN
P22064 TRANSFORMING GROWTH HOM 440 454 KSTHPPPLPAKEEPV (441) 3
FACTOR BETA TIE2-MOUSE Q02858 TYROSINE-PROTEIN KINASE MUS 725 739
SHELRTLPHSPASAD (442) 3 RECEPTOR TJ6_MOUSE P15920 IMMUNE SUPPRESSOR
MUS 81 96 EGEASPPAPPLKHVLE (443) FACTOR J6B7 TLL_DROME P18102
TAILLESS PROTEIN DRO 214 228 ALATRALPPTPPLMA (444) 2 TOP1_HUMAN
P11387 DNA TOPOISOMERASE I (EC HOM 221 237 EHKGPVFAPPYEPLPEN (445)
3 5 99 1 TOPA_HUMAN P11388 DNA TOPOISOMERASE II, HOM 833 849
QRVEPEWYIPIIPMVLI (446) 3 ALPHA ISO TOPB_HUMAN Q02880 DNA
TOPOISOMERASE II, HOM 855 871 QRVEPEWYIPIIPMVLI (447) 3 BETA ISOZ
TRA1_HUMAN P34708 SEX-DETERMINING CAE 1069 1090
PEDDPIYALPPPPPPPAPPRRR 1 3 1 TRANSFORMER PRO (448) TRT1_HUMAN
P13805 TROPONIN T, SLOW HOM 42 57 SRPVVPPLIPPKIPEG (449) 3 SKELETAL
MUSCLE XA1_XENLA P23507 XA-1 PROTEIN PRECURSOR XEN 23 39
GEDSPVFRPPSPPMGPS (450) 2 121 136 FRTGRPLLPIKPEHGR (451) 2
Z01_HUMAN Q07157 TIGHT JUNCTION PROTEIN HOM 1410 1424
IQATPPPPPLPSQYA (452) ZO-1 ZYX_CHICK Q04584 ZYXIN GAL 120 134
AFPSPPPPPPPMFDE (453)
[0338] In Table 16, locus and accession number refer to the
entries' names and accession numbers in GenBank or the Swiss-Prot
database. The two numbers immediately to the left of the displayed
sequences refer to the amino acid positions of the displayed
sequences in the mature proteins. The leftmost of these two numbers
refers to the starting amino acid number of the displayed sequence
in the mature protein. The numbers in parentheses immediately to
the right of the displayed sequences refer to the sequences' SEQ ID
NOs:. The eight columns to the extreme right of Table 16 show the
discrepancies between the displayed sequences and the consensus
motifs of Tables 6-15. The leftmost Src column refers to Class I
motifs; the rightmost Src column refers to Class II motifs.
[0339] It should be apparent to one of ordinary skill that many
other embodiments of the present invention can be contemplated
beyond the preferred embodiments described above but which other
embodiments nevertheless fall within the scope and spirit of the
present invention. Hence, the present invention should not be
construed to be limited to the preferred embodiments described
herein, which serve only to illustrate the present invention, but
only by the claims that follow.
[0340] Also, numerous references are cited throughout the
specification. The complete disclosures of these references are
incorporated by reference herein.
Sequence CWU 0
0
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