U.S. patent application number 12/755046 was filed with the patent office on 2011-02-03 for methods of screening of pp1-interacting polypeptides or proteins, peptides inhibiting pp1c binding to bcl-2 proteins, bcl-xl and bcl-w, and uses thereof.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to XAVIER CAYLA, ALPHONSE GARCIA, ANGELITA REBOLLO.
Application Number | 20110028387 12/755046 |
Document ID | / |
Family ID | 29225746 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110028387 |
Kind Code |
A1 |
GARCIA; ALPHONSE ; et
al. |
February 3, 2011 |
METHODS OF SCREENING OF PP1-INTERACTING POLYPEPTIDES OR PROTEINS,
PEPTIDES INHIBITING PP1c BINDING TO Bcl-2 PROTEINS, BCL-XL AND
BCL-W, AND USES THEREOF
Abstract
The invention relates to methods for identifying novel
PP1-interacting polypeptides and proteins, compounds which are able
to inhibit the binding of PP1c to certain factors naturally
interacting with it, especially proteins of the Bcl-2 family (such
as Bcl-x.sub.L and Bcl-w).
Inventors: |
GARCIA; ALPHONSE; (PARIS,
FR) ; CAYLA; XAVIER; (ROCHECORBON, FR) ;
REBOLLO; ANGELITA; (PARIS, FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, L.L.P.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
PARIS CEDEX 15
FR
|
Family ID: |
29225746 |
Appl. No.: |
12/755046 |
Filed: |
April 6, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10982891 |
Nov 8, 2004 |
7741288 |
|
|
12755046 |
|
|
|
|
PCT/EP03/05453 |
May 6, 2003 |
|
|
|
10982891 |
|
|
|
|
Current U.S.
Class: |
514/3.8 ;
435/184; 435/320.1; 435/7.4; 436/501; 436/86; 506/17; 506/18;
506/9; 514/1.1; 514/15.7; 514/17.7; 514/17.8; 514/2.3; 514/3.7;
514/6.9; 530/387.9; 536/23.5 |
Current CPC
Class: |
G01N 2500/04 20130101;
A61P 31/00 20180101; A61P 31/12 20180101; G01N 2500/02 20130101;
A61P 25/28 20180101; C07K 14/4747 20130101; G01N 2333/916 20130101;
C07K 7/08 20130101; G01N 2500/20 20130101; C40B 30/04 20130101;
A61P 25/16 20180101; A61P 31/18 20180101; C07K 7/06 20130101; A61K
38/1709 20130101; G01N 33/6842 20130101; A61P 9/12 20180101; A61P
3/10 20180101; G01N 33/6845 20130101; C07K 16/18 20130101; G01N
33/505 20130101; C12N 9/16 20130101; G01N 2500/10 20130101; A61P
25/00 20180101 |
Class at
Publication: |
514/3.8 ;
435/7.4; 435/184; 435/320.1; 436/501; 436/86; 506/9; 506/17;
506/18; 514/1.1; 514/2.3; 514/3.7; 514/6.9; 514/15.7; 514/17.7;
514/17.8; 530/387.9; 536/23.5 |
International
Class: |
A61K 38/02 20060101
A61K038/02; G01N 33/573 20060101 G01N033/573; C12N 9/99 20060101
C12N009/99; C12N 15/63 20060101 C12N015/63; G01N 33/68 20060101
G01N033/68; C40B 30/04 20060101 C40B030/04; C40B 40/08 20060101
C40B040/08; C40B 40/10 20060101 C40B040/10; C07K 16/18 20060101
C07K016/18; C07H 21/04 20060101 C07H021/04; A61K 38/17 20060101
A61K038/17; A61P 25/28 20060101 A61P025/28; A61P 3/10 20060101
A61P003/10; A61P 9/12 20060101 A61P009/12; A61P 25/00 20060101
A61P025/00; A61P 31/12 20060101 A61P031/12; A61P 31/00 20060101
A61P031/00; A61P 25/16 20060101 A61P025/16; A61P 31/18 20060101
A61P031/18 |
Foreign Application Data
Date |
Code |
Application Number |
May 7, 2002 |
EP |
02291170 |
Claims
1-23. (canceled)
24. A set of polypeptides having both the motifs M1 and M2 on the
same polypeptide or M1 on one polypeptide and M2 on the other
polypeptide wherein M1 and M2 are mammalian PP1 binding sequences
wherein M1 is RRNFVNKLKPL (SEQ ID NO:93) and M2 is
NQAKKRWFADSTVKVKNVSFEKK (SEQ ID NO:94) or M1 is LDFRNRLQTN (SEQ ID
NO:95) and M2 is KKVKKRVSFAND (SEQ ID NO:96) or M1 is RHFVNKLKPLKS
(SEQ ID NO:97) and M2 is NQAKKRWFADS (SEQ ID NO:98) or M1 is
TCFRPRLRGS (SEQ ID NO:99) and M2 is SQKKKRWFADM (SEQ ID NO:100) or
M1 is GEVFINKGKGF (SEQ ID NO:101) and M2 is RGRQLRVRFATH (SEQ ID
NO:102) or M1 is QVKFRRRREG (SEQ ID NO:103) and M2 is YFKRYQVKFRRR
(SEQ ID NO:104) or M1 is EDFKAKKKEL (SEQ ID NO:105) and M2 is
RITPSYVAFTPE (SEQ ID NO:106) or M1 is SVFMQRLKTNILQ (SEQ ID NO:107)
and M2 is IDEVKNVYFKNFVLKVSWITFLLA (SEQ ID NO:108-109) or M1 is
LQFELRYRPV (SEQ ID NO:110) and M2 is TTKAVMFAK (SEQ ID NO:111) or
M1 is DLFENRKKKN (SEQ ID NO:112) and M2 is VRRVFIM (SEQ ID NO:113)
or M1 is FFKNEKMLY (SEQ ID NO:114) and M2 is RRGSPRVRFEDG (SEQ ID
NO:115).
25. The set of polypeptides according to claim 24, wherein said
peptides are glycosylated, acetylated, phosphorylated or
amidated.
26. A set of isolated nucleic acids encoding a set of peptides
according to claim 24 or a nucleic acid sequence that hybridizes
under stringent conditions to one of said nucleic acids in said set
of peptides.
27. A vector comprising the nucleic acids according to claim
26.
28. A method of identifying a PP1-interacting polypeptide or
protein, comprising: detecting in the sequence said polypeptide or
protein, the presence of two PP1-binding motifs M1 and M2 on the
same polypeptide, wherein the motif M1 and M2 is chosen from one of
the motif pairs in claim 24; and confirming said identified PP-1
interacting polypeptide or protein using a biochemical test.
29. The method of claim 28, wherein said biochemical test consists
in coimmunoprecipitating said identified PP-1 interacting
polypeptide or protein and PP1.
30. A method of screening compounds that inhibit or enhance PP1
activity or change its localization by interaction or that interact
with PP1 regulators, comprising; (a) immobilizing peptides
according to claim 24 on a support; and (b) testing the interaction
of said compounds with the peptides of step (a).
31. A method of screening compounds that interact with PP1,
comprising: i. obtaining antibodies to peptides according to 24 1
and ii. testing the interaction of said compound with said
antibodies.
32. A pharmaceutical compound comprising the set of peptides
according to claim 24.
33. A pharmaceutical composition comprising a nucleic acid encoding
the set of peptides according to claim 24.
34. A pharmaceutical composition comprising the vector of claim
27.
35. A method for inhibiting in vitro the interaction between PP1 c
and Bc1XL, comprising a step of adding a set of peptides having
both the motifs M1 and M2 on the same peptide, wherein the set of
peptides comprises one of the M1 and M2 motif pairs in claim
24.
36. A kit comprising antibodies to the set of peptides according to
claim 24 and reagents for interpreting the interaction.
37. A method of treating or preventing diabetes, high blood
pressure, a neurological disorder, a viral disease or a microbial
infection associated with PP1 said method comprising administering
to a patient in need of such treatment the set of polypeptides
according to claim 24.
38. The method according to claim 37, wherein the neurological
disorder is Parkinson's disease or Alzheimers disease.
39. The method according to claim 37, wherein the viral disease is
HIV-1.
40. Antibodies to the peptides according to claim 24.
41. Antibodies according to claim 40, wherein said antibodies are
monoclonal antibodies.
42. The set of polypeptides according to claim 24, wherein there
are two or more M1 and M2 motifs on the same polypeptide.
43. The set of polypeptides according to claim 42, wherein there
are 2 to 5, M1 and M2 motifs on the same polypeptide.
44. The set of polypeptides according to claim 42, wherein there
are 2 to 10, M1 and M2 motifs on the same polypeptide.
45. The set of polypeptides according to any one of claim 42,
wherein said M1 and M2 motifs have a spacer between them.
46. The set of polypeptides according to claim 45, wherein the
spacer is selected from a sequence of amino acids, a hydrocarbon
chain or a chemical entity which connects the at least two
motifs.
47. The set of polypeptide according to claim 46, wherein said
amino acid spacer has between 1 to 50 amino acids.
48. The set of polypeptide according to claim 46, wherein amino
acid spacer has 36 amino acids.
49. The set of polypeptide according to claim 46, wherein said
spacer contains regulatory sequences between the motifs.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates to methods for identifying novel
PP1-interacting polypeptides and proteins, compounds which are able
to inhibit the binding of PP1c to certain factors naturally
interacting with it, especially proteins of the Bcl-2 family (such
as Bcl-x.sub.L and Bcl-w), and pharmaceutical compositions
comprising the same.
PRIOR ART
[0002] The serine/threonine phosphatases are classified as type 1
(PP1) or type 2 (PP2), depending on their substrate specificity and
sensitivity to inhibitors. PP1 regulates cell cycle progression,
proliferation, transcription, protein synthesis, cytokinesis and
neuronal signaling (Mc Avoy et al, 2001). PP1 is regulated by its
interaction with a variety of protein subunits that target the
catalytic subunit (PP1c) to specific subcellular compartments and
determines its localization, activity and substrate selectivity
(Bollen et al, 2001). PP1 can be regulated by the interaction
between a catalytic subunit and multiple targeting subunits that
allow specific dephosphorylation of diverse cellular targets. The
number of known PP1c targeting subunits is continuously increasing
and to date nearly thirty unique mammalian proteins have been
already identified. PP1 interacting protein include the
glycogen-binding subunits, RGL/GM, GL, PTG/R5/U5, and R6 which
target the phosphatase to glycogen, the myosin-associating
subunits, M110, NIPP-1, p99/PNUTS, and Sds22, which may direct the
phosphatase to the nucleus (Bollen, 2001). Previous studies based
on structural and X-ray crystallography analysis of PP1 interacting
proteins indicated that PP1c binds to distinct known interacting
proteins through a short amino-acid sequence. The [RK]VxF (or
[RK]xVxF) motifs represent a widespread consensus sequence for the
recognition and binding of distinct regulatory subunits and
interacting proteins with PP1c (Egloff et al, 1997; Aggen et al,
2000).
[0003] Using a murine T cell line that can be propagated
independently in the presence of IL-2 or IL-4 (Pitton et al, 1993),
the inventors have described ` that PP1c is a Ras-activated
phosphatase that dephosphorylates Bad (a pro-apoptotic member of
the Bcl-2 protein family) prior to induce apoptosis in response to
IL-2 deprivation (Ayllon et al, 2000). By performing biochemichal
competitive studies, the inventors also recently identified Bcl-2
as a new targeting subunit of PP1c that controls its association to
Bad in IL-2-stimulated cells (Ayllon et al, 2001).
[0004] The Bcl-2 family proteins act as an intracellular checkpoint
in the apoptotic pathway. The Bcl-2 family of proteins is divided
into two functional groups: anti-apoptotic members such as Bcl-2,
Bcl-x.sub.L, Bcl-w, A1 and Mcl-1 and pro-apoptotic members such as
Bax, Bak, Bcl-x.sub.s as well as the BH3-only member Bad (White,
1996; Reed, 1998; Chao and Korsmeyer, 1998). Balance between homo-
and hetero-dimers of Bcl-2 family members may be critical to
maintain cell proliferation or apoptosis (Jacobson, 1997;
Korsmeyer, 1999; Gross et al, 1999). Up- or down-regulation of
these proteins may account for survival of some cell types,
although it is also possible that survival factors use protein
kinases or phosphatases to alter the ability of these proteins to
promote cell survival or apoptosis. Anti-apoptotic Bcl-2 family
members interact with other death agonists of the Bcl-2 family and
with non-Bcl-2 family proteins, including R-Ras, H-Ras, Raf,
caspases, calcineurin and the serine/threonine phosphatase PP1c
(Rebollo et al, 1999; Ayllon et al, 2001). The Bcl-2 family has
been defined by sequence homology based upon specific conserved
motifs termed Bcl-homology regions (BH1, BH2, BH3 and BH4 domains).
BH1, BH2 and BH3 domains have been shown to be important in
homodimerization or heterodimerization and in modulating apoptosis.
Anti-apoptotic molecules have a specific BH4 domain.
[0005] Bad shares identity only in the BH3 domain (Zha et al, 1997)
and forms hetero-dimers with Bcl-2 and Bcl-x (Ottilie et al, 1997).
Upon stimulation of cells with IL-3, NGF and GM-CSF, Bad becomes
serine phosphorylated (Del Peso et al, 1997; Datta et al, 1997),
resulting in association to the 14-3-3 protein and abolishing
interaction with Bcl-x (Hsu et al, 1997). It has been recently
shown that association of 14-3-3 protein to Bad is dependent on
serine 155 phosphorylation of Bad (Datta et al, 2000; Zhou et al,
2000).
[0006] Bcl-w is a pro-survival protein bearing the four conserved
Bcl-2 homology (BH) domains (Gibson et al, 1996). Enforced
expression of Bcl-w, like Bcl-2, renders lymphoid and myeloid cell
lines resistant to apoptosis induced by cytokine deprivation. The
anti-apoptotic molecule Bcl-x.sub.L also contains the four BH
conserved domains (N nez et at, 1994). A second Bcl-x isoform,
Bcl-x.sub.s, encodes a smaller protein of 170 amino acids that
enhances apoptosis (Minn et al, 1996). Bcl-x.sub.L contains a
hydrophobic segment at the C-terminal end that is believed to serve
as a membrane anchor (Boise et al, 1993).
[0007] Apoptosis or programmed cell death is an active process in
which cells induce their self-destruction in response to specific
cell death signals or in the absence of cell survival signals. This
active process, is actually essential in the normal development and
homeostasis of multicellular organisms. It is opposed to necrosis
which is cell death occurring as a result of severe injurious
changes in the environment.
[0008] Various pathologies occur due to a defective or aberrant
regulation of apoptosis in the affected cells of an organism. For
example, defects that result in a decreased level of apoptosis in a
tissue as compared to the normal level required to maintain the
steady-state of the tissue can promote an abnormal increase of the
amount of cells in a tissue. This has been observed in various
cancers, where the formation of tumors occurs because the cells are
not dying at their normal rate. Some DNA viruses such as
Epstein-Barr virus, African swine fever virus and adenovirus, also
inhibit or modulate apoptosis, thereby repressing cell death and
allowing the host cell to continue reproducing the virus.
[0009] To the contrary, a defect resulting in an increase of cell
death in a tissue may be associated with degenerative disorders
wherein cells are dying at a higher rate than they regenerate. This
is observed in various disorders, such as AIDS, senescence, and
neurodegenerative diseases.
[0010] Compounds that modulate positively or negatively apoptosis
can provide means for the treatment or the prevention of these
disorders. As a consequence, the delineation of apoptotic pathways
provides targets for the development of therapeutic agents that can
be used to modulate the response of a cell to apoptotic or cell
survival signals.
[0011] The results disclosed in the present invention indicate that
the anti-apoptotic: members of the Bcl-2 family, Bcl-w and
Bcl-x.sub.L are also targeting subunits of PP1c in IL-4-stimulated
cells. This observation offers a way to a novel general mechanism
of regulation of cell apoptosis that may play a role in the
regulation of pro- or anti-apoptotic molecules in response to cell
death or cell survival signals. The invention is therefore of a
particular importance in the fields of cancer therapy and
neurodegenerative diseases therapy.
[0012] More generally, the present invention provides means to
modulate the interaction between PP1 and the proteins or
polypeptides that bind to it. Therefore, the present invention has
applications in a wide range of fields, since PP1 is involved in
many biological pathways. For example, it is known that a decrease
in phosphorylation of the PP1 complex activates the smooth muscle
myosin light chains and hence relaxes smooth muscles (see, Uehata
et al., 1997). High blood pressure could hence be a target of the
present invention.
[0013] PP1 is a major eukaryotic protein serine/threonine
phosphatase that regulates diverse cellular processes such as cell
cycle, transcription and protein synthesis.
[0014] PP1-regulation can also effect the downstream regulation of
hepatic glycogen synthesis which in turn would lower blood glucose
levels and thus treat diabetes in which hyperglycemia is a severe
problem.
[0015] Moreover, PP1 is involved in several bacterial, viral and
parasitic infections, and inhibiting its interaction with some of
its partners in an infectious context could be beneficial to the
patient.
SUMMARY OF THE INVENTION
[0016] To be concise, the following Summary, the Preferred
Embodiments and the Examples of the present invention are described
below, it being understood that literal word-for-word description
of all of the embodiments and combinations thereof would be
recognized by the person skilled in the art and hence, literally
repetiveness not being necessary. It should be appreciated that the
Examples, as well as the preferred embodiments, the Summary of the
Invention and certain aspects of the prior art, can be combined in
all variations; one aspect of the invention combined with another
irregardless of their place in this description of the entire
specification, without deviating from the present invention, as
recognized by the person skilled in the art, without any
limitations.
[0017] Thus, the present invention relates to a peptide or a set of
peptides which mimicks both the motifs M1 and M2 and which is able
to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions, wherein the motif M1 has the sequence
FXX[RK]X[RK], and the motif M2 has the sequence [RK]VX[FW] or
[RK]XVX[FW], wherein X is any amino acid.
[0018] In another embodiment, the present invention relates to a
set of peptides comprising R (NWGRIVAFFSF) and F (GDEFELRYRRAF)
peptides.
[0019] In yet another embodiment the present invention relates to a
pharmaceutical composition comprising a peptide or a set of
peptides which mimicks both the motifs M1 and M2 and which is able
to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions. Such a composition can, for example,
comprise R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides. Peptides
with modified amino acids (glycosylation, acetylation,
phosphorylation, amidation or derivation by known
protecting/blocking groups) can also be used in the compositions
according to the invention.
[0020] In yet another embodiment the present invention relates to a
pharmaceutical composition comprising a vector comprising a nucleic
acid encoding a peptide or a set of peptides which mimicks both the
motifs M1 and M2.
[0021] In another embodiment the present invention relates to a
method of identifying a PP1-interacting polypeptide or protein,
comprising detecting in the sequence of a polypeptide or protein,
the presence of two PP1-binding motifs M1 and M2.
[0022] In yet another embodiment the present invention discloses a
method of screening compounds that interact with PP1 regulators,
wherein peptides which mimick both the motifs M1 and M2 and which
are able to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions, are immobilized on a support and the
interaction of said compounds with said peptides are tested.
[0023] In yet another embodiment, the present invention relates to
a method of screening compounds that interact with PP1. This method
comprises obtaining antibodies to peptides which mimick both the
motifs M1 and M2 and which are able to inhibit the
Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c
interactions, and testing the interaction of said compound with
said antibodies.
[0024] In another embodiment, the present invention relates to a
method for inhibiting in vitro the interaction between PP1c and
Bcl-x.sub.L or Bcl-w, comprising a step of adding a peptide or a
set of peptides which mimicks both M1 and M2.
[0025] In the above methods, the peptides which mimick both the
motifs M1 and M2 and which are able to inhibit the
Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interaction
can be for example the R (NWGRIVAFFSF) and F (GDEFELRYRRAF)
peptides.
[0026] In yet another embodiment, the present invention relates to
a kit comprising peptides which mimick both the motifs M1 and M2
and which are able to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c,
or the Bcl-2/Bad/PP1c interactions. Such a kit can, for example,
comprise R (NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides. These
peptides may be immobilzed on a solid support.
[0027] In yet another embodiment the present invention relates to a
kit containing antibodies to peptides which mimick both the motifs
M1 and M2 and which are able to inhibit the Bcl-x.sub.L/PP1c, the
Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions. Said antibodies can
for example be raised against R (NWGRIVAFFSF) and F (GDEFELRYRRAF)
peptides.
[0028] In yet another aspect, the present invention relates to a
method for treating animals and vegetables that have any disease
involved in the PP1c pathway, comprising administering to animals
and vegetables in need of such treatment peptides which mimick both
the motifs M1 and M2 and which are able to inhibit the
Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c
interactions.
[0029] In yet another aspect, the present invention relates to a
method for treating an animal that has diabetes or hypertension or
a neurological disorder or a viral infection or a parasitic
infection, comprising administering to an animal in need of such
treatment peptides which mimick both the motifs M1 and M2 and which
are able to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions.
[0030] In the above methods, R (NWGRIVAFFSF) and F (GDEFELRYRRAF)
peptides can for example be administered.
[0031] In another embodiment, the present invention relates to the
use of peptides which mimick both the motifs M1 and M2 and which
are able to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions, to treat diabetes or hypertension or
neurological disorders or a viral infection or a parasitic
infection.
[0032] In yet another embodiment the present invention relates to
the use of peptides which mimick both the motifs M1 and M2 and
which are able to inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or
the Bcl-2/Bad/PP1c interactions, for the preparation of a
medicament to treat diabetes or hypertension or neurological
disorders or a viral infection or a parasitic disease. R
(NWGRIVAFFSF) and F (GDEFELRYRRAF) peptides can for example be used
according to the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1. Association of Bcl-x.sub.L and Bcl-w to PP1c and
Bad.
[0034] A) Cytoplasmic extracts from IL-4-stimulated (60 U/ml) or
-deprived cells were immunoprecipitated with anti-Bcl-x.sub.L or
anti-Bcl-w antibody, transferred to nitrocellulose and blotted with
anti-Bad, anti-PP1c, anti-Bcl-x.sub.L and anti-Bcl-w antibody.
Protein bands were detected using ECL system. Molecular weights of
the corresponding proteins is shown. Similar results were obtained
in three independent experiments. B) Cytoplasmic extracts from
IL-4-stimulated cells were immunoprecipitated with anti-p55 IL-2R
chain antibody, transferred to nitrocellulose and blotted with
anti-Bcl-w, Bcl-x.sub.L or anti-p55 IL-2R antibodies. Protein bands
were detected using the ECL system. C) Cytoplasmic extracts from
freshly isolated thymocytes were immunoprecipitated with anti-Bad,
anti-PP1c or an irrelevant serum and blotted with anti-Bcl-2,
anti-Bcl-x.sub.L, anti-Bad and anti-PP1c. Proteins were detected as
in A. Similar results were obtained in three independent
experiments.
[0035] FIG. 2. Effect of IL-4-deprivation on Bad, PP1c, Bcl-x.sub.L
and Bcl-w expression.
[0036] A) Ts1.alpha..beta. cells were IL-4-stimulated or -deprived
for the times indicated, then lysed. Proteins were transferred to
nitrocellulose and probed with anti-Bcl-x.sub.L, anti-Bcl-w,
anti-Bad and anti-PP1c antibodies. Similar results were obtained in
two independent experiments.
[0037] B) Cytoplasmic extracts from IL-4-stimulated or 24
h-deprived cells were immunoprecipitated with anti-Bcl-x.sub.L or
anti-Bcl-w, separated in a gradient SDS-PAGE gel and blotted with
anti-Pser, anti-Bad, anti-PP1c and, as internal control, with
anti-Bcl-x.sub.L and Anti-Bcl-w. Similar results were obtained in
three independent experiments.
[0038] C) Cytoplasmic extracts from control, IL-4 stimulated or
deprived cells (1.times.10.sup.7) were immunoprecipitated with
anti-Bad antibody and blotted with anti-Bad serine 112, serine 136
and serine 155. As internal control, the blot was developed with
anti-Bad antibody. Similar results were obtained in two independent
experiments. Positive control for serine 112 and 136
phosphorylation of Bad, IL-2 stimulated cells (lane C); positive
control for serine 155 phosphorylation of Bad, Bad-transfected COS
cells (lane C). D) Total extracts (T) of cytoplasmic lysates from
IL-4-stimulated or deprived cells (1.times.10.sup.7) were
immunoprecipitated with anti-PP1c, anti-Raf, anti-Bcl-x.sub.L or
anti-Bad antibody and blotted with anti-14-3-3, anti-PP1c,
anti-Bcl-x.sub.L and anti-Bad. Similar results were obtained in two
independent experiments.
[0039] FIG. 3. Estimation of serine/threonine phosphatase activity
in control or OA-treated Bcl-x, Bad and Bcl-w
immunoprecipitates.
[0040] A) Phosphatase activity was estimated in Bad, Bcl-x.sub.L
and Bcl-w immunoprecipitates from IL-4-stimulated cells using
.sup.32P phosphorylase a as substrate.
[0041] B) Different concentrations of OA were added to Bad or Bcl-w
immunoprecipitates from IL-4 stimulated cells. Phosphatase activity
was estimated using .sup.32P phosphorylase a as substrate. The
reaction was as in A. Similar results were obtained in three
independent experiments. Phosphatase activity is represented as the
percentage of maximal activity in untreated supernatants.
[0042] FIG. 4. Estimation of serine/threonine phosphatase activity
after Bcl-.sub.L and Bcl-w depletion.
[0043] A) Bcl-x.sub.L and Bcl-w were depleted from cytoplasmic
lysates of IL-4 stimulated cells by four sequential
immunoprecipitations. Phosphatase activity was estimated in Bad
immunoprecipitates from control IL-4-stimulated cells or in Bad
immunoprecipitates depleted of Bcl-x.sub.L and Bcl-w after four
sequential immunoprecipitations. Phosphatase activity is
represented as the percentage of the maximal activity detected in
control anti-Bad immunoprecipitates. B) The effect of Bcl-x.sub.L
and Bcl-w depletion in PP1c/Bad association was analyzed.
Cytoplasmic extracts from control IL-4-stimulated cells or
Bcl-x.sub.L, and Bcl-w depleted cytoplasmic extracts were
immunoprecipitated with anti-Bad or anti-PP1c antibody and blotted
with anti-Bad, anti-Bcl-w, anti-Bcl-x.sub.L and anti-PP1c. Similar
results were obtained in three independent experiments. Protein
bands were detected using the ECL system.
[0044] FIG. 5. PP1c binding assay on cellulose-bound Bcl-x.sub.L or
Bcl-w peptides. A) Sequence of F X X R X R motif of Bcl-2,
Bcl-x.sub.L and Bcl-w. B) Membrane with Bcl-x.sub.1 or Bcl-w
peptides containing the R/K X V/I X F or F X X R X R motif, as well
as peptides containing mutated motifs were incubated with purified
PP1c, followed by anti-PP1c antibody and PO-conjugated secondary
antibody. Spots were detected using ECL system. The R/K X V/I X F
and F X X R X R motifs are in bold. Mutated amino acids into the
motif are in bold and underlined. Similar results were obtained in
two independent experiments. Peptide 1 corresponds to the PP1
binding motif of Bcl-x.sub.L and Bcl-w. Peptide 2 corresponds to
the mutated PP1 binding site were V and F residues were mutated to
A.
[0045] FIG. 6. Effect of R, R*, F and R+F peptides on the
interaction Bcl-x.sub.L/PP1c/Bad and Bcl-w/PP1c/Bad.
[0046] A) Cytoplasmic extracts from control IL-4-stimulated cells
were immunoprecipitated with anti-Bad antibody. The interaction
Bcl-x.sub.L/PP1c/Bad and Bcl-w/PP1c/Bad was competed with 1.5 mM of
R, R*, F or R+F peptides for 30 min at room temperature.
Immunoprecipitates were washed, transferred to nitrocellulose and
blotted with anti-Bad, anti-PP1c, anti-Bcl-x.sub.L and anti-Bcl-w.
Similar results were obtained in two independent experiments. For
sequence of peptides, see Materials and Methods or FIG. 5A and
5B.
[0047] B) Cytoplasmic lysates from IL-4-stimulated cells were
immunoprecipitated with anti-Bad antibody. Immunoprecipitates were
treated with 1.5 mM of R, R*, F or R+F peptides for 30 min at room
temperature. Immunoprecipitates were washed and phosphatase
activity estimated using .sup.32P phosphorylase a as substrate.
Similar results were obtained in two independent experiments.
[0048] C) Cytoplasmic lysates from IL-4-stimulated cells were
immunoprecipitated with anti-Bad antibody and then treated with 1.5
mM of R+F peptide or 3 mM of R or F peptide (30 min, room
temperature). Immunoprecipitates were washed and phosphatase
activity estimated as in B.
[0049] FIG. 7. Effect of OA and antisense oligonucleotides on
apoptosis and serine 136 phosphorylation of Bad.
[0050] A) Cells were treated with or without 1 .mu.M OA in the
presence or the absence of IL-4. Cytoplasmic lysates were
immunoprecipitated with anti-Bad antibody, transferred to
nitrocellulose and probed with phospho-Bad ser 136 and anti-Bad,
the latter to verify that OA treatment in vivo does not affect Bad
expression. Protein bands were detected using ECL.
[0051] B) Cells were treated for 6 h with or without 1 .mu.M OA in
the presence or absence of IL-4 and then washed, stained with
ptopidium iodide and analyzed by flow cytometry.
[0052] C) Cells were treated for 24 h with or without 15 .mu.M
sense or antisense oligonucleotide in the presence or the absence
of IL-4. Oligonucleotides were added a 0, 12 and 18 h and then
cells were washed, stained with propidium iodide and analyzed by
flow cytometry. The expression of Bcl-x.sub.L and Bcl-w upon sense
and antisense treatment was analyzed by western blot.
[0053] FIG. 8: A) Two putative PP1 binding motifs in Bcl-2 proteins
Sequence alignment in the vicinity of BH1 and BH3 domain of some
Bcl-2 proteins. These sequences are perfectly conserved in various
species (human, mouse, rat, bovine . . . ).
[0054] B) A role for Serine phosphorylation in PP1 binding
[0055] Six peptides (of 14 AA length) were synthesized and
covalenty linked to a cellulose membrane prior to be analyzed for
PP1-binding as described. A punctual AA mutation in Ser-136 was
introduce in 4 peptides (mutant2, 3,5,6).
[0056] C) Association of P13-K p85 (lane 1), P13-K p110 (lane 2),
HSP70, (lane 3),and CD4 (lane 4) to PP1c.
[0057] IL-4 treated TS1 .alpha..beta. cells (1.times.10.sup.7) were
used for immunoprecipitation as usually described.
Immunoprecipitates were transferred to nitrocellulose, blocked and
incubated with anti-PP1c primary antibody. Membrane was washed and
incubated with PO-conjugated secondary antibody and proteins were
developed using the ECL.
[0058] FIG. 9: Histogram to illustrate the number of proteins and
the number of amino-acids distancing the F-x-x-[RK]-x-[RK] and
[RK]-V-x-[FW]/[RK]-x-V-x-[FW](A)[RK]-V-x-F/[RK]-x-V-x-F(B)
motifs.
PREFERRED EMBODIMENTS OF THE INVENTION
[0059] The following terms that are used throughout the remaining
specification and claims and should be understood to mean, besides
the generic definition, the following more precise definition; it
being understood that the generic definitions are also
included.
[0060] As used herein, the word "aspect" means any technical
feature or element of the claimed invention.
[0061] As used herein, the word "mimick" means close resemblance to
either in structure and/ or function.
[0062] As used herein the word "isolated" means taken from the
natural environment. Isolated does not necessarily mean that what
is taken from the natural environment is 100% purified.
[0063] As used herein the term "biochemical test" means any test
which is capable of identifying an interacting polypeptide or
protein. Examples of a biochemical tests include, but are not
limited to, immunoprecipitation, use of antibodies, either
monoclonal or polyclonal, oligonucleotide probes that can be
labelled with radioactivity or an enzyme, GST pulldown (gel
filtration experiment revealing size of macromolecular complexes)
as described in Ayllon et al EMBO J. 19 2237-2246 (2000), and the
like.
[0064] As used herein, the term "motif" means a particular amino
acid sequence or sequences which are similar and have the same
function in different cellular environments. A motif has some fixed
amino acids and some variable ones.
[0065] As used herein and throughout the entire specification and
in the claims, when several amino acids are bracketed, this means
that the amino acid within the brackets can be one or the other.
Thus [RK] means that the motif can have either R or K.
[0066] As used herein, the word "inhibits" means to prevent the
specific interactions, regardless of the mechanism of this
prevention.
[0067] As used herein a "pharmaceutical composition" includes, but
is not limited to, the peptides of the present invention disclosed
throughout the specification and a pharmaceutically acceptable
carrier. This pharmaceutical composition comprises a
pharmaceutically acceptable amount of the peptides of the present
invention. The pharmaceutically acceptable amount can be estimated
from cell culture assays. For example, a dose can be formulated in
animal models to achieve a circulating concentration range that
includes or encompasses a concentration point or range having the
desired effect in an in vitro system. This information can thus be
used to accurately determine the doses in animals, including
humans.
[0068] The therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms in a patient.
Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or in experimental animals. For example, the LD50 (the dose lethal
to 50% of the population) as well as the ED50 (the dose
therapeutically effective in 50% of the population) can be
determined using methods known in the art. The dose ratio between
toxic and therapeutic effects is the therapeutic index which can be
expressed as the ratio between LD 50 and ED50 compounds that
exhibit high therapeutic indexes.
[0069] The data obtained from the cell culture and animal studies
can be used in formulating a range of dosage of such compounds
which lies preferably within a range of circulating concentrations
that include the ED50 with little or no toxicity.
[0070] The pharmaceutical composition can be administered via any
route such as locally, orally, systemically, intravenously,
intramuscularly, mucosally, using a patch and can be encapsulated
in liposomes, microparticles, microcapsules, and the like. The
pharmaceutical composition can be embedded in liposomes or even
encapsulated. The pharamaceutical composition can also be in a
lyophilized form.
[0071] Any pharmaceutically acceptable carrier or adjuvant can be
used in the pharmaceutical composition. The modulating compound
will be for instance in a soluble form combined with a
pharmaceutically acceptable carrier. The techniques for formulating
and administering these compounds can be found in "Remington's
Pharmaceutical Science" Mack Publication Co., Easton, Pa., latest
edition.
[0072] The mode of administration optimum dosages and galenic forms
can be determined by the criteria known in the art taken into
account the seriousness of the general condition of the mammal,
including the human, the tolerance of the treatment and the side
effects.
[0073] "Pharmaceutical compositions" also include vectors which can
be administered directly in vivo or can be combined with specific
cells that can be or may not be extracted from the animal to be
treated. Types of cells include all eukaryotic and prokaryotic
cells including muscle, heart, liver, lung, brain cells,
thymocytes, blood and the like. Plant and bacterial cells are also
encompassed in the present invention since the PP1 is found in many
different types of plant and bacterial cells. Yeast cells are also
encompassed by the present invention. In fact any cell that
contains PP1 is encompassed by the present invention.
[0074] Thus encompassed by the term "pharmaceutical compositions"
are included all forms of not only classic pharmaceutical
administration, but also include gene therapy.
[0075] By the term "support" is meant any type of object on which
peptides can be immobilized. The type of support includes, but is
not limited to, costar wells, beads, resins, glass chips, membranes
and the like. Any support which can be used to immobilize proteins
or peptides can be used in the methods of the present
invention.
[0076] The term "animal" encompasses any living being which is not
vegetal, including vertebrates such as mammals, birds, reptiles,
amphibians and fish.
[0077] As used herein the terms "polynucleotides", "nucleic acids"
and "oligonucleotides" are used interchangeably and include, but
are not limited to RNA, DNA, RNA/DNA sequences of more than one
nucleotide in either single chain or duplex form. The
polynucleotide sequences of the present invention may be prepared
from any known method including, but not limited to, any synthetic
method, any recombinant method, any ex vivo generation method and
the like, as well as combinations thereof.
[0078] Polynucleotides which can hybridize to any of the
polynucleotides discussed above are also covered by the present
invention. Such polynucleotides are referred to herein as
"hybridizing" polynucleotides. Hybridizing polynucleotides can be
useful as probes or primers, for example.
[0079] According to an embodiment of the present invention, such
hybridizing molecules are at least 10 nucleotides in length.
According to another embodiment, they are at least 25 or at least
50 nucleotides in length.
[0080] In an embodiment, the hybridizing molecules will hybridize
to such molecules under stringent hybridization conditions. One
example of stringent hybridization conditions is where attempted
hybridization is carried out at a temperature of from about
35.degree. C. to about 65.degree. C. using a salt solution which is
about 0.9 molar. However, the skilled person will be able to vary
such conditions as appropriate in order to take into account
variables such as probe length, base composition, type of ions
present, etc.
[0081] By "preventing or treating" is meant to manage a disease or
medical condition or to arrest the onset of a disease or a medical
condition.
[0082] More specifically, the present invention relates to a
peptide or a set of peptides which mimicks both motifs M1 and M2
and which affects the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the
Bcl-2/Bad/PP1c interactions, wherein the motif M1 has the sequence
FXX[RK]X[RK], and the motif M2 has the sequence [RK]VX[FW] or
[RK]XVX[FW], wherein X is any amino acid.
[0083] In a specific embodiment, the present invention relates to
set of peptides comprising the R (NWGRIVAFFSF) peptide and the F
(GDEFELRYRRAF) peptide.
[0084] The present invention does not only encompass the specific
peptides or set of peptides described above, but also modified
peptides in which an amino acid is deleted, added or changed while
retaining the same function of inhibiting the Bcl-x.sub.L/PP1c, the
Bcl-w/PP1c, or the Bcl-2/Bad/PP1c interactions. In particular,
chemical analogs of the amino acids can be used, such as
phosphorylated or thiophosphorylated amino acids. For example, it
has been demonstrated that a phosphorylated Y is similar to the F
residue. Thus, these two amino acids may be interchanged without
effecting the function of the motif.
[0085] Peptidic analogues of the motifs are thus encompassed in the
present invention. These analogues include those structures that
are similar in function but are not identical in composition. An
example of an analogue is a polypeptide that has chemically
modified amino acids which are phosphorylated and cannot be
dephosphorylated by the cellular enzymes.
[0086] The peptides or sets of peptides according to the present
invention, or included in the compositions and kits of the
invention, can encompass two or more motifs on the same molecule,
or 2 to 5 motifs, or 2 to 10 motifs, which motifs are described
above, having a spacer between them. The spacer may be a sequence
of amino acids or a hydrocarbon chain interposed by covalent
linkages. A spacer can also be any chemical entity which can serve
to connect the at least two motifs, and the spacer can also contain
regulatory sequences between the at least two motifs.
[0087] In another embodiment, the spacer of the present invention
has from about 1 to about 50 amino acids. In a particular
embodiment, the spacer has about 36 amino acids.
[0088] In another embodiment, the present invention relates to a
fusion polypeptide or protein containing at least one motif of the
present invention. This fusion polypeptide or protein can be made
according to methods known in the art such as those described in
Sambrook et al, Molecular Cloning A Laboratory Manual 2.sup.nd
Edition (1989). A particular fusion polypeptide or protein of the
invention comprises one or several motifs M1 or M2, linked with at
least one fusogenic peptide which will help the fusion polypeptide
or protein enter target cells. Said fusogenic peptide can be for
example a viral epitope or a ligand to a specific cell receptor
such as chemokine receptors.
[0089] The above motifs, their analogues and modifications can be
synthesized using, for example, an "Applied System" synthesizer or
by Merrifield type solid phase synthesis (See, Merrifield, Adv.
Enzmol. Related Areas Mol. Biol. (1969) 32;221-96 or Merrfield,
Recent Prog. Horm. Res. (1967); 23;451-82) The motifs can also be
recombinantly produced.
[0090] The nucleic acid sequence encoding the motifs can be
inserted into an expression vector which contains the necessary
elements for the transcription and translation of the inserted
peptide/protein-coding sequence. Such transcription elements
include a regulatory region and a promoter. Thus, the nucleic acid
which can encode a marker compound of the present invention is
operably linked to a promoter in the expression vector. The
expression vector can also include a replication origin.
[0091] A wide variety of host/expression vector combinations are
employed in expressing the nucleic acids of the present invention.
Useful expression vectors that can be used include, for example,
segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Suitable vectors include, but are not limited to,
derivatives of SV40 and pcDNA and known bacterial plasmids such as
col EI, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith et
al (1988), pMB9 and derivatives thereof, plasmids such as RP4,
phage DNAs such as the numerous derivatives of phage I such as
NM989, as well as other phage DNA such as M13 and filamentous
single stranded phage DNA; yeast plasmids such as the 2 micron
plasmid or derivatives of the 2 m plasmid, as well as centomeric
and integrative yeast shuttle vectors; vectors useful in eukaryotic
cells such as vectors useful in insect or mammalian cells; vectors
derived from combinations of plasmids and phage DNAs, such as
plasmids that have been modified to employ phage DNA or the
expression control sequences; and the like.
[0092] For example in a baculovirus expression system, both
non-fusion transfer vectors, such as, but not limited to pVL941
(BamHI cloning site Summers), pVL1393 (BamHI, SmaI, XbaI, EcoRI,
NotI, XmaIII, BgIII and PstI cloning sites; Invitrogen), pVL1392
(BgIII, PstI, NotI, XmaIII, EcoRI, XbalI, SmaI and BamHI cloning
site; Summers and Invitrogen) and pBlueBacIII (BamHI, BglII, PstI,
NcoI and HindIII cloning site, with blue/white recombinant
screening, Invitrogen), and fusion transfer vectors such as, but
not limited to, pAc700 (BamHI and KpnI cloning sites, in which the
BamHI recognition site begins with the initiation codon; Summers),
pAc701 and pAc70-2 (same as pAc700, with different reading frames),
pAc360 (BamHI cloning site 36 base pairs downstream of a polyhedrin
initiation codon; Invitrogen (1995)) and pBlueBacHisA, B, C (three
different reading frames with BamHI, BglII, PstI, NcoI and HindIII
cloning site, an N-terminal peptide for ProBond purification and
blue/white recombinant screening of plaques; Invitrogen (220) can
be used.
[0093] Mammalian expression vectors contemplated for use in the
invention include vectors with inducible promoters, such as the
dihydrofolate reductase promoters, any expression vector with a
DHFR expression cassette or a DHFR/methotrexate co-amplification
vector such as pED (PstI, SalI, SbaI, SmaI and EcoRI cloning sites,
with the vector expressing both the cloned gene and DHFR; Kaufman,
1991). Alternatively a glutamine synthetase/methionine sulfoximine
co-amplification vector, such as pEE14 (HindIII, XbalI, SmaI, SbaI,
EcoRI and BclI cloning sites in which the vector expresses
glutamine synthetase and the cloned gene; Celltech). A vector that
directs episomal expression under the control of the Epstein Barr
Virus (EBV) or nuclear antigen (EBNA) can be used such as pREP4
(BamHI, SfiI, XhoI, NotI, NheI, HindIII, NheI, PvuII and KpnI
cloning sites, constitutive RSV-LTR promoter, hygromycin selectable
marker; Invitrogen), pCEP4 (BamHI, SfiI, XhoI, NotI, NheI, HindIII,
NheI, PvuII and KpnI cloning sites, constitutive hCMV immediate
early gene promoter, hygromycin selectable marker; Invitrogen),
pMEP4 (KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI, BamHI cloning
sites, inducible methallothionein IIa gene promoter, hygromycin
selectable marker, Invitrogen), pREP8 (BamHI, XhoI, NofI, HindIII,
NheI and KpnI cloning sites, RSV-LTR promoter, histidinol
selectable marker; Invitrogen), pREP9 (KpnI, NheI, HindIII, NotI,
XhoI, SfiI, BamHI cloning sites, RSV-LTR promoter, G418 selectable
marker, Invitrogen), and pEBVHis (RSV-LTR promoter, hygromycin
selectable marker, N-terminal peptide purifiable via ProBond resin
and cleaved by enterokinase; Invitrogen).
[0094] Selectable mammalian expression vectors for use in the
invention include, but are not limited to, pRc/CMV (HindIII, BstXI,
NotI, SbaI and ApaI cloning sites, G418 selection, Invitrogen),
pRc/RSV (HindII, SpeI, BstXI, NotI, XbaI cloning sites, G418
selection, Invitrogen) and the like. Vaccinia virus mammalian
expression vectors (see, for example Kaufman 1991 that can be used
in the present invention include, but are not limited to, pSC11
(SmaI cloning site, TK- and .beta.-gal selection), pMJ601 (SalI,
SmaI, AfiI, NarI, BspMII, BamHI, ApaI, NheI, SacII, KpnI and
HindIII cloning sites; TK- and .beta.-gal selection), pTKgptF1S
(EcoRI, PstI, SalII, AccI, HindII, SbaI, BamHI and Hpa cloning
sites, TK or XPRT selection) and the like.
[0095] Yeast expression systems that can also be used in the
present include, but are not limited to, the non-fusion pYES2
vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHI, SacI,
KpnI and HindIII cloning sites, Invitrogen), the fusion pYESHisA,
B, C (XbalI, SphI, ShoI, NotI, BstXI, EcoRI, BamHI, SacI, KpnI and
HindIII cloning sites, N-terminal peptide purified with ProBond
resin and cleaved with enterokinase; Invitrogen), pRS vectors and
the like.
[0096] Consequently, mammalian and typically human cells, as well
as bacterial, yeast, fungi, insect, nematode and plant cells an
used in the present invention and may be transfected by the nucleic
acid or recombinant vector as defined herein.
[0097] Examples of suitable cells include, but are not limited to,
VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines such
as ATCC No. CCL61, COS cells such as COS-7 cells and ATCC No. CRL
1650 cells, W138, BHK, HepG2, 3T3 such as ATCC No. CRL6361, A549,
PC12, K562 cells, 293 cells, Sf9 cells such as ATCC No. CRL1711,
Cv1 cells such as ATCC No. CCL70 and JURKAT cells such as ATCC No.
Tib152.
[0098] Other suitable cells that can be used in the present
invention include, but are not limited to, prokaryotic host cells
strains such as Escherichia coli, (e.g., strain DH5-.alpha.),
Bacillus subtilis, Salmonella typhimurium, or strains of the genera
of Pseudomonas, Streptomyces and Staphylococcus, parasites like
Apicomplexan parasites (Plasmodia, Toxoplasma, Cryptosporidia),
Leishmania or Trypanosoma.
[0099] Further suitable cells that can be used in the present
invention include yeast cells such as those of Saccharomyces such
as Saccharomyces cerevisiae or Prombe.
[0100] The above-described motifs and peptides are involved. in
binding Bcl-x.sub.L and Bcl-w to PP1c and are thus targeting
subunits involved in control of all PP1 binding. Thus, due to their
implication in PP1 binding, these motifs are important for the
regulation of any disease concerned with phosphatase regulation in
all types of cells including all eukaryotic and prokaryotic cells
including muscle, heart, liver, lung, brain cells, thymocytes,
blood and the like. Plant and bacterial cells are also encompassed
in the present invention since the PP1 is found in many different
types of plant and bacterial cells. Yeast cells are also
encompassed by the present invention. In fact any cell that
contains PP2 is encompassed by the present invention.
[0101] More importantly, this regulation can effect the downstream
regulation of, for example, hepatic glycogen synthesis which in
turn would lower blood glucose levels and thus treat diabetes in
which hyperglycemia is a severe problem. Thus, the present
invention relates to treating diabetes by administering to an
animal in need of such treatment a pharmaceutically acceptable
amount of the peptides of the present invention which are described
in detail within.
[0102] In yet another embodiment, the present invention relates to
the administration of the peptides of the present invention to
normalize high blood pressure. A decrease in phosphorylation of the
PP1 complex activates the smooth muscle myosin light chains and
hence relaxes smooth muscles (see, Uehata at al., 1997). Therefore,
the present invention also relates to administering to an animal a
pharmaceutically effective amount of the peptides of the present
invention described within to prevent or treat hypertension.
[0103] in yet another embodiment the present invention relates to
treating neurological disorders by modulating neurological
receptors and ion channels. Therefore, the present invention also
relates to treating neurological disorders by administering to an
animal in need of such treatment a pharmaceutically effective
amount of the peptides of the present invention described within to
prevent or treat the neurological disorders, such as Parkinson's
disease.
[0104] More specifically, the present invention relates to treating
Alzheimer's disease. Phosphorylation is known to play a role in
effecting the .beta.-amyloid precursor which causes the plagues in
Alzheimer's disease. Thus, the present invention relates to
treating Alzheimer's disease by administering to an animal in need
of such treatment a pharmaceutically effective amount of the
peptides of the present invention described within to prevent or
treat Alzheimer's disease.
[0105] In yet another embodiment, the present invention relates to
treating viral or microbial infections and more specifically herpes
simplex virus, Myocbacterium tuberculosis and AIDS by administering
to an animal in need of such treatment a pharmaceutically effective
amount of the peptides of the present invention described within to
prevent or treat the viral infection.
[0106] The present invention is especially useful for treating AIDS
since both TAT and reverse transcriptase of the HIV-1 virus could
be PP1 binding proteins.
[0107] In yet another embodiment, the present invention relates to
treating parasitic infections such as malaria, theileria or
cryptosporidium by administering to an animal in need of such
treatment a pharmaceutically effective amount of the peptides of
the present invention described within to prevent or treat these
parasitic infections.
[0108] The above-mentioned treatments involve administering to an
animal in need of such treatment a pharmaceutically acceptable
amount of a peptide or a set of peptides which mimicks both motifs
M1 and M2 and which inhibits the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c,
or the Bcl-2/Bad/PP1c interactions. For example, a set of peptides
comprising the R (NWGRIVAFFSF) peptide and the F (GDEFELRYRRAF)
peptide, analogues of these peptides or their functional
equivalents in a pharmaceutically acceptable vehicle can be
administered to said animal.
[0109] In another embodiment the present invention relates to
pharmaceutical compositions which comprises a peptide or a set of
peptides which mimicks both motifs M1 and M2 and which inhibits the
Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c
interactions. Such a composition can for example comprise the R
(NWGRIVAFFSF) peptide and the F (GDEFELRYRRAF) peptide, analogues
of these peptides or their functional equivalents in a
pharmaceutically acceptable vehicle.
[0110] The pharmaceutically acceptable vehicle includes, but is not
limited to, saline, adjuvants and the like, discussed more
extensively above.
[0111] The present invention is not limited to solely administering
the peptides described within as a "neat" pharmaceutical
composition, but also as a pharmaceutical composition in gene
therapy, using vectors that encode polypeptides according to the
present invention.
[0112] More specifically ex vivo and in vitro gene therapy is part
of the present invention. In this respect, any of the methodologies
relating to gene therapy available within the art can be used in
the practice of the present invention such as those described by
Goldspiel et al Clin. Pharm. 12 pgs. 488-505 (1993).
[0113] Delivery of the therapeutic nucleic acid into a patient can
be direct in vivo gene therapy (i.e., the patient is directly
exposed to the nucleic acid or nucleic acid-containing vector) or
indirect ex vivo gene therapy (i.e., cells are first transformed
with the nucleic acid in vitro and then transplanted into the
patient).
[0114] For example for in vivo gene therapy, an expression vector
containing the nucleic acid is administered in such a manner that
it becomes intracellular, i.e., by infection using a defective or
attenuated retroviral or other viral vectors as described, for
example in U.S. Pat. No. 4,980,286 or by Robbins et al, Pharmacol.
Ther., 80 No. 1 pgs. 35-47 (1998).
[0115] The various retroviral vectors that are known in the art are
such as those described in Miller at al. (Meth. Enzymol. 217 pgs.
581-599 (1993)) which have been modified to delete those retroviral
sequences which are not required for packaging of the viral genome
and subsequent integration into host cell DNA. Also adenoviral
vectors can be used which are advantageous due to their ability to
infect non-dividing cells and such high-capacity adenoviral vectors
are described in Kochanek (Human Gene Therapy, 10, pgs. 2451-2459
(1999)). Chimeric viral vectors that can be used are those
described by Reynolds et al. (Molecular Medecine Today, pgs. 25-31
(1999)). Hybrid vectors can also be used and are described by
Jacoby et al. (Gene Therapy, 4, pgs. 1282-1283 (1997)).
[0116] Direct injection of naked DNA or through the use of
microparticle bombardment (e.g., Gene Gun.RTM.; Biolistic, Dupont)
or by coating it with lipids can also be used in gene therapy.
Cell-surface receptors/transfecting compounds or through
encapsulation in liposomes, microparticles or microcapsules or by
administering the nucleic acid in linkage to a peptide which is
known to enter the nucleus or by administering it in linkage to a
ligand predisposed to receptor-mediated endocytosis (See Wu &
Wu, J. Biol. Chem., 262 pgs. 4429-4432 (1987)) can be used to
target cell types which specifically express the receptors of
interest.
[0117] In another embodiment a nucleic acid ligand compound can be
produced in which the ligand comprises a fusogenic viral peptide
designed so as to disrupt endosomes, thus allowing the nucleic acid
to avoid subsequent lysosomal degradation. The nucleic acid can be
targeted in vivo for cell specific endocytosis and expression by
targeting a specific receptor such as that described in WO92/06180,
WO93/14188 and WO 93/20221. Alternatively the nucleic acid can be
introduced intracellularly and incorporated within the host cell
genome for expression by homologous recombination (See Zijlstra et
al, Nature, 342, pgs. 435-428 (1989)).
[0118] In ex vivo gene therapy, a gene is transferred into cells in
vitro using tissue culture and the cells are delivered to the
patient by various methods such as injecting subcutaneously,
application of the cells into a skin graft and the intravenous
injection of recombinant blood cells such as hematopoietic stem or
progenitor cells.
[0119] Cells into which a nucleic acid can be introduced for the
purposes of gene therapy include, for example, epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes and blood cells. The blood cells that can be used
include, for example, T-lymphocytes, B-lymphocytes, monocytes,
macrophages, neutrophils, eosinophils, megakaryotcytes,
granulocytes, hematopoietic cells or progenitor cells and the
like.
[0120] The polypeptides and complexes of polypeptides of the
invention also find use in raising antibodies. Thus, the present
invention provides antibodies, which can be monoclonal or
polyclonal.
[0121] Thus, the polypeptides and complexes of the invention can be
used as an immunogen to generate antibodies which specifically bind
such an immunogen. Antibodies of the invention include, but are not
limited to polyclonal, monoclonal, bispecific, humanized or
chimeric antibodies, single chain antibodies, Fab fragments and
F(ab') fragments, fragments produced by a Fab expression library,
anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that specifically binds an antigen. The immunoglobulin
molecules of the invention can be of any class (e.g., IgG, IgE,
IgM, IgD and IgA) or subclass of immunoglobulin molecule.
[0122] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
ELISA (enzyme-linked immunosorbent assay). For example, to select
antibodies which recognize a specific domain of a polypeptide of
the invention (e.g., a Selected Interacting Domain), one can assay
generated hybridomas for a product which binds to a polypeptide
fragment containing such domain. For selection of an antibody that
specifically binds a first polypeptide homolog but which does not
specifically bind to (or binds less avidly to) a second polypeptide
homolog, one can select on the basis of positive binding to the
first polypeptide homolog and a lack of binding to (or reduced
binding to) the second polypeptide homolog.
[0123] For preparation of monoclonal antibodies (mAbs) directed
toward a polypeptide of the invention or a fragment or an analog
thereof, any technique which provides for the production of
antibody molecules by continuous cell lines in culture may be used.
For example, the hybridoma technique originally developed by Kohler
and Milstein (1975, Nature 256:495-497), as well as the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
1983, Immunology Today 4:72), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al, 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). Such antibodies can be of any immunoglobulin class
including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The
hybridoma producing the mAbs of the invention can be cultivated in
vitro or in vivo. In an additional embodiment of the invention,
monoclonal antibodies can be produced in germ-free animals
utilizing known technology (PCT/US90/02545, incorporated herein by
reference).
[0124] The monoclonal antibodies include but are not limited to
human monoclonal antibodies and chimeric monoclonal antibodies
(e.g., human-mouse chimeras). A chimeric antibody is a molecule in
which different portions are derived from different animal species,
such as those having a human immunoglobulin constant region and a
variable region derived from a murine mAb. (See, e.g., Cabiliy et
al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No.
4,816397, which are incorporated herein by reference in their
entirety.) Humanized antibodies are antibody molecules from
non-human species having one or more complementarity determining
regions (CDRs) from the non-human species and a framework region
from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat.
No. 5,585,089, which is incorporated herein by reference in its
entirety.)
[0125] Chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European.
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et at, 1988, Science 240:1041-1043; Liu et at,
1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et at, 1987, J.
Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et at, 1987, Canc. Res. 47:999-1005; Wood
et at, 1985, Nature 314:446-449; and Shaw et at, 1988, J. Natl.
Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207;
Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat. No. 5,225,539;
Jones et al., 1986, Nature 321:552-525; Verhoeyan et al, (1988)
Science 239:1534; and Beidler et at, 1988, J. Immunol.
141:4053-4060.
[0126] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chain genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of a BPI of the invention. Monoclonal antibodies
directed against the antigen can be obtained using conventional
hybridoma technology. The human immunoglobulin transgenes harbored
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0127] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al. (1994) Bio/technology 12:899-903).
[0128] The antibodies of the present invention can also be
generated using various phage display methods known in the art. In
phage display methods, functional antibody domains are displayed on
the surface of phage particles which carry the polynucleotide
sequences encoding them. More specifically, such phage can be
utilized to display antigen binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Phage display methods that can be used to make the
antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
Application No. PCT/GB91/01134; PCT Publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0129] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0130] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988).
[0131] The invention further provides for the use of bispecific
antibodies, which can be made by methods known in the art.
Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Milstein et al., 1983, Nature 305:537-539). Because of the random
assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, published 13 May 1993, and in Traunecker et al., 1991,
EMBO J. 10:3655-3659.
[0132] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion can be with an immunoglobulin heavy chain constant domain,
comprising at least part of the hinge, CH2, and CH3 regions.
Generally, the first heavy-chain constant region (CH1) containing
the site necessary for light chain binding, present in at least one
of the fusions. DNAs encoding the immunoglobulin heavy chain
fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide
fragments in embodiments when unequal ratios of the three
polypeptide chains used in the construction provide the optimum
yields. It is, however, possible to insert the coding sequences for
two or all three polypeptide chains in one expression vector when
the expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios are of no particular
significance.
[0133] In another embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690 published Mar. 3, 1994. For
further details for generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 1986, 121:210.
[0134] The invention provides functionally active fragments,
derivatives or analogs of the anti-polypeptide immunoglobulin
molecules. Functionally active means that the fragment, derivative
or analog is able to elicit anti-anti-idiotype antibodies (i.e.,
tertiary antibodies) that recognize the same antigen that is
recognized by the antibody from which the fragment, derivative or
analog is derived. Specifically, in an embodiment, the antigenicity
of the idiotype of the immunoglobulin molecule can be enhanced by
deletion of framework and CDR sequences that are C-terminal to the
CDR sequence that specifically recognizes the antigen. To determine
which CDR sequences bind the antigen, synthetic peptides containing
the CDR sequences can be used in binding assays with the antigen by
any binding assay method known in the art.
[0135] The present invention provides antibody fragments such as,
but not limited to, F(ab')2 fragments and Fab fragments. Antibody
fragments which recognize specific epitopes can be generated by
known techniques. F(ab')2 fragments consist of the variable region,
the light chain constant region and the CH1 domain of the heavy
chain and are generated by pepsin digestion of the antibody
molecule. Fab fragments are generated by reducing the disulfide
bridges of the F(ab')2 fragments. The invention also provides heavy
chain and light chain dimmers of the antibodies of the invention,
or any minimal fragment thereof such as Fvs or single chain
antibodies (SCAs) (e.g., as described in U.S. Pat. No. 4,946,778;
Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl.
Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature
334:544-54), or any other molecule with the same specificity as the
antibody of the invention. Single chain antibodies are formed by
linking the heavy and light chain fragments of the Fv region via an
amino acid bridge, resulting in a single chain polypeptide.
Techniques for the assembly of functional Fv fragments in E. coli
can be used (Skerra et al., 1988, Science 242:1038-1041).
[0136] In other embodiments, the invention provides fusion proteins
of the immunoglobulins of the invention (or functionally active
fragments thereof), for example in which the immunoglobulin is
fused via a covalent bond (e.g., a peptide bond), at either the
N-terminus or the C-terminus to an amino acid sequence of another
protein (or portion thereof, preferably at least 10, 20 or 50 amino
acid portion of the protein) that is not the immunoglobulin. In an
embodiment, the immunoglobulin, or a fragment thereof, is
covalently linked to the other protein at the N-terminus of the
constant domain. As stated above, such fusion proteins may
facilitate purification, increase half-life in vivo, and enhance
the delivery of an antigen across an epithelial barrier to the
immune system.
[0137] The immunoglobulins of the invention include analogs and
derivatives that are either modified, i.e., by the covalent
attachment of any type of molecule as long as such covalent
attachment that does not impair immunospecific binding. For
example, but not by way of limitation, the derivatives and analogs
of the immunoglobulins include those that have been further
modified, e.g., by glycosylation, acetylation, pegylation,
phosphylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications can be carried out by known techniques, including,
but not limited to specific chemical cleavage, acetylation,
formylation, etc. Additionally, the analog or derivative can
contain one or more non-classical amino acids.
[0138] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of the polypeptides
of the invention, e.g., for imaging or radioimaging these proteins,
measuring levels thereof in appropriate physiological samples, in
diagnostic methods, etc. and for radiotherapy.
[0139] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, for
instance, by chemical synthesis or by recombinant expression, and
are preferably produced by recombinant expression technique.
[0140] Recombinant expression of antibodies, or fragments,
derivatives or analogs thereof, requires construction of a nucleic
acid that encodes the antibody. If the nucleotide sequence of the
antibody is known, a nucleic acid encoding the antibody can be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding antibody, annealing
and ligation of those oligonucleotides, and then amplification of
the ligated oligonucleotides by PCR.
[0141] Alternatively, the nucleic acid encoding the antibody can be
obtained by cloning the antibody. If a clone containing the nucleic
acid encoding the particular antibody is not available, but the
sequence of the antibody molecule is known, a nucleic acid encoding
the antibody can be obtained from a suitable source (e.g., an
antibody cDNA library, or cDNA library generated from any tissue or
cells expressing the antibody) by PCR amplification using synthetic
primers hybridizable to the 3' and 5' ends of the sequence or by
cloning using an oligonucleotide probe specific for the particular
gene sequence.
[0142] If an antibody molecule that specifically recognizes a
particular antigen is not available (or a source for a cDNA library
for cloning a nucleic acid encoding such an antibody), antibodies
specific for a particular antigen can be generated by any method
known in the art, for example, by immunizing an animal, such as, a
rabbit, to generate polyclonal antibodies or, by generating
monoclonal antibodies. Alternatively, a clone encoding at least the
Fab portion of the antibody may be obtained by screening Fab
expression libraries (e.g., as described in Huse et al., 1989,
Science 246:1275-1281) for clones of Fab fragments that bind the
specific antigen or by screening antibody libraries (See, e.g.,
Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc.
Natl. Acad. Sci. USA 94:4937).
[0143] Once a nucleic acid encoding at least the variable domain of
the antibody molecule is obtained, it can be introduced into a
vector containing the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.
5,122,464). Vectors containing the complete light or heavy chain
for co-expression with the nucleic acid to allow the expression of
a complete antibody molecule are also available. Then, the nucleic
acid encoding the antibody can be used to introduce the nucleotide
substitution(s) or deletion(s) necessary to substitute (or delete)
the one or more variable region cysteine residues participating in
an intrachain disulfide bond with an amino acid residue that does
not contain a sulfhydyl group. Such modifications can be carried
out by any method known in the art for the introduction of specific
mutations or deletions in a nucleotide sequence, for example, but
not limited to, chemical mutagenesis, in vitro site directed
mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCT
based methods, etc.
[0144] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad.
Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda
et al., 1985, Nature 314:452-454) by splicing genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human antibody constant region, e.g., humanized
antibodies.
[0145] Once a nucleic acid encoding an antibody molecule of the
invention has been obtained, the vector for the production of the
antibody molecule can be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
the protein of the invention by expressing nucleic acid containing
the antibody molecule sequences are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing an antibody molecule coding sequences
and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro. recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. See, for example, the techniques described in
Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual, 2d
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and
Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY).
[0146] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the
invention.
[0147] The host cells used to express a recombinant antibody of the
invention can be either bacterial cells such as Escherichia coli,
or eukaryotic cells, especially for the expression of whole
recombinant antibody molecule. More specifically, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., 198, Gene 45:101; Cockett et al.,
1990, Bio/Technology 8:2).
[0148] A variety of host-expression vector systems can be utilized
to express an antibody molecule of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest can be produced and subsequently purified,
but also represent cells which can, when transformed or transfected
with the appropriate nucleotide coding sequences, express the
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing the antibody
coding sequences; plant cell systems infected with recombinant
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter, the vaccinia virus 7.5K promoter).
[0149] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions comprising an antibody molecule,
vectors which direct the expression of high levels of fusion
protein products that are readily purified may be desirable. Such
vectors include, but are not limited, to the E. coil expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the
antibody coding sequence can be ligated individually into the
vector in frame with the lacZ coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors can also be
used to express foreign polypeptides as fusion proteins with
glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption and binding to a matrix glutathione-agarose beads
followed by elution in the presence of free glutathione. The pGEX
vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0150] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence can be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter). In mammalian host cells, a number of viral-based
expression systems (e.g., an adenovirus expression system) can be
utilized.
[0151] As discussed above, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein.
[0152] For long-term, high-yield production of recombinant
antibodies, stable expression is useful. For example, cells lines
that stably express an antibody of interest can be produced by
transfecting the cells with an expression vector comprising the
nucleotide sequence of the antibody and the nucleotide sequence of
a selectable (e.g., neomycin or hygromycin), and selecting for
expression of the selectable marker. Such engineered cell lines can
be useful in screening and evaluation of compounds that interact
directly or indirectly with the antibody molecule.
[0153] The expression levels of the antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel. The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et at, 1983, Mol. Cell. Biol. 3:257).
[0154] The host cell can be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors can contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector can be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain should be placed before the heavy chain
to avoid an excess of toxic free heavy chain (Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
The coding sequences for the heavy and light chains may comprise
cDNA or genomic DNA.
[0155] Once the antibody molecule of the invention has been
recombinantly expressed, it can be purified by any method known in
the art for purification of an antibody molecule, for example, by
chromatography (e.g., ion exchange chromatography, affinity
chromatography such as with protein A or specific antigen, and
sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins.
[0156] Alternatively, any fusion protein can be readily purified by
utilizing an antibody specific for the fusion protein being
expressed. For example, a system described by Janknecht et al.
allows for the ready purification of non-denatured fusion proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
open reading frame of the gene is translationally fused to an
amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix binding domain for the fusion protein. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni2.sup.+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0157] In another embodiment, antibodies of the invention or
fragments thereof are conjugated to a diagnostic or therapeutic
moiety. The antibodies can be used for diagnosis or to determine
the efficacy of a given treatment regimen. Detection can be
facilitated by coupling the antibody to a detectable substance.
Examples of detectable substances include various enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent materials, radioactive nuclides, positron emitting
metals (for use in positron emission tomography), and
nonradioactive paramagnetic metal ions. See generally U.S. Pat. No.
4,741,900 for metal ions which can be conjugated to antibodies for
use as diagnostics according to the present invention. Suitable
enzymes include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase; suitable prosthetic
groups include streptavidin, avidin and biotin; suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable
luminescent materials include luminol; suitable bioluminescent
materials include luciferase, luciferin, and aequorin; and suitable
radioactive nuclides include .sup.125I, .sup.131I, .sup.111I and
.sup.99Tc.
[0158] Antibodies of the invention or fragments thereof can be
conjugated to a therapeutic agent or drug moiety to modify a given
biological response. The therapeutic agent or drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety can be a protein or polypeptide
possessing a desired biological activity. Such proteins can
include, for example, a toxin such as abrin, ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor, .alpha.-interferon, .beta.-interferon, nerve growth factor,
platelet derived growth factor, tissue plasminogen activator, a
thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or
endostatin; or, a biological response modifier such as a
lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2),
interleukin-6 (IL-6), granulocyte macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
nerve growth factor (NGF) or other growth factor.
[0159] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al, "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (ads.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera at al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0160] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0161] An antibody with or without a therapeutic moiety conjugated
to it can be used as a therapeutic that is administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s).
[0162] The present invention not only relates to the peptides,
their use and administration in pharmaceutically acceptable
vehicles, but also to methods of using the peptides and the motifs
as disclosed within.
[0163] More specifically, the present invention has utilization for
several methods in which the peptides described within are used for
the following purposes: [0164] (1) A method of identifying a
PP1-interacting polypeptide or protein, comprising detecting in the
sequence of said polypeptide or protein, the presence of two
PP1-binding motifs M1 and M2. [0165] (2) A method of screening
compounds that interacts with PP1 regulators, comprising: [0166]
(a) immobilizing peptides described herein on a support; and [0167]
(b) testing the interaction of said compounds with said immobilized
peptides. [0168] (3) A method of screening compounds that interact
with PP1, comprising: [0169] (a) obtaining antibodies to peptides
within the description of the present invention; and [0170] (b)
testing the interaction of said compounds with said antibodies.
[0171] (4) A method for testing molecules that inhibit or enhance
the PP1 activity or change its localization by interaction, said
method comprising: [0172] (a) immobilizing peptides described
within on a support; and [0173] (b) testing the interaction of said
compounds with said immobilized peptides.
[0174] Also encompassed by the present invention are drugs that
after using the methods of the present invention are found to
inhibit and/or enhance PP1 activity, PP1c activity or localization
by interaction.
[0175] Besides methods, the present invention relates to kits. The
kits comprise, but are not limited to, peptides or sets of peptides
which mimick both the motifs M1 and M2 and which are able to
inhibit the Bcl-x.sub.L/PP1c, the Bcl-w/PP1c, or the Bcl-2/Bad/PP1c
interactions, wherein the motif M1 has the sequence FXX[RK]X[RK],
and the motif M2 has the sequence [RK]VX[FW] or [RK]XVX[FW],
wherein X is any amino acid. Such kits can, for example, comprise
the peptides R (NWGRIVAFFSF) and F (GDEFELRYRRAF). These peptides
may be immobilized on any solid support, described above, and which
includes resins, microstar wells, glass chips and the like.
[0176] In yet another embodiment the present invention relates to a
kit containing antibodies to peptides which mimick both the motifs
M1 and M2 and which are able to inhibit the Bcl-x.sub.L/PP1c, the
Bcl-w/PP1c, or the Bc12/Bad/PP1c interactions. These antibodies can
have been raised against the peptides R (NWGRIVAFFSF) and F
(GDEFELRYRRAF) peptides, the analogues of these peptides or their
functional equivalents.
[0177] Besides the peptides or the antibodies, the kits of the
present invention also include reagents for interpreting the
interaction. Such reagents are known in the art and described above
and in Sambrook (supra).
[0178] In yet another embodiment, the present invention concerns a
method of screening cells to find PP1 interactions in the cells,
said method comprising either using antibodies against the motifs
M1 and M2 or peptides including these motifs or their functional
equivalents.
[0179] The present invention thus also relates to a new
phosphatase-derived drug therapy based on the intracellular
delivery of peptides with a sequence or sequences surrounding the
PP1/PP2 binding sites (M1 and M2 motifs) identified in some
interacting proteins. This drug therapy approach, derived from
chemical genetics (Gura, 2000), would specifically inhibit the
interaction of some medically important target proteins with
PP1/PP2A.
[0180] This therapeutic strategy, called "Peptidic knockout" of
PP1/PP2 pathways has the basis in two recent results. The first
result was the identification of a putative PP1 signature. Thus,
from the characterization of two distinct PP1 binding motifs in
Bcl-2 proteins and their existence in most PP1 binding proteins, it
can be concluded that the combinatory presence of the motifs can be
used as a predictive signature for PP1 binding. Thus, the web site
called "PP1 signature" (in preparation at the Institut Pasteur)
that contains all putative PP1 sequences derived from a Swisprot
library can be used to identify putative PP1-binding targets of
interest.
[0181] The second result was the identification of PP2A binding
sites in five proteins. The PP2A binding sites of a viral encoded
(HIV-1) Vpr and a parasitic Ck2a encoded by Theileria were recently
mapped. From the data obtained it can be said that the
intracellular delivery of some peptides mimicking these
PP2A-binding sequences lead to apoptosis in tumors (Hela, Jurkat,
S) or infected cells (Vpr or HIV-1 or Theileria).
Examples
[0182] The following examples can be performed using the materials
and methods described below:
1. Materials and Methods
1.1. Cells and Culture
[0183] Ts1.alpha..beta. is a murine T cell line expressing the
.alpha. and .beta. chains of the IL-2 receptor (Pitton at al, 1993)
that can be propagated independently in IL-2, IL-4 or IL-9. Cells
were cultured in RPMI-1640 supplemented with 5% heat-inactivated
fetal calf serum 2 mM glutamine, 10 mM Hepes, 0.55 mM arginine,
0.24 mM asparagine, 50 .mu.LM 2-ME and 60 U/ml of IL-4.
1.2. Lymphokines, Antibodies, Reagents and Plasmids
[0184] Murine rlL-4 or supernatant of a HeLa subline transfected
with pKCRIL-4.neo was used as a source of murine IL-4.
Anti-Bcl-x.sub.L and anti-Bcl-w antibody were from Calbiochem (La
Jolla, Calif.), Transduction Laboratories (Lexington, Ky.) or
StressGen Biotechnology (Victoria, Canada). Specific anti-PP1c was
from UBI (Lake Placid, N.Y.), Calbiochem or Transduction
Laboratories. Anti-Histones antibody was from Chemicon
International (Temecula, Calif.). Anti-14-3-3 protein antibody was
from UBI (Lake Placid, N.Y.). Anti-Bad serine 112 and 136 were from
New England BioLabs (Beverly, Mass.) and serine 155 was from Cell
Signaling Technology (Beverly, Mass.). Anti-Raf antibody was from
Transduction Laboratories. Anti-Pser and pan-Ras antibody were from
Calbiochem. Recombinant PP1c protein was from Calbiochem. Mito 2813
(anti-mitochondrial pyruvate dehydrogenase) was provided by Dr
Serrano, Centro Nacional de Biotecnologia (Madrid).
1.3. Immunoprecipitation and Western Blot
[0185] Cells (1.times.10.sup.7) were IL-4-stimulated or -deprived
and lysed for 20 min at 4.degree. C. in lysis buffer (50 mM Tris
HCl pH 8, 1% NP-40, 137 mM NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2,
10% glycerol and protease inhibitors cocktail). Digitonin or
detergent free buffers were also used for immunoprecipitation. For
phosphorylation analysis, the buffer was also supplemented with
phosphatase inhibitors cocktail. Lysates were immunoprecipitated
with the Appropriate antibody and Protein A Sepharose was added.
Alternatively, cells were lysed in Laemmli sample buffer and
protein extracts separated by SDS-PAGE, transferred to
nitrocellulose, blocked and incubated with primary antibody.
Membrane was washed and incubated with PO-conjugated secondary
antibody. Proteins were developed using the ECL.
1.4. In Vitro Phosphatase Assay
[0186] IL-4-stimulated cells (1.times.10.sup.7) were lysed in lysis
buffer, supernatants were immunoprecipitated with the corresponding
antibody, followed by incubation with Protein A Sepharose.
Immunoprecipitates were washed with phosphatase buffer (50 mM Tris
HCl, pH 7.5, 0.1% 2-ME, 0.1 mM EDTA and 1 mg/ml BSA) and mixed with
[.sup.32P] phosphorylase a, diluted in phosphatase buffer
supplemented with caffeine. The reaction was incubated (40 min at
30.degree. C.), stopped with 200 .mu.l 20% TCA and centrifuged. A
total of 185 .mu.l of the supernatant were used to estimate the
generation of free phosphate liberated from [.sup.32P]phosphorylase
a.
1.5. Peptide Synthesis
[0187] Peptides comprising the R/K X V/I X F (R) or F X X R X R (F)
motif of Bcl-w and Bcl-x.sub.L, as well as the mutated peptides
(see FIG. 8A for sequence) were prepared by automated spot
synthesis into an aminoderivatized cellulose membrane. Membrane was
blocked, incubated with purified PP1c and, after several washing
steps, incubated with anti-PP1c antibody, followed by PO-conjugated
secondary antibody. Spots were developed using the ECL system.
[0188] R (NWGRIVAFFSF), F (GDEFELRYRRAF) or R* (NWGRIAAAFSF)
peptides were synthetized on an automated multiple peptide
synthesizer using the solid-phase procedure and standard Fmoc
chemistry. The purity and composition of the peptides was confirmed
by reverse-phase high performance liquid chromatography and by
amino acid analysis.
1.6. Protein-Protein Interaction Competition
[0189] The interaction Bcl-w/PP1c and Bcl-x.sub.L/PP1c was competed
by the R, F, or R* peptides. Lysates from IL-4-stimulated cells
were immunoprecipitated with anti-Bad antibody and Protein A
Sepharose was added. The interaction Bcl-w/PP1c and
Bcl-x.sub.L/PP1c was competed by incubation with R, F or R*
peptides (30 min, room temperature). After washing,
immunoprecipitates were either assayed for protein phosphatase
activity or transferred to nitrocellulose and blotted with the
corresponding antibody.
1.7. Sense and Antisense Oligonucleotides
[0190] The phosphothioate analogous of the oligonucleotides from
Bcl-x.sub.L and Bcl-w, including the ATG initiation codon were
purchased from Isogen Bioscience. The sequence of the sense and
antisense oligonucleotides are as follow. Sense Bcl-x.sub.L, ATG
TCT CAG AGC AAC; antisense Bcl-x.sub.L, GTT GCT CTG AGA CAT; sense
Bcl-w, ATG GCG ACC CCA GCC; antisense Bcl-w, GGC TGG GGT CGC
CAT.
2. Experimental Results
[0191] 2.1. Identification of Bcl-w and Bcl-x.sub.L as
PP1c-Interacting Proteins
[0192] It was previously shown that anti-apoptotic molecule Bcl-2
is a targeting subunit of the serine/threonine phosphatase PP1c in
IL-2-stimulated TS1.alpha..beta. cells and that the sequence of
Bcl-2 interacting with PP1c is the R/K X V/I X F motif (Ayllon et
al, 2001). Given that Bcl-x.sub.L and Bcl-w also contain the well
conserved R/K X V/I X F motif observed in Bcl-2, the possibility
that anti-apoptotic molecules Bcl-x.sub.L and Bcl-w may be as well
associated to PP1c in IL-4-stimulated TS1.alpha..beta. cells, which
do not express Bcl-2 and express Bcl-x.sub.L and Bcl-w was
explored. Reciprocal co-immunoprecipitation experiments of
cytoplasmic proteins under IL-4-stimulation or -deprivation
conditions using specific antibodies was performed. PP1c and Bad
were detected by Western blot in anti-Bcl-x.sub.L
immunoprecipitates of IL-4 stimulated cells, decreasing throughout
the starvation period (FIG. 1A). Probing the membrane with
anti-Bcl-x.sub.L antibody showed similar levels in all conditions
analyzed. PP1c and Bad were also detected in anti-Bcl-w
immunoprecipitates of IL-4-stimulated cells, diminishing after
lymphokine deprivation (FIG. 1A). Membrane was also probed with
anti-Bcl-w antibody, showing similar levels. Immunoprecipitation
for cytoplasmic lysates with an irrelevant antibody, anti-p55 IL-2R
chain, was not able to detect those associations (FIG. 1B).
Similarly, Bcl-x.sub.L, Bcl-w and PP1c were detected in Bad
immunoprecipitates and the interaction among these proteins was
also observed by immunoprecipitation of detergent-free lysates as
well as in cytoplasmic proteins isolated by digitonin lysis (data
not shown). These associations were also observed in freshly
isolated thymocytes (FIG. 1C). Given that the number of
Bcl-x.sub.L/PP1c/Bad and Bcl-w/PP1c/Bad complexes decreases after
IL-4-deprivation, down-regulation of the expression of any of the
proteins involved in the formation of the complex was analyzed.
Thus, the total expression of Bcl-x.sub.L, Bcl-w, PP1c and Bad in
IL-4-stimulated or -deprived TS1.alpha..beta. cells was analyzed.
All analyzed proteins were expressed in IL-4-stimulated or
-deprived cells (FIG. 2). As an internal control of protein
loading, membranes were probed with anti-Histones antibody. As the
number of aggregates decreases after IL-4-deprivation without
modification of total expression of the proteins of the complex,
post-translational modifications of Bcl-x.sub.L or Bcl-w may affect
the formation of the trimolecular complex was then analyzed. The
status of serine phosphorylation of Bcl-x.sub.L and Bcl-w was next
analyzed. Cytoplasmic extracts from IL-4-stimulated or -deprived
cells were immunoprecipitated with anti-Bcl-x.sub.L or anti-Bcl-w
and blotted with anti-Pser, anti-PP1c, anti-Bad, anti-Bcl-w and
anti-Bcl-x.sub.L specific antibodies (FIG. 3). Serine
phosphorylation of. Bcl-x.sub.L and Bcl-w was observed in control
IL-4-stimulated cells, decreasing after IL-4 deprivation. In
agreement with results in FIG. 1, the level of Bad and PP1c
associated to Bcl-x.sub.L and Bcl-w diminishes upon lymphokine
deprivation. This result suggests a correlation between serine
phosphorylation of Bcl-x.sub.L and Bcl-w and formation of
trimolecular complexes.
[0193] It was recently shown that IL-2, as well as IL-3, induces
serine phosphorylation of Bad (Ayllon et al, 2000). FIG. 4A shows
that IL-4 induces Bad phosphorylation at serine 136 but not at
serines 112 and 155. Moreover, IL-4-deprivation induces serine 136
dephosphorylation of Bad. IL-2 stimulated cells (C, ser 112 and 136
phosphorylation) or COS (C, ser 155 phosphorylation) cells
overexpressing Bad were used as a positive controls. IL-3-induced
serine phosphorylation of Bad results in its association to the
14-3-3 protein, abolishing interaction with Bcl-x (Zhou et al,
2000). FIG. 4B shows that serine phosphorylation of Bad in response
to IL-4 does not result in binding to 14-3-3 protein. This protein
was detected in total extracts from control IL-4 stimulated cells
(lane T) and was not observed neither in PP1c, nor in Bcl-x.sub.L
or Bad immunoprecipitates of IL-4-stimulated or -deprived cells. As
an internal control, the interaction of Raf and the 14-3-3 protein
in Raf immunoprecipitates is shown (FIG. 4B).
[0194] FIG. 5A shows phosphatase activity in Bcl-x.sub.L, Bcl-w and
Bad immunoprecipitates of IL-4-stimulated cells. The enzymatic
activity in the immunoprecipitates was measured using
.sup.32P-labeled phosphorylase a as substrate. It is interesting to
notice that phosphatase activity detected in Bcl-x.sub.L and Bcl-w
immunoprecipitates nearly corresponds to the phosphatase activity
observed in Bad immunoprecipitates. To confirm that this
phosphatase activity was due to PP1c, enzymatic activity was
estimated in Bad or Bcl-w immunoprecipitates from IL-4-stimulated
cells in the presence of different okadaic acid (OA) concentrations
(FIG. 5B). OA concentrations that inhibit type 2A activity
(10.sup.-9M) had no effect on Bad- or Bcl-w-associated phosphatase
activity in vitro. Addition of 10.sup.-8M OA to Bcl-w or Bad
immunoprecipitates results in .about.50% inhibition of phosphatase
activity, which is strongly reduced after addition of 10.sup.-6M OA
(FIG. 5B). The effect of OA on phosphatase activity was also
estimated in supernatants of Bad and Bcl-w immunoprecipitates. OA
concentrations that had no effect on enzymatic activity in Bad and
Bcl-w immunoprecipitates (10.sup.-9M) shows .about.50% inhibition
in the supematant, as expected from an association of type 1 and
type 2A activities (FIG. 5B). The selective effect of OA suggests
that the phosphatase activity observed in Bad and Bcl-w
immunoprecipitates is PP1c.
2.2. Bcl-w and Bcl-x.sub.L are New Targeting Subunits of PP1c
[0195] It was recently shown that Bcl-2 is a targeting subunit of
PP1c (Ayllon et al, 2001). Given that Bcl-w and Bcl-x.sub.L are
also associated to PP1c and that sequence of binding site of Bcl-2
to PP1c is conserved in Bcl-x.sub.L and Bcl-w, it was hypothesized
that these anti-apoptotic molecules may be new targeting subunits
of PP1c. To test this hypothesis, Bcl-x.sub.L and Bcl-w was
depleted by sequential anti-Bcl-x.sub.L-Bcl-w immunoprecipitation
of cytoplasmic extracts of IL-4 stimulated cells. Supernatant from
the fourth anti-Bcl-x.sub.L+Bcl-w immunoprecipitation was
immunoprecipitated with anti-Bad antibody (5th) and phosphatase
activity estimated (FIG. 6A). Traces of Bad-associated phosphatase
activity were detected in Bcl-x.sub.L and Bcl-w depleted extracts
compared to the high level of activity observed in control anti-Bad
immunoprecipitates of IL-4-stimulated cells. Given that in the
absence of Bcl-x.sub.L and Bcl-w significant Bad-associated
phosphatase activity was not detected, the possibility that these
anti-apoptotic molecules may control targeting of PP1c to Bad was
explored. For this purpose, anti-Bad immunoprecipitations were made
in cytoplasmic extracts of IL-4 stimulated cells or in extracts
depleted of Bcl-x.sub.L and Bcl-w (5th). PP1c, Bcl-x.sub.L and
Bcl-w were detected in control anti-Bad immunoprecipitates and were
not observed in anti-Bad immunoprecipitates from extracts depleted
of Bcl-x.sub.L and Bcl-w (FIG. 6B). In a reciprocal experiment,
Bad, Bcl-x.sub.L and Bcl-w were detected in anti-PP1c
immunoprecipitates of control cells and were not observed in PP1c
immunoprecipitates from extracts depleted of Bcl-x.sub.L and Bcl-w
(FIG. 6B). This result suggests that Bcl-x.sub.L and Bcl-w are
needed for association of PP1c to Bad.
2.3. Determination of Bcl-x.sub.L and Bcl-w Binding Site to
PP1c
[0196] It has been described that R/K X V/I X F motif is shared by
most of the PP1c targeting subunits (21, 23). It was shown that
PP1c targeting subunit Bcl-2 also shares this conserved motif
(Ayllon et al, 2001). Interestingly, Bcl-x.sub.L and Bcl-w
sequences also contain this motif (FIG. 7B). To analyze whether
this sequence of Bcl-x.sub.L and Bcl-w was involved in binding to
PP1c, we generated nitrocellulose-immobilized peptides of
Bcl-x.sub.L and Bcl-w protein containing this motif. Membrane was
incubated with purified PP1c protein. FIG. 7B shows the sequences
interacting with PP1c. The R/K X V/I X F motif, present in
Bcl-x.sub.L and Bcl-w, interact with PP1c and its mutation in
critical V and F residues strongly reduces binding of Bcl-x.sub.L
and Bcl-w to PP1c (FIG. 7B). Analysis of the Bcl-2 binding sites to
PP1c showed, in addition to R/K X V/I X F motif, two sequences
(FSRRYR and FTARGR, that bind PP1c (Ayllon et al, 2001).
Interestingly, similar sequences were as well observed in
Bcl-x.sub.L and Bcl-w (FIG. 7A). FELRYR and FETRFR sequences of
Bcl-x.sub.L and Bcl-w respectively also interacts with PP1c and its
mutation inhibit binding to PP1c, although the affinity depends on
the type of point mutation (FIG. 7B). The interacting consensus F X
X R X R motif was determined by sequence comparison of Bcl-2,
Bcl-x.sub.L and Bcl-w.
[0197] To conclusively confirm that R/K X V/I X F and F X X R X R
motifs are involved in binding of Bcl-x.sub.L and Bcl-w to PP1c,
competition experiments in trimolecular complexes were performed.
Lysates from IL-4-stimulated cells were immunoprecipitated with
anti-Bad antibody and the interaction Bcl-x.sub.L/PP1c and
Bcl-w/PP1c was competed using R* (NWGRIAAAFSF), R (NWGRIVAFFSF) or
F (GDEFELRYRRAF) peptides (FIG. 8A). Bcl-x.sub.L, Bcl-w and PP1c
were detected in control anti-Bad immunoprecipitates, as well as in
anti-Bad immunoprecipitates treated with R* peptide. The amount of
Bcl-x.sub.L, Bcl-w and PP1 c associated to Bad decreases after
competition with F or R peptide, being almost undetectable upon
competition of Bad immunoprecipitates with F+R peptides (FIG. 8A).
Similar level of Bad is observed in control or peptide-treated
anti-Bad immunoprecipitates. Finally, to confirm that Bcl-x.sub.L
and Bcl-w are targeting subunits of PP1c, we estimated phosphatase
activity in control or peptide-treated Bad immunoprecipitates.
Phosphatase activity was detected in control or. R* peptide-treated
immunoprecipitates (FIG. 8B), decreasing upon competition of the
interaction with F or R peptides. Enzymatic activity was strongly
decreased upon competition with R and F peptides. As an internal
control, FIG. 8C shows the phosphatase activity in control or
peptide-treated Bad immunoprecipitates. The concentration of F or R
peptide used was twice the concentration used in F+R
peptide-treated immunoprecipitates. As in FIG. 8B, phosphatase
activity was drastically reduced upon treatment of Bad
immunoprecipitates with F+R peptides. Taken together, these results
illustrate that Bcl-x.sub.L and Bcl-w, as well as Bcl-2, are PP1c
targeting subunits.
2.4. Inhibition of PP1c Enzymatic Activity Blocks Apoptosis
[0198] As 1L-4-deprivation correlates with Bad dephosphorylation
and apoptosis, it was hypothesized that inhibition of phosphatase
activity by okadaic acid (OA) treatment may prevent Bad
dephosphorylation and apoptosis. Treatment of the cells with 1
.mu.M OA in the absence of IL-4 prevented Bad dephosphorylation at
serine 136 (FIG. 9A). No changes in total Bad expression were
observed after OA treatment, suggesting that OA does not affect
protein expression. In addition, IL-4-deprived cells treated with 1
.mu.M OA for 6 h showed significant reduction in the fraction of
apoptotic cells compared with untreated cells (FIG. 9B). Finally,
inhibition of Bcl-x.sub.L and Bcl-w expression by antisense
oligonucleotide treatment of cells also induces apoptosis in
IL-4-stimulated cells (FIG. 9C). The inhibition of Bcl-x.sub.L and
Bcl-w expression upon antisense oligonucleotide treatment was
estimated by Western blot.
3. Bio-Infromatic Results-Predictive Signature for PP1
Interactions: Combinatorial Presence of [RK]VxF or [RK]xVxF and
F-x-x-[RK]-x-[RK] Motifs in Characterized PP1 Binding Proteins
[0199] The combinatorial presence of these two PP1 binding motifs
suggests a general mechanism wherein sequential binding to PP1c
through one of these motifs may favor binding through the second
motif and allow catalytic function. Furthermore these motifs could
also represent a predictive signature to identify new potential PP1
binding proteins. To partially test this concept, a bioinformatic
analysis was performed by using "prose" (Katja Shuerer, IP) program
in the Swissprot Release40 library (October 2001) that contains
101602 non redundant protein sequences.
[0200] As shown in Table 1A, the search for the presence of only
one consensus indicates that 19.47% of the sequences in the library
contain [RK]VxF or [RK]xVxF motifs and 16% contain the Bcl-2-like
motif F-x-x-[RK]-x-[RK]. In contrast, consistently with the notion
of predictive signature, analysis of combinatorial presence of both
PP1 binding motifs [RK]VxF or [RK]xVxF and F-x-x-[RK]-x-[RK]
revealed only 4013 positives sequences, corresponding to 3.94% of
the library (Table 1B). In addition if the F=W equivalency (usually
accepted for [RK]Vx[FW]/[RK]xVx[FW] motifs) is applied, 4783
positives sequences corresponding to 4.7 of the library were found.
In addition, the occasionally equivalency between R/K and Q
slightly increases the number of positives proteins (5769 sequences
corresponding to 5.67%).
[0201] Furthermore, as expected, these sequences include all the
known PP1 interacting proteins of the Bcl-2 family (not shown).
Together this analysis indicates that around 5% of protein
sequences share the two putative PP1 binding motifs in their
sequence. Interestingly, statistical analysis suggests the distance
separating the two PP1 binding motifs is comprises between 0 and
180 aa for 50% of the proteins (FIG. 11). In addition, a more
details analysis reveals a major peak representing 22.3% of
positive sequences (897 sequences for a total of 4013) that
correspond to a 0-50 aa interval between the two motifs. These data
are consistent with the observation that a distance of 36 aa
separates the two binding motifs in BH1 and BH3 domains of
Bcl-2/Bcl-w and Bcl-x.sub.L proteins (not shown).
[0202] On the basis of this analysis, the Institut Pasteur is
creating and will maintain in order a new web site "PP1signature"
which contains all the sequences selected from the Swissprot
Release 40 library corresponding to proteins haboring the two PP1
binding motifs. By simply entering the name of a protein or an
accession number or through blast analysis, everyone will
immediately know if the protein has a putative PP1 signature. In
addition, the user will immediately identify the sequence
encompassing the two putative PP1 binding motifs.
[0203] Most characterised PP1-binding proteins share the two motifs
and can be identified in the web site (table 2). To validate this
proposal of "predictive signature strategy," four candidates were
randomly selected and their association PP1 by simple
co-immunoprecipitation experiments was confirmed (FIG. 10B).
TABLE-US-00001 TABLE 1A POSITIVE ONE MOTIF SEQUENCES % TOTAL
LIBRARY F-x-x-R-x-R 4,074 4 F-x-x[RK]-[RK] 16,260 16 [RK]-V-x-F
8,895 8.76 [RK-x-V-x-F 9,935 9.48 [RK]-V-x-F or 16,273 16.02
[RK]-X-V-x-F [RK]-V-x-[FW] or 19,787 19.48 [RK}-x-V-x-[FW]
TABLE-US-00002 TABLE 1B POSITIVE TWO MOTIFS SEQUENCES % TOTAL
LIBRARY [RK]-V-x-F or 1,025 1 [RK]-x-V-x-F + F-x-x-R-x-R [RK]-V-x-F
or 4,013 3.94 [RK]-x-V-x-F + F-x-x-R-x-R [RK]-V-x-[FW] or 4,783
4.70 [RK]-x-V-x-[FW] + F-x-x-[RK]-x-[RK] [RKQ]-V-x-F/W or 5,769
5.67 [RKQ]-x-V-x-F/W + F-x-x-[RK]-x-[RK]
TABLE-US-00003 TABLE 2A Two putative PPI binding sequences in
characterized PP1-interacting proteins Protein/gene motif 1
Residues motif 2 Residues Yeast G1P2 (homolog of GM) QFERKNEKLD
12-18 LIRSKSVHFDQA 216-227 6320895 GIP2h/YIL045S49933 SLEFLHKPRRLS
55-60 QRSKSVUFD 191-202 *YAL014 L05146 DLFNERRQRR 110-116
MPTRHNVRWEEN 45-56 REG2 6319525 RSWFKARKRRDI 152-157 KPRERHIKFNDN
163-174 Mammalian Mouse PTG AAB49689 RRNFVN KLKPL 28-38
NQAKKRVVFADS 56-67 TVKVKNVSFEKK 149-160 *GL CAA77083 Y LDFRNRLQTN
121-130 KICVKIMVSFAND 56-67 Human R5 4885559 RHFVNKLKPLKS 48-60
NQAKKRVVFADS 79-90 U5 AAC60216 TCFRPRLRGS 101-110 SQKKKRVVFADM
61-77 Splicing factor PSF GEVFINKGKGF 324-334 RGRQLRVRFATH 358-369
P23246 Ribosomal Protein L5 P22451 QVKFRRRREG 17-26 YFKRYQVKFRRR
12-23 GRP-78 P20029 EDFKAKKKEL 615-620 RITPSYVAFTPE 61-72 Human
110pRB P06400 SVFMQRLKTNILQ 758-770 IDEVKNVYFKNF 285-296
VLKVSWITFLLA 190-201 AKAPs (AKAP149/AKAP220/Yotiao) LQFELRYRPV
250-255 TTKAVMFAK 141-145 Hsp-90-.alpha. (Hsp 86) P07901 DLFENRKKKN
353-358 VRRVFIM 367-370 Human MYPT2 4505319 FFKNEKMLY 331-340
RRGSPRVRFEDG 48-59 Human I-2 CAA55475 RQFEMKRKLHY 128-138 Not
detected *These two sequences correspond to a higher degenerated
consensus (F-X-X-R/K-X-R/K/Q) Consensus F-X-X-R/K-X-R/K
R/K-X-V/I-X-F Or R/K-V/I-X-F
Example 4
.beta. Amyloid Precusor as a New PP1 Interacting Protein
[0204] Fibrillar amyloid deposits are defining pathological lesions
in Alzheimer's brain diseases and are thought to mediate neuronal
death. Amyloid is composed of a 39 to 42 amino acid protein
fragment of the amyloid precursor protein (APP). Because depostion
of fibrillar amyloid in vitro has been shown to be dependent upon
the APP concentration, reducing or inhibiting the release of APP is
a therapeutic target.
[0205] The .beta.-amyloid precursor (APP) is overexpressed in PC12
cells treated by NGF, by transfection of a cDNA-encoding the
.beta.-amyloid precursor (APP) according to the methods of Sambrook
et al, supra.
[0206] The cells expressing the .beta.-amyloid precursor are then
incubated with a penetrating peptide corresponding to the punitive
PP1 binding site such as the motif M1 having the sequence
FXX[[RK]X[RK] and the M2 motif [RK]VX[FW] or [RKXVX[FW], where X is
any amino acid.
[0207] The presence of amyloid is tested using monoclonal
antibodies or a
REFERENCES
[0208] Aggen, J. B., Nairn, A. C. and Chamberlin, R., Regulation of
protein phosphatase. Chem. and Biol. 2000. 7: R13-R23.
[0209] Aylion, V., Cayla, X, Garcia, A., Roncal, F; Fernandez, R;
Albar, J. P; Martinez-A, C., Rebollo, A. (2001). Bcl-2 targets
protein phosphatase 1.alpha.. to Bad. J Immunol.; 166:
7345-7352.
[0210] Ayllon, V., Martinez-A., C., Garcia, A., Cayla, X. and
Rebollo, A., Protein phosphatase 1.alpha. is a Ras-activated Bad
phosphatase that regulates IL-2 deprivation-induced apoptosis. EMBO
J. 2000. 19: 2237-2246.
[0211] Beullens M, Van Eynde A, Vulsteke V, Connor J, Shenolikar S,
Stalmans W, Bollen Molecular determinants of nuclear protein
phosphatase-1 regulation by NIPP-1. J Biol Chem. 1999 May 14;
274:14053-61.
[0212] Boise, L. H., Gonzalez-Garcia, M., Postema, C. E., Ding, L.
Y., Lindsten, T., Turka, L. A., Mao, X. H., Nunez, G. and Thompson,
C. B., Bcl-x, a Bcl-2 related gene that functions as a dominant
regulator of apoptotic cell death. Cell 1993. 74: 597-608.
[0213] Bollen M. Combinatorial control of protein phosphatase-1.
TIBS 26 (2001), 426-431
[0214] Chao, D. T. and Korsmeyer, S. J., Bcl-2 family: regulators
of cell death. Annu. Rev. Immunol. 1998. 16: 395-419.
[0215] Colledge, M. and Scott, J. D., AKAPs: from structure to
function. Trends Cell. Biol. 1999. 9: 216-221.
[0216] Datta, S. R., Dukek, H., Tao, X., Masters, S., Fu, H.,
Gotoh, Y. and Greenberg, M. E., Akt phosphorylation of Bad couples
survival signals to the cell intrinsic death machinery. Cell 1997.
91: 231-241.
[0217] Datta, S. R., Katsov, A., Hu, L., Petros, A., Fesik, S. W.,
Yaffe, M. B. and Greenberg, M. E., 14-3-3 proteins and survival
kinases cooperate to inactivate Bad by BH3 domain phosphorylation.
Mol. Cell. 2000. 6: 41-51.
[0218] Del Peso, L., Garcia, M. G., Page, C., Herrera, R. and
Nunez, G., IL-3-induced phosphorylation of Bad through the protein
kinase Aft Science 1997. 278: 687-689.
[0219] Deng, X., Ito, T., Carr, B., Mumby, M. and May, W. S.,
Reversible phosphorylation of Bcl-2 following IL-3 or bryostatin 1
is mediated by direct interaction with protein phosphatase 2A. J.
Biol. Chem. 1998. 273: 34157-34163.
[0220] Egloff, M. P., Johnson, D. J., Moorhead, G., Cohen, P. T.
W., Cohen, P. and Barford, D., Structural basis for the recognition
of regulatory subunits by the catalytic subunit of protein
phosphatase 1. EMBO J. 1997. 16: 1876-1887.
[0221] Gibson, L., Holmgreen, S., Huang, D. C. S, Bernard, O.,
Copeland, N. G., Jenkins, N. A., Sutherland, G. R, Baker, E.,
Adams, J. M. and Cory, S., Bcl-w, a novel member of the Bcl-2
family promotes cell survival. Oncogene 1996. 13: 665-675.
[0222] Gross, A., McDonnell, J. M. and Korsmeyer, S. J, Bcl-2 gene
family and the regulation of programmed cell death. Genes Dev.
1999. 13: 1899-1911
[0223] Gura, T. (2000). "A chemistry set for life." Nature
407(6802): 282-4.
[0224] Hsu, H. Y., Kaipai, L., Zhu, L. and Hsueh, A .J.,
Interference of Bad induced apoptosis in mammalian cells by 14-3-3
isoforms and P11. Mol. Endocrinol. 1997. 11: 1858-1867.
[0225] Ito, T., Deng, X., Carr, B. and May, W. S., Bcl-2
phosphorylation required for antiapoptotic function. J. Biol. Chem.
1997. 272: 11671-11673.
[0226] Jacobson, M. D., Apoptosis: Bcl-2-related proteins get
connected, Curr. Biol. 1997. 7: 277-281.
[0227] Korsmeyer, S. J, Bcl-2 family and the regulation of
programmed cell death. Cancer Res. 1999. 59:1693s-1700s.
[0228] McAvoy T, Allen P B, Obaishi H, Nakanishi H, Takai Y,
Greengard P, Nairn A C, Hemmings H C Jr. Regulation of neurabin I
interaction with protein phosphatase 1 by phosphorylation.
Biochemistry. 1999. 38:12943-9.
[0229] Minn, A. J., Boise, L. H. and Thompson, C. B., Bcl-xs
antagonizes the protective effects of Bcl-xL. J. Biol. Chem. 1996.
271: 6306-6312.
[0230] N nez, G., Merino, R., Grillot, D. and Gonzalez-Garcia, M.,
Bcl-2 and Bcl-x: regulatory swiches for lymphoid death and
survival. Immunot Today 1994. 15: 582-588.
[0231] Ottilie, S. J., Diaz, L., Home, W., Chang, J., Wang, Y.,
Wilson, G., Chag, S., Weeks, S., Fritz, L. C. and Oltersdofr, T.,
Dimerization properties of human Bad. J. Biol. Chem. 1997. 272:
30866-30890.
[0232] Pitton, C., Rebollo, A., Van Snick, J., Theze, J. and
Garcia, A., High affinity and intermediate affinity forms of the
expressed in an IL-9-dependent murine T cell line deliver
proliferative signals via differences in their transduction
pathways. Cytokine 1993. 5: 362-371.
[0233] Rebollo, A., Perez-Sala, D. and Martinez-A., C., Bcl-2
differentially targets K-, N- and H-Ras to mitochondria in IL-2
supplemented or deprived cells: implications in prevention of
apoptosis. Oncogene 1999. 18: 4930-4939.
[0234] Reed, J. C, Bcl-2 family proteins. Oncogene 1998. 17:
3225-3236. Sattler M, Liang H, Nettesheim D, Meadows R P, Harlan J
E, Eberstadt M, Yoon H S, Shuker S B, Chang B S, Minn A J, Thompson
C B, Fesik S W, Structure of Bcl-xL-Bak peptide complex:
recognition between regulators of apoptosis. Science. 1997.
275(5302):983-986.
[0235] Shenolikar, S., Protein serine/threonine phosphatases, new
avenues for cell regulation. Annu. Rev. Cell Biol. 1994. 10: 55-86.
White, E., Life, death and the pursuit of apoptosis. Genes Dev.
1996. 10: 1-15.
[0236] Yaffe M B., Rittinger Katrin, Volinia S., Caron P R., Aitken
A., Leffers H., Smerdon S J., Cantley L. C, The Structural Basis
for 14-3-3:Phosphopeptide Binding Specificity implicated 14-3-3 as
a key regulator of signal transductionevents. Cell 1997, 91:
961-971.
[0237] Zha, J., Harada, H., Osipov, K., Jockel, J., Waksman, G. and
Korsmeyer, S. J., Serine phosphorylation of death agonist Bad in
response to survival factor results in binding to 14-3-3 protein.
J. Biol. Chem. 1997. 272: 24101-24104.
[0238] Zhou, X. M., Liu, Y., Payne, G., Lutz, R. J. and Chittenden,
T., Growth factors inactivate the cell death promoter Bad by
phosphorylation of its BH3 domain on serine 155. J. Biol. Chem.
2000. 275: 25046-25051.
[0239] Zolnierowicz, S. and Bollen, M. Protein phosphorylation and
protein phosphatases. EMBO J. 2000. 19: 483-488.
Sequence CWU 1
1
11615PRTArtificial SequenceSynthetic Peptide 1Arg Xaa Val Xaa Phe1
525PRTArtificial SequenceSynthetic Peptide 2Lys Xaa Val Xaa Phe1
535PRTArtificial SequenceSynthetic Peptide 3Arg Xaa Ile Xaa Phe1
545PRTArtificial SequenceSynthetic Peptide 4Lys Xaa Ile Xaa Phe1
556PRTArtificial SequenceSynthetic Peptide 5Phe Xaa Xaa Arg Xaa
Arg1 5611PRTArtificial SequenceSynthetic Peptide 6Asn Trp Gly Arg
Ile Val Ala Phe Phe Ser Phe1 5 10712PRTArtificial SequenceSynthetic
Peptide 7Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe1 5
10811PRTArtificial SequenceSynthetic Peptide 8Asn Trp Gly Arg Ile
Ala Ala Ala Phe Ser Phe1 5 10915DNAArtificial SequenceSynthetic
Construct 9atgtctcaga gcaac 151015DNAArtificial SequenceSynthetic
Construct 10gttgctctga gacat 151115DNAArtificial SequenceSynthetic
Construct 11atggcgaccc cagcc 151215DNAArtificial SequenceSynthetic
Construct 12ggctggggtc gccat 15136PRTArtificial sequenceSynthetic
Peptide 13Phe Ser Arg Arg Tyr Arg1 5146PRTArtificial
SequenceSynthetic Peptide 14Phe Thr Ala Arg Gly Arg1
5156PRTArtificial SequenceSynthetic Peptide 15Phe Glu Leu Arg Tyr
Arg1 5166PRTArtificial SequenceSynthetic Peptide 16Phe Glu Thr Arg
Phe Arg1 51710PRTSaccharomyces cerevisiae 17Gln Phe Glu Arg Lys Asn
Glu Lys Leu Asp1 5 101812PRTSaccharomyces cerevisiae 18Leu Ile Arg
Ser Lys Ser Val His Phe Asp Gln Ala1 5 101912PRTSaccharomyces
cerevisiae 19Ser Leu Glu Phe Leu His Lys Pro Arg Arg Leu Ser1 5
10209PRTSaccharomyces cerevisiae 20Gln Arg Ser Lys Ser Val His Phe
Asp1 52110PRTSaccharomyces cerevisiae 21Asp Leu Phe Asn Glu Arg Arg
Gln Arg Arg1 5 102212PRTSaccharomyces cerevisiae 22Met Pro Thr Arg
His Asn Val Arg Trp Glu Glu Asn1 5 102312PRTSaccharomyces
cerevisiae 23Arg Ser Trp Phe Lys Ala Arg Lys Arg Arg Asp Ile1 5
102412PRTSaccharomyces cerevisiae 24Lys Pro Arg Glu Arg His Ile Lys
Phe Asn Asp Asn1 5 102511PRTMus musculus 25Arg Arg Asn Phe Val Asn
Lys Leu Lys Pro Leu1 5 102612PRTMus musculus 26Asn Gln Ala Lys Lys
Arg Val Val Phe Ala Asp Ser1 5 102712PRTMus musculus 27Thr Val Lys
Val Lys Asn Val Ser Phe Glu Lys Lys1 5 102810PRTArtificial
SequenceSynthetic Peptide 28Leu Asp Phe Arg Asn Arg Leu Gln Thr
Asn1 5 102912PRTArtificial SequenceSynthetic Peptide 29Lys Lys Val
Lys Lys Arg Val Ser Phe Ala Asn Asp1 5 103012PRTHomo sapiens 30Arg
His Phe Val Asn Lys Leu Lys Pro Leu Lys Ser1 5 103112PRTHomo
sapiens 31Asn Gln Ala Lys Lys Arg Val Val Phe Ala Asp Ser1 5
103210PRTArtificial SequenceSynthetic Peptide 32Thr Cys Phe Arg Pro
Arg Leu Arg Gly Ser1 5 103312PRTArtificial SequenceSynthetic
Peptide 33Ser Gln Lys Lys Lys Arg Val Val Phe Ala Asp Met1 5
103411PRTArtificial SequenceSynthetic Peptide 34Gly Glu Val Phe Ile
Asn Lys Gly Lys Gly Phe1 5 103512PRTArtificial SequenceSynthetic
Peptide 35Arg Gly Arg Gln Leu Arg Val Arg Phe Ala Thr His1 5
103610PRTArtificial SequenceSynthetic Peptide 36Gln Val Lys Phe Arg
Arg Arg Arg Glu Gly1 5 103712PRTArtificial SequenceSynthetic
Peptide 37Tyr Phe Lys Arg Tyr Gln Val Lys Phe Arg Arg Arg1 5
103810PRTArtificial SequenceSynthetic Peptide 38Glu Asp Phe Lys Ala
Lys Lys Lys Glu Leu1 5 103912PRTArtificial SequenceSynthetic
Peptide 39Arg Ile Thr Pro Ser Tyr Val Ala Phe Thr Pro Glu1 5
104013PRTHomo sapiens 40Ser Val Phe Met Gln Arg Leu Lys Thr Asn Ile
Leu Gln1 5 104112PRTHomo sapiens 41Ile Asp Glu Val Lys Asn Val Tyr
Phe Lys Asn Phe1 5 104212PRTHomo sapiens 42Val Leu Lys Val Ser Trp
Ile Thr Phe Leu Leu Ala1 5 104310PRTArtificial SequenceSynthetic
Peptide 43Leu Gln Phe Glu Leu Arg Tyr Arg Pro Val1 5
10449PRTArtificial SequenceSynthetic Peptide 44Thr Thr Lys Ala Val
Met Phe Ala Lys1 54510PRTArtificial SequenceSynthetic Peptide 45Asp
Leu Phe Glu Asn Arg Lys Lys Lys Asn1 5 10467PRTArtificial
SequenceSynthetic Peptide 46Val Arg Arg Val Phe Ile Met1
5479PRTHomo sapiens 47Phe Phe Lys Asn Glu Lys Met Leu Tyr1
54812PRTHomo sapiens 48Arg Arg Gly Ser Pro Arg Val Arg Phe Glu Asp
Gly1 5 104911PRTHomo sapiens 49Arg Gln Phe Glu Met Lys Arg Lys Leu
His Tyr1 5 105012PRTArtificial SequenceSynthetic Peptide 50Asn Trp
Gly Arg Ile Val Ala Phe Phe Ser Phe Gly1 5 105112PRTArtificial
SequenceSynthetic Peptide 51Asn Trp Gly Arg Ile Ala Ala Ala Phe Ser
Phe Gly1 5 105212PRTArtificial SequenceSynthetic Peptide 52Gly Asp
Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe1 5 105312PRTArtificial
SequenceSynthetic Peptide 53Gly Asp Glu Gly Glu Leu Gly Tyr Gly Arg
Ala Phe1 5 105412PRTArtificial SequenceSynthetic Peptide 54Gly Asp
Glu Ser Glu Leu Ser Tyr Ser Arg Ala Phe1 5 105512PRTArtificial
SequenceSynthetic Peptide 55Gly Asp Glu Phe Glu Leu Gly Tyr Gly Arg
Ala Phe1 5 105612PRTArtificial SequenceSynthetic Peptide 56Gly Asp
Glu Phe Glu Leu Ser Tyr Ser Arg Ala Phe1 5 105712PRTArtificial
SequenceSynthetic Peptide 57Gly Asp Glu Gly Glu Leu Arg Tyr Arg Arg
Ala Phe1 5 105812PRTArtificial SequenceSynthetic Peptide 58Gly Asp
Glu Ser Glu Leu Arg Tyr Arg Arg Ala Phe1 5 105912PRTArtificial
SequenceSynthetic Peptide 59Gly Asp Glu Gly Glu Leu Gly Tyr Arg Arg
Ala Phe1 5 106012PRTArtificial SequenceSynthetic Peptide 60Gly Asp
Glu Ser Glu Leu Ser Tyr Arg Arg Ala Phe1 5 106113PRTArtificial
SequenceSynthetic Peptide 61Pro Asn Trp Gly Arg Leu Val Ala Phe Phe
Val Phe Gly1 5 106213PRTArtificial SequenceSynthetic Peptide 62Pro
Asn Trp Gly Arg Leu Ala Ala Ala Phe Val Phe Gly1 5
106313PRTArtificial SequenceSynthetic Peptide 63Gly Asp Glu Phe Glu
Thr Arg Phe Arg Arg Thr Phe Ser1 5 106413PRTArtificial
SequenceSynthetic Peptide 64Gly Asp Glu Gly Glu Thr Gly Phe Gly Arg
Thr Phe Ser1 5 106513PRTArtificial SequenceSynthetic Peptide 65Gly
Asp Glu Phe Glu Thr Gly Phe Gly Arg Thr Phe Ser1 5
106613PRTArtificial SequenceSynthetic Peptide 66Gly Asp Glu Phe Glu
Thr Arg Phe Gly Arg Thr Phe Ser1 5 106713PRTArtificial
SequenceSynthetic Peptide 67Gly Asp Glu Gly Glu Thr Gly Phe Arg Arg
Thr Phe Ser1 5 106813PRTArtificial SequenceSynthetic Peptide 68Gly
Asp Glu Gly Glu Thr Arg Phe Arg Arg Thr Phe Ser1 5
106923PRTArtificial SequenceSynthetic Peptide 69Val Lys Gln Ala Leu
Arg Glu Ala Gly Asp Phe Glu Leu Arg Tyr Arg1 5 10 15Arg Ala Phe Ser
Asp Thr Ser 207022PRTArtificial SequenceSynthetic Peptide 70Glu Leu
Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe1 5 10 15Ser
Phe Gly Gly Ala Leu 207123PRTArtificial SequenceSynthetic Peptide
71Arg Ala Ala Gly Asp Glu Phe Glu Thr Arg Arg Arg Thr Phe Ser Asp1
5 10 15Leu Ala Ala Gln Leu His Val 207219PRTArtificial
SequenceSynthetic Peptide 72Glu Leu Phe Gln Gly Gly Pro Asn Trp Gly
Arg Leu Val Ala Phe Phe1 5 10 15Val Gly Ala7324PRTArtificial
SequenceSynthetic Peptide 73His Thr Leu Arg Gln Ala Gly Asp Asp Phe
Ser Arg Arg Tyr Arg Arg1 5 10 15Asp Phe Ala Glu Met Ser Ser Gln
207420PRTArtificial SequenceSynthetic Peptide 74Glu Leu Phe Arg Asp
Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe1 5 10 15Glu Phe Gly Gly
207530PRTArtificial SequenceSynthetic Peptide 75Phe Arg Gly Arg Ser
Arg Ser Ala Pro Pro Asn Leu Trp Ala Ala Gln1 5 10 15Arg Tyr Gly Arg
Glu Leu Arg Arg Met Ser Asp Glu Glu Phe 20 25 30766PRTArtificial
SequenceSynthetic Peptide 76Phe Arg Gly Arg Ser Arg1
5777PRTArtificial SequenceSynthetic Peptide 77Arg Ser Arg Ser Ser
Ala Pro1 57813PRTArtificial SequenceSynthetic Peptide 78Glu Glu Glu
Leu Ser Phe Arg Gly Arg Ser Arg Ser Ala1 5 107913PRTArtificial
SequenceSynthetic Peptide 79Glu Glu Glu Leu Glu Phe Arg Gly Arg Ser
Arg Ser Ala1 5 108013PRTArtificial SequenceSynthetic Peptide 80Glu
Glu Glu Leu Gly Phe Arg Gly Arg Ser Arg Ser Ala1 5
108112PRTArtificial SequenceSynthetic Peptide 81Arg Gln Ala Gly Asp
Asp Phe Ser Arg Arg Tyr Arg1 5 108212PRTArtificial
SequenceSynthetic Peptide 82Arg Gln Ala Gly Asp Asp Phe Glu Arg Arg
Tyr Arg1 5 108312PRTArtificial SequenceSynthetic Peptide 83Arg Gln
Ala Gly Asp Asp Phe Gly Arg Arg Tyr Arg1 5 10847PRTArtificial
SequenceSynthetic Peptide 84Arg Ser Arg Ser Ser Ala Pro1
58510PRTArtificial SequenceSynthetic Peptide 85Gln Phe Glu Arg Lys
Asn Glu Lys Leu Asp1 5 108612PRTArtificial SequenceSynthetic
Peptide 86Leu Ile Arg Ser Lys Ser Val His Phe Asp Gln Ala1 5
108712PRTArtificial SequenceSynthetic Peptide 87Ser Leu Glu Phe Leu
His Lys Pro Arg Arg Leu Ser1 5 10889PRTArtificial SequenceSynthetic
Peptide 88Gln Arg Ser Lys Ser Val His Phe Asp1 58910PRTArtificial
SequenceSynthetic Peptide 89Asp Leu Phe Asn Glu Arg Arg Gln Arg
Arg1 5 109012PRTArtificial SequenceSynthetic Peptide 90Met Pro Thr
Arg His Asn Val Arg Trp Glu Glu Asn1 5 109112PRTArtificial
SequenceSynthetic Peptide 91Arg Ser Trp Phe Lys Ala Arg Lys Arg Arg
Asp Ile1 5 109212PRTArtificial SequenceSynthetic Peptide 92Lys Pro
Arg Glu Arg His Ile Lys Phe Asn Asp Asn1 5 109311PRTArtificial
SequenceSynthetic Peptide 93Arg Arg Asn Phe Val Asn Lys Leu Lys Pro
Leu1 5 109412PRTArtificial SequenceSynthetic Peptide 94Asn Gln Ala
Lys Lys Arg Val Val Phe Ala Asp Ser1 5 109510PRTArtificial
SequenceSynthetic Peptide 95Leu Asp Phe Arg Asn Arg Leu Gln Thr
Asn1 5 109612PRTArtificial SequenceSynthetic Peptide 96Lys Lys Val
Lys Lys Arg Val Ser Phe Ala Asn Asp1 5 109712PRTArtificial
SequenceSynthetic Peptide 97Arg His Phe Val Asn Lys Leu Lys Pro Leu
Lys Ser1 5 109812PRTArtificial SequenceSynthetic Peptide 98Asn Gln
Ala Lys Lys Arg Val Val Phe Ala Asp Ser1 5 109910PRTArtificial
SequenceSynthetic Peptide 99Thr Cys Phe Arg Pro Arg Leu Arg Gly
Ser1 5 1010012PRTArtificial SequenceSynthetic Peptide 100Ser Gln
Lys Lys Lys Arg Val Val Phe Ala Asp Met1 5 1010111PRTArtificial
SequenceSynthetic Peptide 101Gly Glu Val Phe Ile Asn Lys Gly Lys
Gly Phe1 5 1010212PRTArtificial SequenceSynthetic Peptide 102Arg
Gly Arg Gln Leu Arg Val Arg Phe Ala Thr His1 5 1010310PRTArtificial
SequenceSynthetic Peptide 103Gln Val Lys Phe Arg Arg Arg Arg Phe
Gly1 5 1010412PRTArtificial SequenceSynthetic Peptide 104Tyr Phe
Lys Arg Lys Gln Val Lys Phe Arg Arg Arg1 5 1010510PRTArtificial
SequenceSynthetic Peptide 105Glu Asp Phe Lys Ala Lys Lys Lys Glu
Leu1 5 1010612PRTArtificial SequenceSynthetic Peptide 106Arg Ile
Thr Pro Ser Tyr Val Ala Phe Thr Pro Glu1 5 1010713PRTArtificial
SequenceSynthetic Peptide 107Ser Val Phe Met Gln Arg Leu Lys Thr
Asn Ile Leu Gln1 5 1010812PRTArtificial SequenceSynthetic Peptide
108Ile Asp Glu Val Lys Asn Val Tyr Phe Lys Asn Phe1 5
1010912PRTArtificial SequenceSynthetic Peptide 109Val Leu Lys Val
Ser Trp Ile Thr Phe Leu Leu Ala1 5 1011010PRTArtificial
SequenceSynthetic Peptide 110Leu Gln Phe Glu Leu Arg Tyr Arg Pro
Val1 5 101119PRTArtificial SequenceSynthetic Peptide 111Thr Thr Lys
Ala Val Met Phe Ala Lys1 511210PRTArtificial SequenceSynthetic
Peptide 112Asp Leu Phe Glu Asn Arg Lys Lys Lys Asn1 5
101137PRTArtificial SequenceSynthetic Peptide 113Val Arg Arg Val
Phe Ile Met1 51149PRTArtificial SequenceSynthetic Peptide 114Phe
Phe Lys Asn Glu Lys Met Lys Tyr1 511512PRTArtificial
SequenceSynthetic Peptide 115Arg Arg Gly Ser Pro Arg Val Arg Phe
Glu Asp Gly1 5 1011611PRTArtificial SequenceSynthetic Peptide
116Arg Gln Phe Glu Met Lys Arg Lys Leu His Tyr1 5 10
* * * * *