U.S. patent application number 10/119099 was filed with the patent office on 2002-10-10 for smad2 phosphorylation and interaction with smad4.
Invention is credited to Dijke, Peter Ten, Engstrom, Ulla, Heldin, Carl-Henrik, Piek, Ester, Souchelnytskyi, Serhiy, Tamaki, Kiyoshi, Wernstedt, Christer.
Application Number | 20020146774 10/119099 |
Document ID | / |
Family ID | 26725451 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020146774 |
Kind Code |
A1 |
Souchelnytskyi, Serhiy ; et
al. |
October 10, 2002 |
Smad2 phosphorylation and interaction with Smad4
Abstract
The invention describes amino acid residues of the Smad2 protein
which are important for phosphorylation and activity, and Smad2
polypeptide fragments and biologically functional variants thereof.
Included and dominant-negative variants of Smad2 and antibodies
relating thereto. Also included are nucleic acids which encode such
variants. Antibodies which selectively bind pathway-restricted Smad
proteins phosphorylated at the C-terminal tail also are provided.
Methods and products for using such nucleic acids and polypeptides
also are provided.
Inventors: |
Souchelnytskyi, Serhiy;
(Uppsala, SE) ; Tamaki, Kiyoshi; (Uppsala, SE)
; Engstrom, Ulla; (Uppsala, SE) ; Wernstedt,
Christer; (Uppsala, SE) ; Piek, Ester;
(Uppsala, SE) ; Dijke, Peter Ten; (Uppsala,
SE) ; Heldin, Carl-Henrik; (Uppsala, SE) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2211
US
|
Family ID: |
26725451 |
Appl. No.: |
10/119099 |
Filed: |
April 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10119099 |
Apr 9, 2002 |
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09552138 |
Apr 19, 2000 |
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6368829 |
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09552138 |
Apr 19, 2000 |
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09082039 |
May 20, 1998 |
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6103869 |
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60047807 |
May 20, 1997 |
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60081313 |
Apr 10, 1998 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/4702 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C07K 014/715; C12P
021/02; C12N 005/06; C07H 021/04 |
Claims
1. An isolated Smad2 polypeptide comprising a polypeptide having
the amino acid sequence of SEQ ID NO:2 or its human homolog except
that the polypeptide includes a mutation comprising a non-serine
amino acid located at one or more of amino acids 464, 465 and
467.
2. The isolated Smad2 polypeptide of claim 1 wherein the mutation
is located at positions selected from the group consisting of 464;
465; 467; 464 and 465; 464 and 467; 465 and 467; and 464, 465 and
467.
3. The isolated Smad2 polypeptide of claim 1 wherein the mutation
is selected from the group consisting of Ser464A; Ser465A; Ser467A;
Ser464A and Ser465A; Ser464A and Ser467A; Ser465A and Ser467A; and
Ser464A, Ser465A and Ser465A.
4. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser464A.
5. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser465A.
6. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser467A.
7. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser464A and Ser465A.
8. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser464A and Ser467A.
9. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser465A and Ser467A.
10. The isolated Smad2 polypeptide of claim 3, wherein the mutation
is Ser464A, Ser465A and Ser467A.
11. An isolated nucleic acid molecule which encodes the isolated
polypeptide of any of claims 1-10.
12. An expression vector comprising the isolated nucleic acid
molecule of claim 11 operably linked to a promoter.
13. A host cell transformed or transfected with the expression
vector of claim 12.
14. An isolated Smad4 binding polypeptide comprising the amino acid
sequence of SEQ ID NO:3.
15. The isolated Smad4 binding polypeptide of claim 14, wherein the
isolated polypeptide comprises a polypeptide selected from the
group consisting of the 4 most C-terminal amino acids of SEQ ID
NO:2, the 5 most C-terminal amino acids of SEQ ID NO:2, the 6 most
C-terminal amino acids of SEQ ID NO:2, the 7 most C-terminal amino
acids of SEQ ID NO:2, the 8 most C-terminal amino acids of SEQ ID
NO:2, the 9 most C-terminal amino acids of SEQ ID NO:2, the 10 most
C-terminal amino acids of SEQ ID NO:2, the 11 most C-terminal amino
acids of SEQ ID NO:2 , the 12 most C-terminal amino acids of SEQ ID
NO:2, the 13 most C-terminal amino acids of SEQ ID NO:2, the 14
most C-terminal amino acids of SEQ ID NO:2, the 15 most C-terminal
amino acids of SEQ ID NO:2, the 16 most C-terminal amino acids of
SEQ ID NO:2, the 17 most C-terminal amino acids of SEQ ID NO:2, the
18 most C-terminal amino acids of SEQ ID NO:2, the 19 most
C-terminal amino acids of SEQ ID NO:2, and the 20 most C-terminal
amino acids of SEQ ID NO:2.
16. The isolated Smad4 binding polypeptide of claim 15, wherein the
isolated polypeptide comprises the amino acid sequence of SEQ ID
NO:4.
17. The isolated Smad4 binding polypeptide of claim 14, 15 or 16,
wherein the isolated polypeptide is phosphorylated on one or more
amino acids selected from the group consisting of Ser464, Ser465,
Ser467, Ser464/Ser465, Ser464/Ser467, Ser465/Ser467 and
Ser464/Ser465/Ser467.
18. An isolated polypeptide which binds selectively the polypeptide
of any of claim 1, 2, 14, 15, 16 or 17 provided that the isolated
polypeptide is not T.beta.R-I, T.beta.R-II or Smad4.
19. The isolated polypeptide of claim 18, wherein the isolated
polypeptide is an antibody.
20. The isolated polypeptide of claim 18, wherein the isolated
polypeptide is an antibody fragment selected from the group
consisting of a Fab fragment, a F(ab).sub.2 fragment or a fragment
including a CDR3 region selective for a Smad2 polypeptide.
20B. The isolated polypeptide of claim 18, wherein the isolated
polypeptide is a monoclonal antibody.
21. A method for inhibiting TGF-.beta. signal transduction in a
mammalian cell, comprising contacting the mammalian cell with an
amount of an inhibitor of phosphorylation of endogenous Smad2
effective to reduce TGF-.beta. signal transduction in the mammalian
cell.
22. The method of claim 21, wherein the inhibitor is a dominant
negative Smad2 polypeptide.
23. The method of claim 22 wherein the dominant negative Smad2 is
the polypeptide of any of claim 1, 2 or 3.
24. A composition comprising: the polypeptide of claim 1, and a
pharmaceutically acceptable carrier.
25. A method for decreasing TGF-.beta. signal transduction activity
in a subject comprising administering to a subject in need of such
treatment an agent that selectively binds to a TGF-.beta. receptor,
in an amount effective to decrease TGF-.beta. signal transduction
activity in the subject.
26. The method of claim 25, wherein the agent is an isolated
polypeptide as claimed in any of claim 1, 2 or 3.
27. A method for identifying lead compounds for a pharmacological
agent useful in the diagnosis or treatment of disease associated
with Smad2/TGF-.beta. receptor interaction, comprising forming a
mixture comprising a Smad2 polypeptide as claimed in claim 1, a
TGF-.beta. receptor, and a candidate pharmacological agent,
incubating the mixture under conditions which, in the absence of
the candidate pharmacological agent, permit a first amount of
specific binding of the TGF-.beta. receptor by the Smad2
polypeptide, and detecting a test amount of the specific binding of
the TGF-.beta. receptor by the Smad2 polypeptide, wherein reduction
of the test amount of specific binding of the the Smad2 polypeptide
relative to the first amount of specific binding of the the Smad2
polypeptide indicates that the candidate pharmacological agent is a
lead compound for a pharmacological agent which disrupts the
Smad2/TGF-.beta. receptor interaction, and wherein increase of the
test amount of specific binding relative to the first amount of
specific binding indicates that the candidate pharmacological agent
is a lead compound for a pharmacological agent which enhances the
Smad2/TGF-.beta. receptor interaction.
28. The method of claim 27, wherein the Smad2 polypeptide is the
polypeptide of claim 3.
29. A method for identifying lead compounds for a pharmacological
agent useful in the diagnosis or treatment of disease associated
with TGF-.beta. mediated Smad2 signal transduction activity,
comprising forming a mixture comprising a wild type Smad2
polypeptide, a TGF-.beta. receptor, and a candidate pharmacological
agent comprising a mutated Smad2 polypeptide, incubating the
mixture under conditions which, in the absence of the candidate
pharmacological agent, permit a first amount of TGF-.beta. mediated
phosphorylation of the wild type Smad2 polypeptide, and detecting a
test amount of TGF-.beta. mediated phosphorylation of the wild type
Smad2 polypeptide, wherein reduction of the test amount of the
phosphorylation of the wild type Smad2 polypeptide relative to the
first amount of the phosphorylation of the wild type Smad 2
polypeptide indicates that the candidate pharmacological agent is a
lead compound for a pharmacological agent which disrupts the
TGF-.beta. mediated Smad2 signal transduction activity.
30. The method of claim 29, wherein the mutated Smad2 polypeptide
is the polypeptide of claim 3 or TGF-.beta. receptor binding
fragments thereof.
31. An isolated polypeptide which binds selectively to at least one
pathway-restricted Smad polypeptide or fragment thereof having the
C-terminal amino acid sequence of SEQ ID NO:5, wherein the
pathway-restricted Smad polypeptide or fragment thereof is
phosphorylated on at least one serine residue of SEQ ID NO:5,
provided that the isolated polypeptide is not T.beta.R-I,
T.beta.R-II or Smad4.
32. The isolated polypeptide of claim 31, wherein the pathway
restricted Smad polypeptide is selected from the group consisting
of Smad1, Smad3, Smad5 and Smad9.
33. The isolated polypeptide of claim 31, wherein the at least one
serine residue is selected from the group consisting of the second
serine residue, the third serine residue and the second and third
serine residues.
34. The isolated polypeptide of claim 31, wherein the isolated
polypeptide is an antibody.
35. The isolated polypeptide of claim 34, wherein the isolated
polypeptide is an antibody fragment selected from the group
consisting of a Fab fragment, a F(ab).sub.2 fragment or a fragment
including a CDR3 region selective for a pathway-restricted Smad
polypeptide.
36. The isolated polypeptide of claim 34, wherein the antibody is a
monoclonal antibody.
37. The isolated polypeptide of claim 34, wherein the antibody
binds selectively to one of the pathway restricted Smad
polypeptides.
38. A method for determining the amount of a pathway-restricted
Smad polypeptide having a phosphorylated C-terminal serine residue
in a biological sample, comprising contacting the biological sample
with an isolated polypeptide that selectively binds at least one
phosphorylated serine of the amino acid sequence as set forth in
SEQ ID NO:5, determining the binding of the isolated polypeptide to
the pathway-restricted Smad polypeptide, and comparing the binding
to a control as a determination of the amount of a
pathway-restricted Smad polypeptide having a phosphorylated
C-terminal serine residue in the biological sample.
39. The method of claim 38, wherein the pathway-restricted Smad
polypeptide is selected from the group consisting of Smad1, Smad2,
Smad3, Smad5, and Smad9.
40. The method of claim 38, wherein the isolated polypeptide is an
antibody.
41. The method of claim 40, wherein the antibody selectively binds
a pathway-restricted Smad polypeptide having at least one
phosphorylated C-terminal serine residue selected from the group
consisting of the second serine residue of SEQ ID NO:5, the third
serine residue of SEQ ID NO:5 and the second and third serine
residues of SEQ ID NO:5.
42. A method for identifying lead compounds for a pharmacological
agent which modulate phosphorylation of the C-terminal serine
residues of a pathway-restricted Smad polypeptide, comprising
forming a mixture comprising a pathway-restricted Smad polypeptide
having at least one C-terminal serine residue, a TGF-.beta.
superfamily receptor or receptor complex capable of phosphorylating
the at least one C-terminal serine residue, and a candidate
pharmacological agent, incubating the mixture under conditions
which, in the absence of the candidate pharmacological agent,
permit a first amount of phosphorylation of the at least one
C-terminal serine residue by the TGF-.beta. superfamily receptor or
receptor complex, and detecting a test amount of the
phosphorylation of the at least one C-terminal serine residue by
the TGF-.beta. superfamily receptor or receptor complex, wherein
reduction of the test amount of phosphorylation relative to the
first amount of phosphorylation indicates that the candidate
pharmacological agent is a lead compound for a pharmacological
agent which reduces phosphorylation of the at least one C-terminal
serine residue of a pathway-restricted Smad polypeptide, and
wherein an increase of the test amount of phosphorylation relative
to the first amount of phosphorylation indicates that the candidate
pharmacological agent is a lead compound for a pharmacological
agent which increases the phosphorylation of the C-terminal serine
residues of a pathway-restricted Smad polypeptide.
43. The method of claim 42, wherein the pathway-restricted Smad
polypeptide is selected from the group consisting of Smad1, Smad2,
Smad3, Smad5, and Smad9.
44. The method of claim 42, wherein the at least one C-terminal
serine residue is selected from the group consisting of the second
serine residue of SEQ ID NO:5, the third serine residue of SEQ ID
NO:5 and the second and third serine residues of SEQ ID NO:5.
45. The method of claim 42, wherein the step of forming a mixture
comprising a pathway-restricted Smad polypeptide, a TGF-.beta.
superfamily receptor or receptor complex, and a candidate
pharmacological agent comprises contacting a cell which includes a
pathway-restricted Smad polypeptide and a TGF-.beta. receptor or
receptor complex with a candidate pharmacological agent.
46. The method of claim 42 or claim 45, wherein the step of forming
a mixture further comprises adding a ligand which activates the
TGF-.beta. superfamily receptor or receptor complex.
47. A method for reducing heteromeric Smad protein complex
formation in a cell, comprising providing an antibody which
selectively binds a phosphorylated pathway-restricted Smad
polypeptide or a nucleic acid encoding the antibody, and contacting
the cell with an amount of the antibody or the nucleic acid
encoding the antibody sufficient to reduce formation of the
heteromeric Smad protein complex in the cell.
48. The method of claim 47, wherein the pathway-restricted Smad
polypeptide is selected from the group consisting of Smad1, Smad2,
Smad3, Smad5, and Smad9.
49. The method of claim 47, wherein the antibody selectively binds
a pathway-restricted Smad polypeptide having at least one
phosphorylated C-terminal serine residue selected from the group
consisting of the second serine residue of SEQ ID NO:5, the third
serine residue of SEQ ID NO:5 and the second and third serine
residues of SEQ ID NO:5.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
09/552,138, filed Apr. 19, 2000, now pending, which is a divisional
of application Ser. No. 09/082,039, filed May 20, 1998, now U.S.
Pat. No. 6,103,869, which claims priority under 35 U.S.C. .sctn.119
from U.S. provisional application serial No. 60/047,807, filed May
20, 1997, and from U.S. provisional application serial No.
60/081,313, filed Apr. 10, 1998.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acids and encoded
polypeptides which interact with the TGF-.beta. receptor complex
and which is a negative regulator of TGF-.beta. signaling. The
invention also relates to agents which bind the nucleic acids or
polypeptides. The invention further relates to methods of using
such nucleic acids and polypeptides in the treatment and/or
diagnosis of disease.
BACKGROUND OF THE INVENTION
[0003] During mammalian embryogenesis and adult tissue homeostasis
transforming growth factor .beta. (TGF-.beta.) performs pivotal
tasks in intercellular communication (Roberts et al., Growth
Factors, 8:1-9, 1993). The cellular effects of theis pleiotropic
factor are exerted by ligand-induced hetero-oligomerization of two
distantly related type I and type II serine/threonine kinase
receptors, T.beta.R-I and T.beta.R-II, respectively (Lin and
Lodish, Trends Cell Biol., 11:972-978.,1993; Derynck, Trends
Biochem. Sci., 19:548-553, 1994; Massague and Weis-Garcia, Cancer
Surv. 27:41-64, 1996; ten Dijke et al., Curr. Opin. Cell Biol.,
8:139-145, 1996). The two receptors, which both are required for
signaling, act in sequence; T.beta.R-I is a substrate for the
constitutively active T.beta.R-II kinase (Wrana et al., Nature,
370:341-347, 1994; Wieser et al., EMBO J, 14:2199-2208, 1995).
TGF-.beta. forms part of a large family of structurally related
proteins which include activins and bone morphogenetic proteins
(BMPs) that signal in a similar fashion, each employing distinct
complexes of type I and type II serine/threonine kinase receptors
(Lin and Lodish, 1993; Derynck, 1994; Massague and Weis-Garcia,
1996; ten Dijke et al., 1996).
[0004] Genetic studies of TGF-.beta.-like signaling pathways in
Drosophila and Caenorhabditis elegans have led to the
identification of mothers against dpp (Mad) (Sekelsky et al.,
Genetics, 139:1347-1358, 1995) and sma (Savage et al., Proc. Natl.
Acad. Sci. USA, 93:790-794, 1996) genes, respectively. The products
of these related genes perform essential functions downstream of
TGF-.beta.-like ligands acting via serine/threonine kinase
receptors in these organisms (Wiersdorf et al., Development,
122:2153-2163,1996; Newfeld et al., Development, 122:2099-2108,
1996; Hoodless et al., Cell, 85:489-500, 1996). Vertebrate homologs
of Mad and sma have been termed Smads (Derynck et al., Cell,
87:173, 1996) or MADR genes (Wrana and Attisano, Trends Genet.,
12:493-496, 1996). Genetic alterations in Smad2 and Smad4/DPC4 have
been found in specific tumor subsets, and thus Smads may function
as tumor suppressor genes (Hahn et al., Science, 271:350-353, 1996;
Riggins et al., Nature Genet., 13:347-349, 1996; Eppert et al.,
Cell, 86:543-552, 1996). Smad proteins share two regions of high
similarity, termed MH1 and MH2 domains, connected with a variable
proline-rich sequence (Massague, Cell 85:947-950, 1996; Derynck and
Zhang, Curr. Biol., 6:1226-1229, 1996). The C-terminal part of
Smad2, when fused to a heterologous DNA-binding domain, was found
to have transcriptional activity (Liu et al., Nature, 381:620-623,
1996; Meersseman et al., Mech. Dev., 61:127-140, 1997). The intact
Smad2 protein when fused to a DNA-binding domain, was latent, but
transcriptional activity was unmasked after stimulation with ligand
(Liu et al., 1996).
[0005] Different Smads specify different responses using functional
assays in Xenopus. Whereas Smad1 induces ventral mesoderm, a
BMP-like response, Smad2 induces dorsal mesoderm, an
activin/TGF-.beta.-like response (Graff et al., Cell, 85:479-487,
1996; Baker and Harland, Genes & Dev., 10:1880-1889 1996;
Thomsen, Development, 122:2359-2366, 1996). Upon ligand stimulation
Smads become phosphorylated on serine and threonine residues; BMP
stimulates Smad1 phosphorylation, whereas TGF-.beta. induces Smad2
and Smad3 phosphorylation (Hoodless et al., 1996; Liu et al., 1996;
Eppert et al., 1996; Lechleider et al., J. Biol. Chem.,
271:17617-17620, 1996; Yingling et al., Proc. Natl. Acad. Sci. USA,
93:8940-8944, 1996; Zhang et al., Nature, 383:168-172, 1996;
Macas-Silva et al., Cell, 87:1215-1224, 1996; Nakao et al., J.
Biol. Chem., 272:2896-2900, 1996).
[0006] Smad4 is a common component of TGF-.beta., activin and BMP
signaling (Lagna et al., Nature, 383:832-836, 1996; Zhang et al.,
Curr. Biol., 7:270-276, 1997; de Winter et al., Oncogene,
14:1891-1900, 1997). Smad4 phosphorylation has thus far been
reported only after activin stimulation of transfected cells (Lagna
et al., 1996). After stimulation with TGF-.beta. or activin Smad4
interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric
complex of Smad4 and Smad1 has been observed (Lagna et al., 1996).
Upon ligand stimulation, Smad complexes translocate from the
cytoplasm to the nucleus (Hoodless et al., 1996; Liu et al., 1996;
Baker and Harland, 1996; Macas-Silva et al., 1996), where they, in
combination with DNA-binding proteins, may regulate gene
transcription (Chen et al., Nature, 383:691-696, 1996).
SUMMARY OF THE INVENTION
[0007] The invention provides isolated Smad2 polypeptides and
agents which bind such polypeptides, including antibodies. The
invention also provides isolated nucleic acid molecules which
encode the foregoing polypeptides, unique fragments of those
molecules, expression vectors containing the foregoing, and host
cells transfected with those molecules. The foregoing can be used
in the diagnosis or treatment of conditions characterized by
TGF-.beta. signal transduction. The invention also-provides methods
for identifying pharmacological agents useful in the diagnosis or
treatment of such conditions. Here, the identification of Smad2
amino acid residues phosphorylated in vivo is reported.
[0008] According to one aspect of the invention, an isolated Smad2
polypeptide is provided. The polypeptide has the amino acid
sequence of SEQ ID NO:2 or its human homolog except that the
polypeptide includes a mutation comprising a non-serine amino acid
located at one or more of amino acids 464, 465 and 467. In certain
embodiments, the isolated Smad2 polypeptide compises a mutation
which is located at a position or positions selected from the group
consisting of 464; 465; 467; 464 and 465; 464 and 467; 465 and 467;
and 464, 465 and 467. Preferably the isolated Smad2 polypeptide
comprises a mutation or mutations of the serine residues to alanine
residues (e.g., Ser465A) or aspartic acid residues (e.g., Ser465D)
such as those selected from the group consisting of Ser464A;
Ser465A; Ser467A; Ser464A and Ser465A; Ser464A and Ser467A; Ser465A
and Ser467A; Ser464A, Ser465A and Ser465A; Ser465D; Ser467D; and
Ser465D and Ser467D.
[0009] In other embodiments, the foregoing isolated polypeptide
consists of a fragment or variant of the foregoing which retains
the activity of the foregoing.
[0010] According to still another aspect of the invention, nucleic
acid molecules which encode the foregoing polypeptides are
provided. The nucleic acids can be composed of natural and/or
non-natural nucleotides and linked with natural and/or non-natural
internucleoside bonds.
[0011] According to still another aspect of the invention, the
invention involves expression vectors, and host cells transformed
or transfected with such expression vectors, comprising the nucleic
acid molecules described above.
[0012] According to another aspect of the invention, there are
provided isolated Smad4 binding polypeptides comprising the amino
acid sequence of SEQ ID NO:3, which selectively bind a Smad4
protein or fragment thereof, provided that the isolated polypeptide
is not-wild type Smad2. In certain embodiments, the isolated Smad4
binding polypeptide comprises the C-terminal 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of SEQ ID
NO:2. Preferably, the isolated Smad4 binding polypeptide of
comprises the amino acid sequence of SEQ ID NO:4. In other
preferred embodiments, the foregoing isolated Smad4 binding
polypeptides are phosphorylated on one or more amino acids selected
from the group consisting of Ser464, Ser465, Ser467, Ser464/Ser465,
Ser464/Ser467, Ser465/Ser467 and Ser464/Ser465/Ser467.
[0013] According to yet another aspect of the invention, the
invention involves an isolated polypeptide which binds selectively
any of the foregoing isolated polypeptides, provided that the
isolated polypeptide is not T.beta.R-I, T.beta.R-II or Smad4. In
preferred embodiments, the isolated binding polypeptides include
antibodies and fragments of antibodies (e.g., Fab, F(ab).sub.2, Fd
and antibody fragments which include a CDR3 region which binds
selectively to the polypeptides of the invention). Particularly
preferred antibodies include monoclonal antibodies.
[0014] In another aspect of the invention, an isolated polypeptide
is provided which binds selectively to at least one
pathway-restricted Smad polypeptide or fragment thereof having the
C-terminal amino acid sequence of SEQ ID NO:5. The
pathway-restricted Smad polypeptide or fragment thereof is
phosphorylated on at least one serine residue of SEQ ID NO:5, and
the isolated polypeptide is not T.beta.R-I, T.beta.R-II or Smad4.
In certain embodiments, the pathway restricted Smad polypeptide is
selected from the group consisting of Smad1, Smad3, Smad5 and
Smad9. In other embodiments, the at least one serine residue is
selected from the group consisting of the second serine residue,
the third serine residue and the second and third serine residues.
In preferred embodiments, the isolated binding polypeptides include
antibodies and fragments of antibodies (e.g., Fab, F(ab).sub.2, Fd
and antibody fragments which include a CDR3 region which binds
selectively to pathway-restricted Smad polypeptides). Particularly
preferred antibodies include monoclonal antibodies. In certain
embodiments, the antibody or antibody fragment binds selectively to
one of the pathway restricted Smad polypeptides.
[0015] According to still another aspect of the invention, methods
for inhibiting TGF-.beta. signal transduction in a mammalian cell
are provided. The methods involve contacting a mammalian cell with
an amount of an inhibitor of phosphorylation of endogenous Smad2
effective to reduce TGF-.beta. signal transduction in the mammalian
cell. In certain embodiments of the foregoing methods, the
inhibitor is a dominant negative Smad2 polypeptide, such as the
foregoing Smad2 polypeptides which include a mutation at one or
more of residues 464, 465 and 467.
[0016] The invention in still another aspect provides compositions
comprising a Smad2 polypeptide which includes a mutation at one or
more of residues 464, 465 and 467, and a pharmaceutically
acceptable carrier.
[0017] The invention in a further aspect involves a method for
decreasing TGF-.beta. signal transduction activity in a subject. An
agent that selectively binds to a TGF-.beta. receptor and blocks
TGF-.beta. signaling is administered to a subject in need of such
treatment, in an amount effective to decrease Smad2 TGF-.beta.
signal transduction activity in the subject. Preferred agents are
Smad2 polypeptides which include a mutation at one or more of
serines 464, 465 and 467, particularly those which are substituted
with alanines or aspartic acids.
[0018] According to another aspect of the invention, methods are
provided for identifying lead compounds for a pharmacological agent
useful in the diagnosis or treatment of disease associated with
Smad2/TGF-.beta. receptor interaction. The methods involve forming
a mixture of a Smad2 polypeptide comprising a mutation at one or
more of serines 464, 465 and 467, a TGF-.beta. receptor, and a
candidate pharmacological agent. The mixture is incubated under
conditions which, in the absence of the candidate pharmacological
agent, permit a first amount of specific binding of the TGF-.beta.
receptor by the Smad2 polypeptide. A test amount of the specific
binding of the TGF-.beta. receptor by the Smad2 polypeptide then is
detected. Detection of a reduction in the foregoing activity in the
presence of the candidate pharmacological agent indicates that the
candidate pharmacological agent is a lead compound for a
pharmacological agent which disrupts the Smad2/TGF-.beta. receptor
interaction. Detection of an increase in the foregoing activities
in the presence of the candidate pharmacological agent indicates
that the candidate pharmacological agent is a lead compound for a
pharmacological agent which enhances Smad2/TGF-.beta. receptor
interaction. Preferably the Smad2 polypeptide comprises a mutation
or mutations selected from the group consisting of Ser464A;
Ser465A; Ser467A; Ser464A and Ser465A; Ser464A and Ser467A; Ser465A
and Ser467A; Ser464A, Ser465A and Ser465A; Ser465D; Ser467D; and
Ser465D and Ser467D.
[0019] According to a further aspect of the invention, methods are
provided for identifying lead compounds for a pharmacological agent
useful in the diagnosis or treatment of disease associated with
TGF-.beta.-mediated Smad2 signal transduction activity. The methods
involve forming a mixture of a wild type Smad2 polypeptide, a
TGF-.beta. receptor, and a candidate pharmacological agent
comprising a mutated Smad2 polypeptide, preferably having a
mutation at one or more of serines 464, 465 and 467. The mixture is
incubated under conditions which, in the absence of the candidate
pharmacological agent, permit a first amount of TGF-.beta. mediated
phosphorylation of the wild type Smad2 polypeptide. A test amount
of TGF-.beta. mediated phosphorylation of the wild type Smad2
polypeptide then is detected. Detection of a reduction in the test
amount of TGF-.beta. mediated phosphorylation of the wild type
Smad2 polypeptide relative to the first amount of TGF-.beta.
mediated phosphorylation of the wild type Smad2 polypeptide
indicates that the candidate pharmacological agent is a lead
compound for a pharmacological agent which disrupts the TGF-.beta.
mediated Smad2 signal transduction activity. Preferably the mutated
Smad2 polypeptide comprises a mutation or mutations selected from
the group consisting of Ser464A; Ser465A; Ser467A; Ser464A and
Ser465A; Ser464A and Ser467A; Ser465A and Ser467A; Ser464A, Ser465A
and Ser465A; Ser465D; Ser467D; and Ser465D and Ser467D, or
TGF-.beta. receptor binding fragments thereof.
[0020] According to another aspect of the invention, methods for
determining the amount of a pathway-restricted Smad polypeptide
having a phosphorylated C-terminal serine residue in a biological
sample are provided. The methods include contacting the biological
sample with an isolated polypeptide that selectively binds at least
one phosphorylated serine of the amino acid sequence as set forth
in SEQ ID NO:5, determining the binding of the isolated polypeptide
to the pathway-restricted Smad polypeptide, and comparing the
binding to a control as a determination of the amount of a
pathway-restricted Smad polypeptide having a phosphorylated
C-terminal serine residue in the biological sample. The biological
sample can be a biological extract such as in vitro extract, or can
be tissue sample for in vivo or in vitro immunohistochemistry
analysis. In certain embodiments, the pathway-restricted Smad
polypeptide is selected from the group consisting of Smad1, Smad2,
Smad3, Smad5, and Smad9. In preferred embodiments, the isolated
binding polypeptides include antibodies and fragments of
antibodies, including monoclonal antibodies. In certain of the
preferred embodiments, the antibody or antibody fragment
selectively binds a pathway-restricted Smad polypeptide having at
least one phosphorylated C-terminal serine residue selected from
the group consisting of the second serine residue of SEQ ID NO:5,
the third serine residue of SEQ ID NO:5 and the second and third
serine residues of SEQ ID NO:5.
[0021] Accordig to yet another aspect of the invention, methods for
identifying lead compounds for a pharmacological agent which
modulate phosphorylation of the C-terminal serine residues of a
pathway-restricted Smad polypeptide are provided. The methods
include forming a mixture comprising a pathway-restricted Smad
polypeptide having at least one C-terminal serine residue, a
TGF-.beta. superfamily receptor or receptor complex capable of
phosphorylating the at least one C-terminal serine residue, and a
candidate pharmacological agent. The mixture is incubated under
conditions which, in the absence of the candidate pharmacological
agent, permit a first amount of phosphorylation of the at least one
C-terminal serine residue by the TGF-.beta. superfamily receptor or
receptor complex. A test amount of the phosphorylation of the at
least one C-terminal serine residue by the TGF-.beta. superfamily
receptor or receptor complex then is detected. A reduction of the
test amount of phosphorylation relative to the first amount of
phosphorylation indicates that the candidate pharmacological agent
is a lead compound for a pharmacological agent which reduces
phosphorylation of the at least one C-terminal serine residue of a
pathway-restricted Smad polypeptide. An increase of the test amount
of phosphorylation relative to the first amount of phosphorylation
indicates that the candidate pharmacological agent is a lead
compound for a pharmacological agent which increases the
phosphorylation of the C-terminal serine residues of a
pathway-restricted Smad polypeptide. In certain embodiments, the
pathway-restricted Smad polypeptide is selected from the group
consisting of Smad1, Smad2, Smad3, Smad5, and Smad9. In other
embodiments, the at least one C-terminal serine residue is selected
from the group consisting of the second serine residue of SEQ ID
NO:5, the third serine residue of SEQ ID NO:5 and the second and
third serine residues of SEQ ID NO:5. In still other embodiments,
the step of forming a mixture comprising a pathway-restricted Smad
polypeptide, a TGF-.beta. superfamily receptor or receptor complex,
and a candidate pharmacological agent includes contacting a cell
which includes a pathway-restricted Smad polypeptide and a
TGF-.beta. receptor or receptor complex with a candidate
pharmacological agent. In preferred embodiments of the foregoing
methods, the step of forming a mixture further comprises adding a
ligand which activates the TGF-.beta. superfamily receptor or
receptor complex.
[0022] In another aspect of the invention methods for reducing
heteromeric Smad protein complex formation in a cell is provided.
The methods include providing an antibody which selectively binds a
phosphorylated pathway-restricted Smad polypeptide or a nucleic
acid encoding the antibody, and contacting the cell with an amount
of the antibody or the nucleic acid encoding the antibody
sufficient to reduce formation of the heteromeric Smad protein
complex in the cell. In certain embodiments, the pathway-restricted
Smad polypeptide is selected from the group consisting of Smad1,
Smad2, Smad3, Smad5, and Smad9. In other embodiments, the antibody
selectively binds a pathway-restricted Smad polypeptide having at
least one phosphorylated C-terminal serine residue selected from
the group consisting of the second serine residue of SEQ ID NO:5,
the third serine residue of SEQ ID NO:5 and the second and third
serine residues of SEQ ID NO:5.
[0023] Use of the foregoing compounds in the preparation of a
medicament also in provided.
[0024] These and other aspects of the invention will be described
in further detail in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 depicts T.beta.R-mediated phosphorylation of
Smad2.
[0026] FIG. 2 shows the identification of Ser465 and Ser467 as the
major in vivo phosphorylation sites in Smad2.
[0027] FIG. 3 demonstrates that phosphorylation of Ser465 requires
that Ser467 is phosphorylated.
[0028] FIG. 4 shows the dominant-negative effect on TGF-p-mediated
transciptional response of Smad2 mutants with serine residues 464,
465 and/or 467 replaced with alanine or aspartic acid residues.
[0029] FIG. 5 demonstrates the association of wild-type and mutant
Smad2 with T.beta.R-I.
[0030] FIG. 6 shows that phosphorylated peptides derived from the
C-terminus of Smad2 bind to Smad4.
[0031] FIG. 7 demonstrates the specificity of a Smad2 phospho tail
antibody for phosphorylated Smad2.
[0032] FIG. 8 shows that the Smad2 phospho tail antibody recognizes
Smad2 phosphorylated in TGF-.beta. stimulated cells.
BRIEF DESCRIPTION OF THE SEQUENCES
[0033] SEQ ID NO:1 is the nucleotide sequence of the mouse Smad2
cDNA (accession number U60530).
[0034] SEQ ID NO:2 is the amino acid sequence of the mouse Smad2
protein (accession number U60530).
[0035] SEQ ID NO:3 is the amino acid sequence of the four amino
acid fragment of the Smad2 C-terminus which binds Smad4.
[0036] SEQ ID NO:4 is the amino acid sequence of the fourteen amino
acid fragment of the Smad2 C-terminus which binds Smad4.
[0037] SEQ ID NO:5 is the amino acid sequence of the C-terminal
motif of the pathway-restricted Smads.
[0038] SEQ ID NO:6 is the amino acid sequence of a peptide used to
prepare polyclonal antibodies to phosphorylated Smad2.
[0039] SEQ ID NO:7 is the amino acid sequence of another
phosphorylated Smad2 peptide used for preparation of
antibodies.
[0040] SEQ ID NO:8 is the amino acid sequence of the C-terminus of
Smad1, Smad3, Smad5 and Smad9 proteins.
[0041] SEQ ID NO:9 is the amino acid sequence of the phosphorylated
peptide used for preparation of antibodies to Smad1, Smad3, Smad5
and Smad9.
[0042] SEQ ID NO:10 is the amino acid sequence of the fourteen
amino acid fragment of the Smad1 protein C-terminus.
[0043] SEQ ID NO:11 is the amino acid sequence of the fourteen
amino acid fragment of the Smad3 protein C-terminus.
[0044] SEQ ID NO:12 is the amino acid sequence of the fourteen
amino acid fragment of the Smad5 protein C-terminus.
[0045] SEQ ID NO:13 is the amino acid sequence of the fourteen
amino acid fragment of the Smad9 protein C-terminus.
[0046] SEQ ID NO:14 is the nucleotide sequence of the human Smad2
mRNA (accession number AF027964).
[0047] SEQ ID NO:15 is the amino acid sequence of the human Smad2
protein (accession number AF027964).
DETAILED DESCRIPTION OF THE INVENTION
[0048] We present the identification of TGF-.beta.-mediated
phosphorylation of Smad2 at two serine residues in the C-terminus
of the protein, i.e., Ser465 and Ser467. Phosphorylation of Ser465
required that Ser467 was phosphorylated. Mutation of Ser465 and/or
Ser467 in Smad2 to alanine residues resulted in dominant negative
inhibition of TGF-.beta. signaling, as did mutation of Ser464 which
is not an in vivo phosphorylation site. These Smad2 mutants were
found to interact stably with an activated TGF-.beta. receptor
complex, in contrast to wild-type Smad2, which interacts only
transiently. A peptide containing the four C-terminal amino acid
residues of Smad2 with Ser465 and Ser467 phosphorylated, bound in
vitro to a glutathione S-transferase-Smad4 fusion protein with
higher affinity that the corresponding non-phosphorylated peptide.
Binding to GST-Smad4 was strongly enhanced when a longer C-terminal
peptide (14 amino acid residues) was used, and binding was in this
case seen also in the absence of phosphorylation.
[0049] The invention thus involves in one aspect Smad2
polypeptides, genes encoding those polypeptides, functional
modifications and variants of the foregoing, useful fragments of
the foregoing, as well as therapeutics relating thereto. The
invention also involves the recognition that pathway-restricted
Smad proteins (including Smad1, Smad2, Smad3, Smad5, and Smad9)
share a conserved sequence at the C-terminus (SEQ ID NO:5), and
therefore the invention relates to all of the pathway-restricted
Smad proteins.
[0050] The invention provides isolated polypeptides, such as
variants of the Smad2 polypeptides described previously (e.g.,
Nakao et al., 1996; SEQ ID NO:2). In particular, mutants of the
Smad2 polypeptide (e.g. the mouse protein is represented by SEQ ID
NO:2) and fragments thereof are provided. Such polypeptides are
useful, for example, alone for the modulation of TGF-.beta. signal
transduction or as fusion proteins to generate antibodies, e.g.,
for use as components of an immunoassay.
[0051] The peptides of the invention are isolated peptides. As used
herein, with respect to peptides, the term "isolated peptides"
means that the peptides are substantially pure and are essentially
free of other substances with which they may be found in nature or
in vivo systems to an extent practical and appropriate for their
intended use. In particular, the peptides are sufficiently pure and
are sufficiently free from other biological constituents of their
hosts cells so as to be useful in, for example, producing
pharmaceutical preparations or sequencing. Because an isolated
peptide of the invention may be admixed with a pharmaceutically
acceptable carrier in a pharmaceutical preparation, the peptide may
comprise only a small percentage by weight of the preparation. The
peptide is nonetheless substantially pure in that it has been
substantially separated from the substances with which it may be
associated in living systems.
[0052] As used herein, Smad2 polypeptides include polypeptides
which contain one or more modifications to the primary amino acid
sequence of Smad2 (e.g. SEQ ID NO:2), e.g. mutant "variants" of
"wild-type" Smad2. Modifications which create a Smad2 mutant can be
made to a Smad2 polypeptide 1) to reduce or eliminate an activity
of a Smad2 polypeptide, such as TGF-.beta. receptor mediated
phosphorylation; 2) to enhance a property of a Smad2 polypeptide,
such as binding to T.beta.R-I or protein stability in an expression
system or the stability of protein-protein binding; or 3) to
provide a novel activity or property to a Smad2 polypeptide, such
as addition of an antigenic epitope or addition of a detectable
moiety. Modifications to a Smad2 polypeptide are typically made to
the nucleic acid which encodes the Smad2 polypeptide, and can
include deletions, point mutations, truncations, amino acid
substitutions and additions of amino acids or non-amino acid
moieties. Alternatively, modifications can be made directly to the
polypeptide, such as by cleavage, addition of a linker molecule,
addition of a detectable moiety, such as biotin, addition of a
fatty acid, and the like. Modifications also embrace fusion
proteins comprising all or part of the Smad2 amino acid sequence.
Fragments of Smad2 which retain characteristic activities such as
the TGF-.beta. receptor binding or interaction activity, the
inhibition of wild-type Smad2 activity and the ability to decrease
TGF-.beta.-mediated signaling and gene expression. One of ordinary
skill in the art can readily determine which fragments retain such
activities by preparing fragments of non-wild-type Smad2 using
standard procedures such as PCR, restriction endonucleiase
digestion, or site-directed mutagenesis, and then testing such
fragments for non-wild-type Smad2 activities as described
herein.
[0053] Smad2 polypeptides also include polypeptides referred to
herein as "Smad4 binding polypeptides". Such Smad2 polypeptides
comprise the portion of Smad 2 which binds to Smad4. As
demonstrated below in the examples, Smad4 binding polypeptides can
be as small as four amino acids in length, and as large as
wild-type Smad2 or larger. It has been discovered that the
C-terminus of Smad2 directs the binding of Smad2 to Smad4 and thus
preferred examples of Smad4 binding polypeptides include the
C-terminal end of Smad2. Examples include the polypeptides of SEQ
ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Other Smad4 binding
polypeptides which include SEQ ID NO:3 and additional amino acids
of Smad2 C-terminus are contemplated. As will be apparent to one of
ordinary skill in the art, Smad4 binding polypeptides also can
include non-Smad amino acid sequences.
[0054] It has been discovered that the Smad4 binding polypeptide
has increased activity when phosphorylated. Thus, it is preferable
to prepare and use phosphorylated Smad4 binding polypeptides as
exemplified below. One of ordinary skill in the art can prepare
such polypeptides by using methods and materials well known in the
art.
[0055] In general, mutants include pathway-restricted Smad
polypeptides such as Smad2 polypeptides which are modified
specifically to alter a feature of the polypeptide related to its
physiological activity. For example, the experimental evidence
provided below demonstrates that serines 465 and 467 are the in
vivo phosphorylation sites on Smad2. It has also been demonstrated
that serine 464 is important for certain Smad2 activities. Thus
mutations to one or more of these serine residues can create a
Smad2 polypeptide which has different activity than the wild-type
Smad2. Such mutants are useful for, inter alia, modulation of
TGF-.beta. signal transduction in vitro and in vivo. Smad2 mutants
can be used to modulate TGF-.beta. activity for a variety of
therapeutic and experimental uses as provided elsewhere herein.
Other uses will be apparent to one of ordinary skill in the
art.
[0056] As demonstrated in the examples below, substitution of
alanine residues or aspartic acid residues for the aforementioned
serines in Smad2 results in mutant Smad2 polypeptides with varying
degrees of activity with respect to TGF-.beta. receptor
interaction, reduction of TGF-.beta. mediated gene expression,
reduction of phosphorylation of wild-type Smad2, and the like. It
will be clear to one of ordinary skill in the art that other
mutations can be made with equal facility using techniques which
are standard in the art. Such mutations are not limited to point
mutations but also embrace larger deletions and truncations.
[0057] In general, variants include Smad2 polypeptides which are
modified specifically to alter a feature of the polypeptide
unrelated to its physiological activity. For example, cysteine
residues can be substituted or deleted to prevent unwanted
disulfide linkages. Similarly, certain amino acids can be changed
to enhance expression of a Smad2 polypeptide by eliminating
proteolysis by proteases in an expression system (e.g., dibasic
amino acid residues in yeast expression systems in which KEX2
protease activity is present). In addition, mutations which do not
result in additional altered activity can be incorporated to enable
ease of detection, greater stability, or other properties at the
discretion of the artisan.
[0058] Mutations of a nucleic acid which encode a
pathway-restricted Smad polypeptide such as a Smad2 polypeptids
preferably preserve the amino acid reading frame of the coding
sequence, and preferably do not create regions in the nucleic acid
which are likely to hybridize to form secondary structures, such a
hairpins or loops, which can be deleterious to expression of the
variant polypeptide.
[0059] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the polypeptide. Variant
pathway-restricted Smad polypeptides such as Smad2 polypeptides are
then expressed and tested for one or more activities to determine
which mutation provides a variant polypeptide with the desired
properties. Further mutations can be made to variants (or to
non-variant Smad2 polypeptides) which are silent as to the amino
acid sequence of the polypeptide, but which provide preferred
codons for translation in a particular host. The preferred codons
for translation of a nucleic acid in, e.g., E. coli, are well known
to those of ordinary skill in the art. Still other mutations can be
made to the noncoding sequences of a Smad2 gene or cDNA clone to
enhance expression of the polypeptide. The activity of variants of
pathway-restricted Smad polypeptides such as Smad2 polypeptides can
be tested by cloning the gene encoding the variant Smad polypeptide
into a bacterial or mammalian expression vector, introducing the
vector into an appropriate host cell, expressing the variant Smad
polypeptide, and testing for a functional capability of the Smad
polypeptides as disclosed herein. For example, the variant Smad2
polypeptide can be tested for inhibition of TGF-.beta. signaling
activity as disclosed in the Examples, or for inhibition of Smad2
phosphorylation or for binding to Smad4 as is also disclosed
herein. Preparation of other variant polypeptides may favor testing
of other activities, such as anitbody binding, as will be known to
one of ordinary skill in the art.
[0060] The skilled artisan will also realize that conservative
amino acid substitutions may be made in pathway-restricted Smad
polypeptides such as Smad2 polypeptides to provide functionally
equivalent variants of the foregoing polypeptides, i.e, the
variants retain the functional capabilities of the Smad2
polypeptides. As used herein, a "conservative amino acid
substitution" refers to an amino acid substitution which does not
alter the relative charge or size characteristics of the protein in
which the amino acid substitution is made. Variants can be prepared
according to methods for altering polypeptide sequence known to one
of ordinary skill in the art such as are found in references which
compile such methods, e.g. Molecular Cloning: A Laboratory Manual,
J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. Exemplary functionally equivalent
variants of the Smad2 polypeptides include conservative amino acid
substitutions of SEQ ID NOs:2, 3 and 4. Conservative substitutions
of amino acids include substitutions made amongst amino acids
within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R,
H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
[0061] Conservative amino-acid substitutions in the amino acid
sequence of pathway-restricted Smad polypeptides such as Smad2
polypeptides to produce functionally equivalent variants of Smad2
polypeptides typically are made by alteration of the nucleic acid
encoding Smad2 polypeptides (e.g., SEQ ID NOs:2, 3 and 4). Such
substitutions can be made by a variety of methods known to one of
ordinary skill in the art. For example, amino acid substitutions
may be made by PCR-directed mutation, site-directed mutagenesis
according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci.
U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene
encoding a Smad2 polypeptide. Where amino acid substitutions are
made to a small unique fragment of a pathway-restricted Smad
polypeptide (e.g. a Smad2 polypeptide), such as a Smad4 binding
polypeptide, the substitutions can be made by directly synthesizing
the peptide. The activity of functionally equivalent fragments of
Smad2 polypeptides can be tested by cloning the gene encoding the
altered Smad2 polypeptide into a bacterial or mammalian expression
vector, introducing the vector into an appropriate host cell,
expressing the altered Smad2 polypeptide, and testing for a
functional capability of the Smad2 polypeptides as disclosed
herein. Peptides which are chemically synthesized can be tested
directly for function, e.g., for binding to Smad4.
[0062] Homologs and alleles of the Smad2 nucleic acids of the
invention can be identified by conventional techniques. Thus, an
aspect of the invention is those nucleic acid sequences which code
for Smad2 polypeptides and which hybridize to a nucleic acid
molecule consisting of the coding region of SEQ ID NO: 1 or SEQ ID
NO: 14, under stringent conditions. The term "stringent conditions"
as used herein refers to parameters with which the art is familiar.
Nucleic acid hybridization parameters may be found in references
which compile such methods, e.g. Molecular Cloning: A Laboratory
Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. More specifically, stringent
conditions, as used herein, refers, for example, to hybridization
at 65.degree. C. in hybridization buffer (3.5.times. SSC, 0.02%
Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5
mM NaH.sub.2PO.sub.4(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M
sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl
sulphate; and EDTA is ethylenediaminetetracetic acid. After
hybridization, the membrane upon which the DNA is transferred is
washed at 2.times. SSC at room temperature and then at 0.1.times.
SSC/0.1.times. SDS at temperatures up to 65.degree. C.
[0063] There are other conditions, reagents, and so forth which can
used, which result in a similar degree of stringency. The skilled
artisan will be familiar with such conditions, and thus they are
not given here. It will be understood, however, that the skilled
artisan will be able to manipulate the conditions in a manner to
permit the clear identification of homologs and alleles of Smad2
nucleic acids of the invention. The skilled artisan also is
familiar with the methodology for screening cells and libraries for
expression of such molecules which then are routinely isolated,
followed by isolation of the pertinent nucleic acid molecule and
sequencing.
[0064] In general homologs and alleles typically will share at
least 40% nucleotide identity and/or at least 50% amino acid
identity to SEQ ID NO:1 and SEQ ID NO:2 (or SEQ ID NO: 14 and SEQ
ID NO: 15), respectively, in some instances will share at least 50%
nucleotide identity and/or at least 65% amino acid identity and in
still other instances will share at least 60% nucleotide identity
and/or at least 75% amino acid identity. Watson-Crick complements
of the foregoing nucleic acids also are embraced by the
invention.
[0065] In screening for Smad2 nucleic acids, a Southern blot may be
performed using the foregoing conditions, together with a
radioactive probe. After washing the membrane to which the DNA is
finally transferred, the membrane can be placed against X-ray film
to detect the radioactive signal.
[0066] The invention also includes degenerate nucleic acids which
include alternative codons to those present in the native
materials. For example, serine residues are encoded by the codons
TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is
equivalent for the purposes of encoding a serine residue. Thus, it
will be apparent to one of ordinary skill in the art that any of
the serine-encoding nucleotide triplets may be employed to direct
the protein synthesis apparatus, in vitro or in vivo, to
incorporate a serine residue into an elongating Smad2 polypeptide.
Similarly, nucleotide sequence triplets which encode other amino
acid residues include, but are not limited to: CCA, CCC, CCG and
CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine
codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT
(asparagine codons); and ATA, ATC and ATT (isoleucine codons).
Other amino acid residues may be encoded similarly by multiple
nucleotide sequences. Thus, the invention embraces degenerate
nucleic acids that differ from the biologically isolated nucleic
acids in codon sequence due to the degeneracy of the genetic
code.
[0067] In one set of embodiments, the nucleic acids of the
invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester internucleoside linkage. These nucleic acids may be
prepared by art recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
[0068] In preferred embodiments, however, the nucleic acids of the
invention also may include "modified" oligonucleotides. That is,
the nucleic acids may be modified in a number of ways which do not
prevent their transcription or translation but which enhance their
stability or targeting or which otherwise enhance their therapeutic
effectiveness.
[0069] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
internucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
[0070] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. The present invention, thus,
contemplates pharmaceutical preparations containing modified
nucleic acids that encode Smad2 polypeptides, together with
pharmaceutically acceptable carriers.
[0071] Nucleic acids encoding the pathway-restricted Smad
polypeptides such as Smad2 polypeptides of the invention (including
TGF-.beta. receptor binding and Smad4 binding polypeptides) may be
administered as part of a pharmaceutical composition. Such a
pharmaceutical composition may include the nucleic acids in
combination with any standard physiologically and/or
pharmaceutically acceptable carriers which are known in the art.
The compositions should be sterile and contain a therapeutically
effective amount of the nucleic acids in a unit of weight or volume
suitable for administration to a patient. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredients. The term "physiologically acceptable"
refers to a non-toxic material that is compatible with a biological
system such as a cell, cell culture, tissue, or organism. The
characteristics of the carrier will depend on the route of
administration. Physiologically and pharmaceutically acceptable
carriers include diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other materials which are well known in the art.
As used herein, a "vector" may be any of a number of nucleic acids
into which a desired sequence may be inserted by restriction and
ligation for transport between different genetic environments or
for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids, phagemids and virus genomes. A
cloning vector is one which is able to replicate in a host cell,
and which is further characterized by one or more endonuclease
restriction sites at which the vector may be cut in a determinable
fashion and into which a desired DNA sequence may be ligated such
that the new recombinant vector retains its ability to replicate in
the host cell. In the case of plasmids, replication of the desired
sequence may occur many times as the plasmid increases in copy
number within the host bacterium or just a single time per host
before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase. An expression vector is one into which a
desired DNA sequence may be inserted by restriction and ligation
such that it is operably joined to regulatory sequences and may be
expressed as an RNA transcript. Vectors may further contain one or
more marker sequences suitable for use in the identification of
cells which have or have not been transformed or transfected with
the vector. Markers include, for example, genes encoding proteins
which increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase or alkaline phosphatase), and genes
which visibly affect the phenotype of transformed or transfected
cells, hosts, colonies or plaques (e.g., green fluorescent
protein). Preferred vectors are those capable of autonomous
replication and expression of the structural gene products present
in the DNA segments to which they are operably joined.
[0072] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operablyjoined if induction of a promoter in the 5' is regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0073] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. Especially, such 5'
non-transcribed regulatory sequences will include a promoter region
which includes a promoter sequence for transcriptional control of
the operably joined gene. Regulatory sequences may also include
enhancer sequences or upstream activator sequences as desired. The
vectors of the invention may optionally include 5' leader or signal
sequences. The choice and design of an appropriate vector is within
the ability and discretion of one of ordinary skill in the art.
[0074] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered by the introduction
into the cells of heterologous DNA (RNA) encoding Smad2 polypeptide
or fragment or variant thereof. That heterologous DNA (RNA) is
placed under operable control of transcriptional elements to permit
the expression of the heterologous DNA in the host cell.
[0075] Preferred systems for mRNA expression in mammalian cells are
those such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.)
that contain a selectable marker such as a gene that confers G418
resistance (which facilitates the selection of stably transfected
cell lines) and the human cytomegalovirus (CMV) enhancer-promoter
sequences. Additionally, suitable for expression in primate or
canine cell lines is the pCEP4 vector (Invitrogen), which contains
an Epstein Barr virus (EBV) origin of replication, facilitating the
maintenance of plasmid as a multicopy extrachromosomal element.
Another expression vector is the pEF-BOS plasmid containing the
promoter of polypeptide Elongation Factor 1a, which stimulates
efficiently transcription in vitro. The plasmid is described by
Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use
in transfection experiments is disclosed by, for example, Demoulin
(Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred
expression vector is an adenovirus, described by
Stratford-Perricaudet, which is defective for E1 and E3 proteins
(J. Clin. Invest. 90:626-630, 1992). The use of the adenovirus as
an Adeno.P1A recombinant is disclosed by Warnier et al., in
intradermal injection in mice for immunization against P1A (Int. J.
Cancer, 67:303-310, 1996).
[0076] The invention also embraces so-called expression kits, which
allow the artisan to prepare a desired expression vector or
vectors. Such expression kits include at least separate portions of
each of the previously discussed coding sequences. Other components
may be added, as desired, as long as the previously mentioned
sequences, which are required, are included.
[0077] The invention also permits the construction of Smad2 mutant
gene "transgenics" in cells and in animals, providing materials for
studying certain aspects of TGF-.beta. signal transduction.
[0078] The invention as described herein has a number of uses, some
of which are described elsewhere herein. First, the invention
permits preparation and isolation of the Smad2 polypeptides with
non-wild-type activities such as reduced or even ablated
phosphorylation. A variety of methodologies well-known to the
skilled practitioner can be utilized to obtain isolated Smad2
molecules. The polypeptide may be purified from cells which
naturally produce the polypeptide by chromatographic means or
immunological recognition. Alternatively, an expression vector may
be introduced into cells to cause production of the polypeptide. In
another method, mRNA transcripts may be microinjected or otherwise
introduced into cells to cause production of the encoded
polypeptide. Translation of mRNA in cell-free extracts such as the
reticulocyte lysate system also may be used to produce polypeptide.
Those skilled in the art also can readily follow known methods for
isolating Smad2 polypeptides. These include, but are not limited
to, immunochromotography, HPLC, size-exclusion chromatography,
ion-exchange chromatography and immune-affinity chromatography.
[0079] The identification of the in vivo phosphorylation sites and
Smad4 binding interface of pathway-restricted Smad polypeptides
such as Smad2 polypeptides also makes it possible for the artisan
to diagnose and modulate disorders characterized by aberrant
TGF-.beta. signaling activity. These methods involve in certain
embodiments contacting the Smad polypeptides of the invention to
cells suspected of having elevated TGF-.beta. signaling. The Smad
polypeptides of the invention act as dominant-negative inhibitors
of TGF-.beta. mediated phosphorylation of wild-type Smad and
binding of pathway-restricted Smad polypeptides such as Smad2
polypeptides and Smad4 and thus can modulate elevated TGF-.beta.
signaling.
[0080] The invention also makes it possible isolate proteins such
as T.beta.R-I and Smad4 by the binding of such proteins to
pathway-restricted Smad polypeptides such as Smad2 polypeptides as
disclosed herein. The identification of this binding also permits
one of skill in the art to block the binding of pathway-restricted
Smad polypeptides such as Smad2 polypeptides to other proteins,
such as T.beta.R-I and Smad4. Binding of the proteins can be
effected by introducing into a biological system in which the
proteins bind (e.g., a cell) a polypeptide including a Smad
T.beta.R-I binding site in an amount sufficient to block the
binding. The identification of a Smad4 binding site in Smad2 also
enables one of skill in the art to prepare modified proteins, using
standard recombinant DNA techniques, which can bind to proteins
such as Smad4.
[0081] The invention further provides methods for reducing or
increasing TGF-.beta. signal transduction in a cell. Such methods
are useful in vitro for altering the TGF-.beta. signal
transduction, for example, in testing compounds for potential to
block aberrant TGF-.beta. signal transduction or increase deficient
TGF-.beta. signal transduction. In vivo, such methods are useful
for modulating growth, e.g., to treat cancer. Increasing TGF-.beta.
signal transduction in a cell by, e.g., introducing a dominant
negative Smad2 polypeptide in the cell, can be used to provide a
model system for testing the effects of putative inhibitors of
TGF-.beta. signal transduction. Such methods also are useful in the
treatment of conditions which result from excessive or deficient
TGF-.beta. signal transduction. TGF-.beta. signal transduction can
be measured by a variety of ways known to one of ordinary skill in
the art, such as the reporter systems described in the Examples.
Various modulators of the activity of pathway-restricted Smad
polypeptides such as Smad2 can be screened for effects on
TGF-.beta. signal transduction using the methods disclosed herein.
The skilled artisan can first determine the modulation of a Smad2
activity, such as TGF-.beta. signaling activity, and then apply
such a modulator to a target cell or subject and assess the effect
on the target cell or subject. For example, in screening for
modulators of Smad2 useful in the treatment of cancer, cells in
culture can be contacted with Smad2 modulators and the increase or
decrease of growth or focus formation of the cells can be
determined according to standard procedures. Smad2 activity
modulators can be assessed for their effects on other TGF-.beta.
signal transduction downstream effects by similar methods in many
cell types.
[0082] The invention also provides, as previously noted, "dominant
negative" polypeptides derived from Smad2. A dominant negative
polypeptide is an inactive variant of a protein, which, by
interacting with the cellular machinery, displaces an active
protein from its interaction with the cellular machinery or
competes with the active protein, thereby reducing the effect of
the active protein. For example, a dominant negative receptor which
binds a ligand but does not transmit a signal in response to
binding of the ligand can reduce the biological effect of
expression of the ligand. Likewise, a dominant negative
catalytically-inactive kinase which interacts normally with target
proteins but does not phosphorylate the target proteins can reduce
phosphorylation of the target proteins in response to a cellular
signal. Similarly, a dominant negative transcription factor which
binds to a promoter site in the control region of a gene but does
not increase gene transcription can reduce the effect of a normal
transcription factor by occupying promoter binding sites without
increasing transcription.
[0083] The end result of the expression of a dominant negative
polypeptide in a cell is a reduction in function of active
proteins. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a protein, and using standard
mutagenesis techniques to create one or more dominant negative
variant polypeptides. For example, given the teachings contained
herein of mutant Smad2 polypeptides, one of ordinary skill in the
art can modify the sequence of the Smad2 polypeptide (SEQ ID NO:2)
by site-specific mutagenesis, scanning mutagenesis, partial gene
deletion or truncation of the Smad2 cDNA, and the like. See, e.g.,
U.S. Pat. No. 5,580,723 and Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. The skilled artisan then can test the population of
mutagenized polypeptides for diminution in a selected activity
and/or for retention of such an activity (e.g., Smad2 reduction of
TGF-.beta. signaling activity). Other similar methods for creating
and testing dominant negative variants of a protein will be
apparent to one of ordinary skill in the art.
[0084] Dominant negative pathway-restricted Smad polypeptides
(e.g., Smad2 polypeptides) include variants in which the in vivo
phosphorylation sites are mutated or in which a portion of the
Smad4 binding site has been mutated or deleted to reduce or
eliminate Smad2 interaction with the Smad4. One of ordinary skill
in the art can readily prepare and test Smad2 mutant and variants
bearing mutations or deletions for diminution of selected
activities.
[0085] The invention also involves agents such as polypeptides
which bind to pathway-restricted Smad polypeptides such as the
Smad2 polypeptides of the invention and to complexes of Smad
polypeptides and binding partners such as T.beta.R-I or Smad4. Such
binding agents can be used, for example, in screening assays to
detect the presence or absence of Smad polypeptides and complexes
of Smad polypeptides and their binding partners and in purification
protocols to isolate Smad polypeptides and complexes of Smad
polypeptides and their binding partners. Such agents also can be
used to inhibit the native activity of the Smad polypeptides or
their binding partners, for example, by binding to such
polypeptides, or their binding partners or both.
[0086] The invention, therefore, embraces peptide binding agents
which, for example, can be antibodies or fragments of antibodies
having the ability to selectively bind to Smad2 polypeptides of the
invention. Antibodies include polyclonal and monoclonal antibodies,
prepared according to conventional methodology (e.g., Kohler and
Milstein, Nature, 256:495, 1975). As exemplified below, preferred
antibodies include antibodies which bind selectively to the
phosphorylated tail of Smad2 (Smad2 phospho tail antibodies). Other
preferred antibodies include similar phospho tail-specific
antibodies for other pathway-restricted Smad proteins (e.g. Smad1,
Smad3, Smad5, Smad9). Included in the foregoing are antibodies
raised against the four amino acid C-terminal motif of
pathway-restricted Smads (SEQ ID NO:5), and antibodies raised
against longer phosphorylated fragments of Smad proteins which
recognized individual Smad proteins (e.g. SEQ ID NO:4 and
corresponding sequences in other pathway-restricted Smads).
[0087] An "antibody" as used herein includes human monoclonal
antibodies or functionally active fragments thereof having human
constant regions and a Smad protein binding CDR3 region from a
mammal of a species other than a human.
[0088] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modem Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0089] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FRI through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDR3). The CDRs, and in particular the CDR3
regions, and more particularly the heavy chain CDR3, are largely
responsible for antibody specificity.
[0090] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. Thus,
for example, PCT International Publication Number WO 92/04381
teaches the production and use of humanized murine RSV antibodies
in which at least a portion of the murine FR regions have been
replaced by FR regions of human origin. Such antibodies, including
fragments of intact antibodies with antigen-binding ability, are
often referred to as "chimeric" antibodies.
[0091] Humanized monoclonal antibodies may be made by any method
known in the art. Humanized monoclonal antibodies, for example, may
be constructed by replacing the non-CDR regions of a non-human
mammalian antibody with similar regions of human antibodies while
retaining the epitopic specificity of the original antibody. For
example, non-human CDRs and optionally some of the framework
regions may be covalently joined to human FR and/or Fc/pFc' regions
to produce a functional antibody. There are entities in the United
States which will synthesize humanized antibodies from specific
murine antibody regions commercially, such as Protein Design Labs
(Mountain View Calif.).
[0092] European Patent Application 0239400, the entire contents of
which is hereby incorporated by reference, provides an exemplary
teaching of the production and use of humanized monoclonal
antibodies in which at least the CDR portion of a murine (or other
non-human mammal) antibody is included in the humanized antibody.
Briefly, the following methods are useful for constructing a
humanized CDR monoclonal antibody including at least a portion of a
mouse CDR. A first replicable expression vector including a
suitable promoter operably linked to a DNA sequence encoding at
least a variable domain of an Ig heavy or light chain and the
variable domain comprising framework regions from a human antibody
and a CDR region of a murine antibody is prepared. Optionally a
second replicable expression vector is prepared which includes a
suitable promoter operably linked to a DNA sequence encoding at
least the variable domain of a complementary human Ig light or
heavy chain respectively. A cell line is then transformed with the
vectors. Preferably the cell line is an immortalized mammalian cell
line of lymphoid origin, such as a myeloma, hybridoma, trioma, or
quadroma cell line, or is a normal lymphoid cell which has been
immortalized by transformation with a virus. The transformed cell
line is then cultured under conditions known to those of skill in
the art to produce the humanized antibody.
[0093] As set forth in European Patent Application 0239400 several
techniques are well known in the art for creating the particular
antibody domains to be inserted into the replicable vector. For
example, the DNA sequence encoding the domain may be prepared by
oligonucleotide synthesis. Alternatively a synthetic gene lacking
the CDR regions in which four framework regions are fused together
with suitable restriction sites at the junctions, such that double
stranded synthetic or restricted subcloned CDR cassettes with
sticky ends could be ligated at the junctions of the framework
regions. Another method involves the preparation of the DNA
sequence encoding the variable CDR containing domain by
oligonucleotide site-directed mutagenesis. Each of these methods is
well known in the art. Therefore, those skilled in the art may
construct humanized antibodies containing a murine CDR region
without destroying the specificity of the antibody for its
epitope.
[0094] In preferred embodiments, the humanized antibodies of the
invention are human monoclonal antibodies including at least the
Smad protein binding CDR3 region of the deposited monoclonal
antibody. As noted above, such humanized antibodies may be produced
in which some or all of the FR regions of deposited monoclonal
antibody have been replaced by homologous human FR regions. In
addition, the Fc portions may be replaced so as to produce IgA or
IgM as well as human IgG antibodies bearing some or all of the CDRs
of the Smad phospho tail antibodies. Of particular importance is
the inclusion of the Smad phospho tail protein binding CDR3 region
and, to a lesser extent, the other CDRs and portions of the
framework regions of the Smad phospho tail antibodies. Such
humanized antibodies will have particular clinical utility in that
they will specifically recognize Smad proteins, particularly
phosphorylated forms, but will not evoke an immune response in
humans against the antibody itself.
[0095] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab').sub.2, Fab, Fv
and Fd fragments; chimeric antibodies in which the Fc and/or FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric
F(ab').sub.2 fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences.
[0096] As used herein the term "functionally active antibody
fragment" means a fragment of an antibody molecule including a Smad
protein binding region of the invention which retains the Smad
binding functionality of an intact antibody having the same
specificity as the antibodies disclosed herein. Such fragments are
also well known in the art and are regularly employed both in vitro
and in vivo. In particular, well-known functionally active antibody
fragments include but are not limited to F(ab').sub.2, Fab, Fv and
Fd fragments of antibodies. These fragments which lack the Fc
fragment of intact antibody, clear more rapidly from the
circulation, and may have less non-specific tissue binding than an
intact antibody (Wahl et al., J Nucl. Med. 24:316-325 (1983)). For
example, single-chain antibodies can be constructed in accordance
with the methods described in U.S. Pat. No. 4,946,778 to Ladner et
al. Such single-chain antibodies include the variable regions of
the light and heavy chains joined by a flexible linker moiety.
Methods for obtaining a single domain antibody ("Fd") which
comprises an isolated variable heavy chain single domain, also have
been reported (see, for example, Ward et al., Nature 341:644-646
(1989), disclosing a method of screening to identify an antibody
heavy chain variable region (V.sub.H single domain antibody) with
sufficient affinity for its target epitope to bind thereto in
isolated form). Methods for making recombinant Fv fragments based
on known antibody heavy chain and light chain variable region
sequences are known in the art and have been described, e.g., Moore
et al., US Pat. No. 4,462,334. Other references describing the use
and generation of antibody fragments include e.g., Fab fragments
(Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier,
Amsterdam, 1985)), Fv fragments (Hochman et al., Biochemistry 12:
1130 (1973); Sharon et al., Biochemistry 15: 1591 (1976); Ehrilch
et al., U.S. Pat. No. 4,355,023) and portions of antibody molecules
(Audilore-Hargreaves, U.S. Pat. No. 4,470,925). Thus, those skilled
in the art may construct antibody fragments from various portions
of intact antibodies without destroying the specificity of the
antibodies for the Smad protein epitope.
[0097] Functionally active antibody fragments also encompass
"humanized antibody fragments." As one skilled in the art will
recognize, such fragments could be prepared by traditional
enzymatic cleavage of intact humanized antibodies. If, however,
intact antibodies are not susceptible to such cleavage, because of
the nature of the construction involved, the noted constructions
can be prepared with immunoglobulin fragments used as the starting
materials; or, if recombinant techniques are used, the DNA
sequences, themselves, can be tailored to encode the desired
"fragment" which, when expressed, can be combined in vivo or in
vitro, by chemical or biological means, to prepare the final
desired intact immunoglobulin fragment.
[0098] Thus, the invention in certain aspects involves polypeptides
of numerous size and type that bind specifically to
pathway-restricted Smad polypeptides (including Smad4 binding
polypeptides), and complexes of Smad polypeptides and their binding
partners. These polypeptides may be derived also from sources other
than antibody technology. For example, such polypeptide binding
agents can be provided by degenerate peptide libraries which can be
readily prepared in solution, in immobilized form or as phage
display libraries. Combinatorial libraries also can be synthesized
of peptides containing one or more amino acids. Libraries further
can be synthesized of peptoids and non-peptide synthetic
moieties.
[0099] Phage display can be particularly effective in identifying
binding peptides useful according to the invention, e.g. those
which bind to phosphorylated portions of pathway restricted Smad
polypeptides, particularly the C-terminal amino acid motif set
forth in SEQ ID NO:5, with or without additional amino acids to
confer additional specificity to antibody binding. Briefly, one
prepares a phage library (using e.g. m13, fd, or lambda phage),
displaying inserts from 4 to about 80 amino acid residues using
conventional procedures. The inserts may represent, for example, a
completely degenerate or biased array. One then can select
phage-bearing inserts which bind to the Smad2 polypeptide, e.g.
having one or more phosphorylated serines in the C-terminal tail,
or other similarly phosphorylated pathway-restricted Smad
polypeptides. This process can be repeated through several cycles
of reselection of phage that bind to the Smad polypeptide. Repeated
rounds lead to enrichment of phage bearing particular sequences.
DNA sequence analysis can be conducted to identify the sequences of
the expressed polypeptides. The minimal linear portion of the
sequence that binds to the Smad polypeptide can be determined. One
can repeat the procedure using a biased library containing inserts
containing part or all of the minimal linear portion plus one or
more additional degenerate residues upstream or downstream thereof.
Yeast two-hybrid screening methods also may be used to identify
polypeptides that bind to the Smad polypeptides. Thus, the Smad2
polypeptides of the invention, other phosphorylated
pathway-restricted Smads and fragments thereof, can be used to
screen peptide libraries, including phage display libraries, to
identify and select peptide binding partners of the Smad
polypeptides of the invention. Such molecules can be used, as
described, for screening assays, for purification protocols, for
interfering directly with the functioning of Smad polypeptides and
for other purposes that will be apparent to those of ordinary skill
in the art.
[0100] A Smad2 polypeptide, or a fragment thereof, also can be used
to isolate the native binding partners, including, e.g., the
TGF-.beta. receptor complex and Smad4. Isolation of such binding
partners may be performed according to well-known methods. For
example, isolated Smad2 polypeptides can be attached to a substrate
(e.g., chromatographic media, such as polystyrene beads, or a
filter), and then a solution suspected of containing the TGF-.beta.
receptor complex may be applied to the substrate. If a TGF-.beta.
receptor complex which can interact with Smad2 polypeptides is
present in the solution, then it will bind to the substrate-bound
Smad2 polypeptide. The TGF-.beta. receptor complex then may be
isolated. Other proteins which are binding partners for Smad2, such
as other Smads or activin receptor complexes, may be isolated by
similar methods without undue experimentation.
[0101] It will also be recognized that the invention embraces the
use of the Smad2 cDNA sequences in expression vectors, as well as
to transfect host cells and cell lines, be these prokaryotic (e.g.,
E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast
expression systems and recombinant baculovirus expression in insect
cells). Especially useful are mammalian cells such as human, mouse,
hamster, pig, goat, primate, etc. They may be of a wide variety of
tissue types, and include primary cells and cell lines. Specific
examples include keratinocytes, peripheral blood leukocytes, bone
marrow stem cells and embryonic stem cells. The expression vectors
require that the pertinent sequence, i.e., those nucleic acids
described supra, be operably linked to a promoter.
[0102] When administered, the therapeutic compositions of the
present invention are administered in pharmaceutically acceptable
preparations. Such preparations may routinely contain
pharmaceutically acceptable concentrations of salt, buffering
agents, preservatives, compatible carriers, supplementary immune
potentiating agents such as adjuvants and cytokines and optionally
other therapeutic agents.
[0103] The therapeutics of the invention can be administered by any
conventional route, including injection or by gradual infusion over
time. The administration may, for example, be oral, intravenous,
intraperitoneal, intramuscular, intracavity, subcutaneous, or
transdermal. When antibodies are used therapeutically, a preferred
route of administration is by pulmonary aerosol. Techniques for
preparing aerosol delivery systems containing antibodies are well
known to those of skill in the art. Generally, such systems should
utilize components which will not significantly impair the
biological properties of the antibodies, such as the paratope
binding capacity (see, for example, Sciarra and Cutie, "Aerosols,"
in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp
1694-1712; incorporated by reference). Those of skill in the art
can readily determine the various parameters and conditions for
producing antibody aerosols without resort to undue
experimentation. When using antisense preparations of the
invention, slow intravenous administration is preferred.
[0104] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
[0105] The preparations of the invention are administered in
effective amounts. An effective amount is that amount of a
pharmaceutical preparation that alone, or together with further
doses, produces the desired response. In the case of treating
cancer, the desired response is inhibiting the progression of the
cancer. This may involve only slowing the progression of the
disease temporarily, although more preferably, it involves halting
the progression of the disease permanently. This can be monitored
by routine methods or can be monitored according to diagnostic
methods of the invention discussed herein.
[0106] The invention also contemplates gene therapy. The procedure
for performing ex vivo gene therapy is outlined in U.S. Pat. No.
5,399,346 and in exhibits submitted in the file history of that
patent, all of which are publicly available documents. In general,
it involves introduction in vitro of a functional copy of a gene
into a cell(s) of a subject which contains a defective copy of the
gene, and returning the genetically engineered cell(s) to the
subject. The functional copy of the gene is under operable control
of regulatory elements which permit expression of the gene in the
genetically engineered cell(s). Numerous transfection and
transduction techniques as well as appropriate expression vectors
are well known to those of ordinary skill in the art, some of which
are described in PCT application WO95/00654. In vivo gene therapy
using vectors such as adenovirus, retroviruses, herpes virus, and
targeted liposomes also is contemplated according to the
invention.
[0107] The invention further provides efficient methods of
identifying pharmacological agents or lead compounds for agents
active at the level of a cellular function modulatable by
apathway-restricted Smad polypeptide or fragment such as a Smad2
polypeptide or fragment thereof. In particular, such functions
include TGF-.beta. superfamily signal transduction and formation of
TGF-.beta. superfamily receptor-Smad2 and Smad2-Smad4 protein
complexes. Generally, the screening methods involve assaying for
compounds which interfere with a Smad activity such as TGF-.beta.
superfamily receptor-Smad binding, etc. Such methods are adaptable
to automated, high throughput screening of compounds. The target
therapeutic indications for pharmacological agents detected by the
screening methods are limited only in that the target cellular
function be subject to modulation by alteration of the formation of
a complex comprising a pathway-restricted Smad polypeptide such as
a Smad2 polypeptide of the invention or fragment thereof and one or
more natural Smad intracellular binding targets, such as a
TGF-.beta. superfamily receptor. Target indications include
cellular processes modulated by TGF-.beta., BMP and/or activin
signal transduction following receptor-ligand binding.
[0108] A wide variety of assays for pharmacological agents are
provided, including, labeled in vitro protein-protein binding
assays, electrophoretic mobility shift assays, immunoassays,
cell-based assays such as two- or three-hybrid screens, expression
assays, etc. For example, three-hybrid screens are used to rapidly
examine the effect of transfected nucleic acids on the
intracellular binding of pathway-restricted Smad or Smad fragments
to specific intracellular targets. The transfected nucleic acids
can encode, for example, combinatorial peptide libraries or
antisense molecules. Convenient reagents for such assays, e.g.,
GAL4 fusion proteins, are known in the art. An exemplary cell-based
assay involves transfecting a cell with a nucleic acid encoding a
Smad2 polypeptide fused to a GAL4 DNA binding domain and a nucleic
acid encoding a Smad4 domain which interacts with Smad2 fused to a
transcription activation domain such as VP16. The cell also
contains a reporter gene operably linked to a gene expression
regulatory region, such as one or more GAL4 binding sites.
Activation of reporter gene transcription occurs when the Smad2 and
Smad4 fusion polypeptides bind such that the GALA DNA binding
domain and the VP16 transcriptional activation domain are brought
into proximity to enable transcription of the reporter gene. Agents
which modulate a Smad2 polypeptide mediated cell function are then
detected through a change in the expression of reporter gene.
Methods for determining changes in the expression of a reporter
gene are known in the art.
[0109] Pathway-restricted Smad fragments used in the methods, when
not produced by a transfected nucleic acid are added to an assay
mixture as an isolated polypeptide. Pathway-restricted Smad
polypeptides such as Smad2 polypeptides preferably are produced
recombinantly, although such polypeptides may be isolated from
biological extracts. Recombinantly produced Smad polypeptides
include chimeric proteins comprising a fusion of a Smad protein
with another polypeptide, e.g., a polypeptide capable of providing
or enhancing protein-protein binding, sequence specific nucleic
acid binding (such as GAL4), enhancing stability of the Smad
polypeptide under assay conditions, or providing a detectable
moiety, such as green fluorescent protein or Flag epitope as
provided in the examples below.
[0110] The assay mixture is comprised of a natural intracellular
Smad binding target such as a TGF-.beta. superfamily receptor or
fragment thereof capable of interacting with a Smad polypeptide.
While natural Smad binding targets may be used, it is frequently
preferred to use portions (e.g., peptides or nucleic acid
fragments) or analogs (i.e., agents which mimic the Smad binding
properties of the natural binding target for purposes of the assay)
of the Smad binding target so long as the portion or analog
provides binding affinity and avidity to the Smad fragment
measurable in the assay.
[0111] The assay mixture also comprises a candidate pharmacological
agent. Typically, a plurality of assay mixtures are run in parallel
with different agent concentrations to obtain a different response
to the various concentrations. Typically, one of these
concentrations serves as a negative control, i.e., at zero
concentration of agent or at a concentration of agent below the
limits of assay detection. Candidate agents encompass numerous
chemical classes, although typically they are organic compounds.
Preferably, the candidate pharmacological agents are small organic
compounds, i.e., those having a molecular weight of more than 50
yet less than about 2500, preferably less than about 1000 and, more
preferably, less than about 500. Candidate agents comprise
functional chemical groups necessary for structural interactions
with polypeptides and/or nucleic acids, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups and more preferably at
least three of the functional chemical groups. The candidate agents
can comprise cyclic carbon or heterocyclic structure and/or
aromatic or polyaromatic structures substituted with one or more of
the above-identified functional groups. Candidate agents also can
be biomolecules such as peptides, saccharides, fatty acids,
sterols, isoprenoids, purines, pyrimidines, derivatives or
structural analogs of the above, or combinations thereof and the
like. Where the agent is a nucleic acid, the agent typically is a
DNA or RNA molecule, although modified nucleic acids as defined
herein are also contemplated.
[0112] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides, synthetic organic
combinatorial libraries, phage display libraries of random
peptides, and the like. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant and animal
extracts are available or readily produced. Additionally, natural
and synthetically produced libraries and compounds can be readily
be modified through conventional chemical, physical, and
biochemical means. Further, known pharmacological agents may be
subjected to directed or random chemical modifications such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs of the agents.
[0113] A variety of other reagents also can be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-nucleic acid
binding. Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease, inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
[0114] The mixture of the foregoing assay materials is incubated
under conditions whereby, but for the presence of the candidate
pharmacological agent, the Smad2 polypeptide specifically binds the
cellular binding target, a portion thereof or analog thereof. The
order of addition of components, incubation temperature, time of
incubation, and other perimeters of the assay may be readily
determined. Such experimentation merely involves optimization of
the assay parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4 C and 40 C.
Incubation times preferably are minimized to facilitate rapid, high
throughput screening, and typically are between 0.1 and 10
hours.
[0115] After incubation, the presence or absence of specific
binding between the Smad2 polypeptide and one or more binding
targets is detected by any convenient method available to the user.
For cell free binding type assays, a separation step is often used
to separate bound from unbound components. The separation step may
be accomplished in a variety of ways. Conveniently, at least one of
the components is immobilized on a solid substrate, from which the
unbound components may be easily separated. The solid substrate can
be made of a wide variety of materials and in a wide variety of
shapes, e.g., microtiter plate, microbead, dipstick, resin
particle, etc. The substrate preferably is chosen to maximum signal
to noise ratios, primarily to minimize background binding, as well
as for ease of separation and cost.
[0116] Separation may be effected for example, by removing a bead
or dipstick from a reservoir, emptying or diluting a reservoir such
as a microtiter plate well, rinsing a bead, particle,
chromotograpic column or filter with a wash solution or solvent.
The separation step preferably includes multiple rinses or washes.
For example, when the solid substrate is a microtiter plate, the
wells may be washed several times with a washing solution, which
typically includes those components of the incubation mixture that
do not participate in specific bindings such as salts, buffer,
detergent, non-specific protein, etc. Where the solid substrate is
a magnetic bead, the beads may be washed one or more times with a
washing solution and isolated using a magnet.
[0117] Detection may be effected in any convenient way for
cell-based assays such as two- or three-hybrid screens. The
transcript resulting from a reporter gene transcription assay of
Smad2 polypeptide interacting with a target molecule typically
encodes a directly or indirectly detectable product, e.g.,
.beta.-galactosidase activity, luciferase activity, and the like.
For cell free binding assays, one of the components usually
comprises, or is coupled to, a detectable label. A wide variety of
labels can be used, such as those that provide direct detection
(e.g., radioactivity, luminescence, optical or electron density,
etc). or indirect detection (e.g., epitope tag such as the FLAG
epitope, enzyme tag such as horseseradish peroxidase, etc.). The
label may be bound to a Smad binding partner, or incorporated into
the structure of the binding partner.
[0118] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels may be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates, strepavidin-biotin
conjugates, etc. Methods for detecting the labels are well known in
the art.
[0119] The invention provides Smad-specific binding agents, methods
of identifying and making such agents, and their use in diagnosis,
therapy and pharmaceutical development. For example, Smad2-specific
pharmacological agents are useful in a variety of diagnostic and
therapeutic applications, especially where disease or disease
prognosis is associated with improper utilization of a pathway
involving Smad2, e.g., TGF-.beta. induced phosphorylation of Smad2,
Smad4-Smad2 complex formation, etc. Novel Smad-specific binding
agents include pathway restricted Smad-specific antibodies (e.g.
phospho tail antibodies) and other natural intracellular binding
agents identified with assays such as two hybrid screens, and
non-natural intracellular binding agents identified in screens of
chemical libraries and the like.
[0120] In general, the specificity of Smad2 binding to a binding
agent is shown by binding equilibrium constants. Targets which are
capable of selectively binding a Smad polypeptide preferably have
binding equilibrium constants of at least about 10.sup.7 M.sup.-1,
more preferably at least about 10.sup.8 M.sup.-1, and most
preferably at least about 10.sup.9 M.sup.-1. The wide variety of
cell based and cell free assays may be used to demonstrate
pathway-restricted Smad-specific binding. Cell based assays include
one, two and three hybrid screens, assays in which Smad-mediated
transcription is inhibited or increased, etc. Cell free assays
include Smad-protein binding assays, immunoassays, etc. Other
assays useful for screening agents which bind pathway-restricted
Smad polypeptides such as Smad2 polypeptides include fluorescence
resonance energy transfer (FRET), and electrophoretic mobility
shift analysis (EMSA).
[0121] Various techniques may be employed for introducing nucleic
acids of the invention into cells, depending on whether the nucleic
acids are introduced in vitro or in vivo in a host. Such techniques
include transfection of nucleic acid-CaPO.sub.4 precipitates,
transfection of nucleic acids associated with DEAE, transfection
with a retrovirus including the nucleic acid of interest, liposome
mediated transfection, and the like. For certain uses, it is
preferred to target the nucleic acid to particular cells. In such
instances, a vehicle used for delivering a nucleic acid of the
invention into a cell (e.g., a retrovirus, or other virus; a
liposome) can have a targeting molecule attached thereto. For
example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell can be bound to or incorporated within the nucleic
acid delivery vehicle. For example, where liposomes are employed to
deliver the nucleic acids of the invention, proteins which bind to
a surface membrane protein associated with endocytosis may be
incorporated into the liposome formulation for targeting and/or to
facilitate uptake. Such proteins include capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular half
life, and the like. Polymeric delivery systems also have been used
successfully to deliver nucleic acids into cells, as is known by
those skilled in the art. Such systems even permit oral delivery of
nucleic acids.
EXAMPLES
[0122] Materials and Methods
[0123] Cell Lines
[0124] COS-1 cells and mink lung epithelial (Mv1Lu) cells were
obtained from American Type Culture Collection. Cells were cultured
in DMEM (GIBCO-BRL) with 10% fetal bovine serum, 100 units/ml of
penicillin and 50 .mu.g/ml of streptomycin.
[0125] Constructs and Cell Transfection
[0126] Expression of plasmids for T.beta.R-I, T.beta.R-II and Smad2
were previously described (ten Dijke et al., Science, 264:101-104,
1994; Nakao et al., 1996). Smad2 mutants were made by a polymerase
chain reaction (PCR)-directed approach and subcloned in pcDNA3
vector. Mutations and sequences of the exchanged restriction
fragments in Smad2 cDNA were confirmed by DNA sequencing. Transient
transfections were performed using a DEAE-dextran protocol, as
described (Crcamo et al., Mol. Cell. Biol., 14:3810-3821,
1994).
[0127] [.sup.32P]orthophosphate Labeling, Tryptic Phosphopeptide
Mapping, Two-Dimensional Phosphoamino Acid Analysis and Automated
Edman Degradation
[0128] [.sup.32P]orthophosphate labeling, tryptic phosphopeptide
mapping, two-dimensional phosphoamino acid analysis and automated
Edman degradation were performed as previously described
(Souchelnytskyi et al., EMBO J, 15:6231-6240, 1996). In brief,
subconfluent cells were labeled in phosphate-free medium containing
0.5% dialyzed FCS, 20 mM HEPES, pH 7.2, and
[.sup.32P]orthophosphate (1.0 mCi/ml). After stimulation with
TGF-.beta.I for 60 min, the cells were washed and lysed in lysis
buffer (20 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.5% Triton X-100, 5 mM
NaF, 10 mM Na.sub.4P.sub.2O.sub.7, 1 mM Na.sub.3O.sub.4, 1 mM
phenylmethylsulphonyl fluoride (PMSF), 100 U/ml aprotinin). The
cell lysates were subjected to immunoprecipitation using the SED
antisera against Smad2 (SED; Nakao et al., 1996);
immunoprecipitates were then subjected to SDS-PAGE and transferred
to a nitrocellulose membrane. The phosphorylated Smad2 proteins
were excised from the filter and digested in situ with trypsin
(modified sequencing grade; Promega). Two-dimensional
phosphopeptide mapping was done using the Hunter thin-layer
electrophoresis (HTLE-7000; CBS Scientific), essentially as
described by Boyle et al. (Methods Enzymol., 201:110-149, 1991).
First dimension electrophoresis was performed in pH 1.9 buffer
(formic acid/glacial acid/water; 44:156:1800; v/v/v) for 27 min at
2000 V, and chromatography in the second dimension in isobutyric
acid/n-butanol/pyridine/glacial acetic acid/water (1250:38:96: 58:
558; v/v/v/v/v). After exposure, phosphopeptides were eluted from
the plates in pH 1.9 buffer and lyophilized; aliquots of the
samples were then subjected to two-dimensional phosphoamino acid
analysis and automated Edman degradation in parallel. For
radiochemical sequencing, the eluted phosphopeptides were coupled
to Sequelon-AA membrane (Millipore) by use of carbodiimide
coupling, according to standard procedures as described by the
manufacturer, and Edman degradation was performed using an Applied
Biosystems sequencer (Model 477A). Released phenylthiohydantoin
amino acid-derivatives from each cycle were spotted onto thin-layer
chromatography plates. The radioactivity in each spot was
quantitated by exposure on a FujiX Bio-Imager.
[0129] Iodination of TGF-.beta.I and Affinity Cross-Linking
[0130] TGF-.beta. was iodinated using the chloramine T method
according to Frolik et al. (J. Biol. Chem., 259:10995-11000, 1984).
Cross-linking was performed as previously described (Franzn et al.,
Cell, 75:681-692, 1993). Complexes of Smad2 and TGF-.beta.
receptors, cross-linked with .sup.125I-TGF-.beta., were
immunoprecipitated with Smad2 antiserum (SED; Nakao et al., 1996)
and subjected to SDS-PAGE. Gels were dried and exposed on a FujiX
Bio-Imager. To determine equality of T.beta.R's expression,
aliquots of cell lysates were subjected to immunoprecipitation
using anti-T.beta.R-I antibodies, and analyzed by SDS-PAGE and
autoradiography as previously described (Franzn et al., 1993). To
determine the expression of Smad proteins, cell lysates were
separated by SDS-PAGE, electrotransferred to nitrocellulose
membrane, immunoblotted with the Smad2 antiserum and developed
using an enhanced chemiluminescence detection system
(Amersham).
[0131] Transcriptional Response Assay
[0132] Mv1Lu cells were transiently transfected with p3TP-Lux
(Crcamo et al., Mol. Cell. Biol., 15:1573-1581, 1995) in the
absence or presence of Smad2 expression plasmids by using the
DEAE-dextran method. After transfection cells were incubated for 24
h in DMEM with 10% FBS, and then incubated with 0.1% FBS for 5 h,
after which TGF-.beta.1 was added. Luciferase activity in the cell
lysate was measured after 22-24 h using the luciferase assay system
(Promega Biotech), according to the manufacturer's protocol using
an LKB Luminometer (LKB Bromma).
[0133] Peptide Synthesis and Coupling to Solid Support
[0134] The peptides KKKSSMS (SEQ ID NO:6) and KKKYTQMGSPSVRCSSMS
(SEQ ID NO:7) were synthesized using an Applied Biosystems peptide
synthesizer (model ABI 430A) by Fmoc chemistry. The corresponding
peptides with the two most C-terminal serine residues
phosphorylated were synthesized using phosphorylated F-moc serine
residue derivatives during synthesis. The peptides were analyzed by
plasma desorption mass spectrometry using an Applied Biosystems Bio
Ion 20 instrument. Peptide fractions were freeze-dried and stored
under dry conditions. Peptides were coupled to activated CNBr
Sepharose 4B (Pharmacia) through primary amino groups according to
the manufacturer's protocol; the three lysines at the N-terminus
were included to facilitate efficient coupling. The efficiency of
coupling was determined by measuring the OD.sub.280 (or OD.sub.215
for short peptides) of the peptide solution before and after
coupling. The coupling efficiencies of all four peptides were
nearly 100%.
[0135] Preparation of GST-Fusion Proteins
[0136] cDNA for Smads were cloned into pGEX vectors (Pharmacia
LKB), and fusion proteins were prepared and absorbed essentially as
described (Smith and Johnson, Gene, 63:31-40, 1988).
[0137] Association of Phosphorylated and Nonphosphorylated Smad2
peptides with GST Fusion Proteins
[0138] Phosphorylated and nonphosphorylated Smad2-derived
C-terminal peptides were incubated with GST-Smad fusion proteins in
phosphate buffered saline containing 1% Triton (PBS-T) with 0.1%
BSA overnight at 4.degree. C. After washing five times with PBS-T
with 0.1% BSA and twice with PBS-T, the samples were subjected to
SDS-PAGE. Proteins were electrotransferred to nitrocellulose
membrane and immunoblotted with GST antiserum (gift from Aino
Ruusala) and developed using an enhanced chemiluminescence
detection system (Amersham).
Example 1
Mapping of in vivo Phosphorylation Sites in Smad2
[0139] TGF-.beta. receptor activation leads to phosphorylation of
Smad2 on serine and threonine residues (Eppert et al., 1996;
Macas-Silva et al., 1996; Nakao et al., 1996). In order to localize
the phosphorylated residues in Smad2, two-dimensional tryptic
phosphopeptide mapping was performed. Untransfected Mv1Lu cells,
Mv1Lu cells transiently transfected with Smad2 alone, and COS-1
cells transiently transfected with Smad2 together with T.beta.R-I
and T.beta.R-II, were labeled with [.sup.32P]orthophosphate and
incubated with or without TGF-.beta.1. Thereafter, cell lysates
were subjected to immunoprecipitation using a Smad2 antiserum,
followed by SDS-PAGE and autoradiography. Plates were exposed and
analyzed by using a FujiX Bio-Imager. Sample application points are
shown by small black squares. In the absence of TGF-.beta., Smad2
was only phosphorylated at low stoichiometry; phosphorylation was
dramatically enhanced after ligand-stimulation (FIG. 1A).
[0140] The Smad2 phosphoproteins of the experiment shown in FIG. 1A
were transferred to a membrane, cut out (from lanes 3 (B), 4 (C), 5
(E) and 6 (F)) and subjected to tryptic digestion. After digestion
with trypsin, peptides were resolved by high-voltage
electrophoresis and thin-layer chromatography. Analysis of the
two-dimensional maps of .sup.32P-labeled phosphopeptides revealed
about 16 spots of different intensities (FIG. 1B, C, D). In the
absence of ligand-stimulation, a broad smear (spot 18) and few
faint spots were seen. The abundantly phosphorylated peptide (spot
18) was immunoprecipitated with antiserum raised against a peptide
from a sequence in a large tryptic peptide that covers almost
completely the proline-rich linker sequence between the MH1 and MH2
domains. This phosphopeptide contained phosphoserine as well as
phosphothreonine.
[0141] Stimulation by TGF-.beta. led to the appearance and
induction of multiple spots; in particular, seven highly negatively
charged phosphopeptides were seen (spots 5, 6, 6n, 12a, 12b, 12c
and 15). Phosphopeptide maps of endogenous and overexpressed Smad2
in Mv1Lu cells were identical, albeit signal intensity was higher
on maps of overexpressed Smad2. Overexpression of Smad2 with
T.beta.R-I and T.beta.R-II in COS-1 cells and incubation with
ligand also led to stimulation of Smad2 phosphorylation (FIG. 1A).
Tryptic phosphopeptide maps of Smad2 phosphorylated in vivo in
these cells revealed a nearly identical pattern as compared to
phosphorylated Smad2 from Mv1Lu cells (FIG. 1E, F, G). Phosphoamino
acid analysis of spots 1 through 18, revealed that all were
phosphorylated on serine resides except for spot 11 and 18 that
contained both phosphoserine and phosphothreonine residues (data
not shown). None of the spots contained phosphotyrosine.
[0142] The phosphopeptide corresponding to spot 15 in FIG. 1F was
subjected to phosphoamino acid analysis. The migration of
phosphorylated serine (Sp), threonine (Tp) and tyrosine (Yp), used
as standards, is shown. To identify the positions of phosphorylated
residues in the phosphopeptides, the release of radioactivity upon
Edman degradation of peptides extracted from the two-dimensional
chromatography plate was determined. The elution positions of
.sup.32P-labeled amino acids are shown and aligned to the sequence
of the single possible Smad2-derived tryptic peptide containing
serine residues in the third and fifth position. The peptide
corresponding to spot 15, which contained only phosphoserine,
yielded .sup.32P-radioactivity release in the third and fifth
cycles, with some trailing in the following cycles which is
characteristic for this method. This indicates that Ser465 and
Ser467 in the C-terminus of Smad2 were phosphorylated, since the
corresponding tryptic peptide is the only one in the Smad2 with
serine residues in position three and five FIG. 2A, B). The
carboxy-terminal tryptic peptide also contained another
phosphorylatable amino acid, i.e., Ser464. However, radiochemical
sequencing and phosphoamino acid analysis did not reveal
phosphoserine at position two in phosphopeptides with the migration
position expected if Ser464 would have been phosphorylated alone or
in combinations with Ser465 and Ser467. Thus, Ser464 in Smad2
appears not to be phosphorylated in response to
TGF-.beta.-stimulation.
[0143] In order to confirm that Ser465 and Ser467 are
phosphorylation sites in Smad2, these residues and the neighboring
Ser464 were mutated to alanine residues singly and in combinations.
Wild-type Smad2 (W.T.), as well as Smad2/S464A (S464A),
Smad2/S465,467A (S465,467A) and Smad2/S464,465,467A (S464,465,467A)
mutants were subjected to two-dimensional phosphopeptide mapping.
COS-1 cells were transiently transfected with Smad2 or Smad2
mutants together with T.beta.R-I and T.beta.R-II, and were labeled
with [.sup.32P]orthophosphate. After treatment with TGF-.beta.,
wild-type Smad2 or Smad2 mutants were immunoprecipitated and
subjected to two-dimensional tryptic phosphopeptide mapping. Sample
application points are shown by black squares. The arrows show the
migration position of the Ser465 and Ser467 containing tryptic
peptide. Analysis of two-dimensional tryptic phosphopeptide maps of
wild-type and mutant Smad2 from .sup.32P-labeled
TGF-.beta.-stimulated COS-1 cells, revealed that spot 15,
corresponding to a peptide with Ser465 and Ser467 phosphorylated,
was not seen in the phosphopeptide map of the Smad2/S465A mutant
contained spot 15; the map of the Smad2S454A mutant was in fact
identical to that of wild-type Smad2. Moreover, the phosphopeptide
map of the triple Smad2/S464, 465, 467A mutant also lacked spot 15,
and was identical to that of the Smad2/S465, 467A mutant. Thus,
Ser465 and Ser467 are in vivo phosphorylation sites in Smad2.
[0144] FIG. 2D shows a schematic illustration of Smad2 and
C-terminal sequences of Smad1, Smad2, Smad3 and Smad5. Conserved
residues are boxed. The C-terminal motif SS(M/V)S (SEQ ID NO: 5) is
found in Smad1, Smad2, Smad3, Smad5 and Smad9, and therefore, it is
possible that all these Smads are direct substrates for their
appropriate activated receptors. In accordance with this
possibility a phosphopeptide map of Smad3 from
TGF-.beta.-stimulated cells revealed phosphopeptides with similar
migration as spot 15 of Smad2; no such spot was found in a map of
Smad4, which lacks the C-terminal SS(M/V)S motif.
Example 2
Phosphorylation of Ser465 Requires that Ser467 is
Phosphorylated
[0145] Since Ser465 and Ser467 are located in close proximity of
each other, the possibility that they are phosphorylated
sequentially was examined. T.beta.R-I-mediated phosphorylation of
Smad2 mutants in which Ser465 or Ser467 were converted to alanine
or aspartic acid residues was characterized in transfected COS-1
cells. Two-dimensional tryptic phosphopeptide mapping of wild-type
Smad2 and Smad2/S465A, Smad2/S465D, Smad2/S467A and Smad2/S467D
mutants after co-expression with TGF-.beta. receptors and
stimulation with TGF-.beta., performed as described above, showed
loss of spot 15 in all cases. The arrows indicate the migration
position of the C-terminal tryptic peptides, which is absent on the
map of the Smad2/S467A mutant. Notably, a new spot appeared in the
maps of the Smad2/S465A, Smad2/S465D and Smad2/S467D mutants, but
not in the map of the Smad2/S467A mutant (FIG. 3A). The new peptide
had a shorter migration distance in electrophoresis at pH 1.9
compared to that of spot 15, which is in agreement with a lower
degree of phosphorylation of the novel phosphopeptide and suggested
that it was phosphorylated at one rather than two residues. Edman
degradation elution profiles of the new phosphopeptides appeared on
maps of the Smad2/S465A and Smad2/S467D mutants are shown in FIG.
3B. The .sup.32P-radioactivity released in each cycle was measured.
The amino acid sequences of the C-terminals of the Smad2 mutants
are presented along with the fraction numbers. When radiochemical
sequencing of the novel spot from the map of the Smad2/S465A mutant
was performed, radioactivity eluted at the fifth cycle, which is
consistent with the expected phosphorylation of the C-terminal
tryptic peptide at Ser467 after mutation of Ser465. The fact that
the new spot was not seen in the map of the Smad2/S467A mutant
suggests that phosphorylation of Ser465 requires phosphorylation of
Ser467. The finding that the map of a Smad2/S467D mutant showed the
novel spot, suggested that introduction of a negative charge at
position 467 could rescue the phosphorylation of Ser465.
Radiochemical sequencing of the spot revealed that the peak of
radioactivity eluted at the third cycle. This result indicates that
Ser465 was phosphorylated in the C-terminal peptide of the
Smad2/S467D mutant, and suggests that the requirement for
phosphorylation at Ser467 was bypassed by the introduction of a
negative charge at this residue.
Example 3
Mutation of Ser464, Ser465 and/or Ser467 in Smad2 Interferes with
TGF-.beta.-Mediated Signaling
[0146] To reveal the importance of Ser464, Ser465 and Ser467 for
TGF-.beta. signaling, the ability of Smad2 mutants, in which these
residues were replaced with alanine or aspartic acid residues
singly or in combinations, to block the TGF-.beta.-mediated
transcriptional response was measured using a p3TP-Lux reporter
plasmid (FIG. 4). Mv1Lu cells were transiently transfected with
p3TP-Lux plasmid alone or in the presence of Smad2 or Smad2 mutant
expression plasmids. Luciferase activity was determined before
(.box-solid.) or after (.quadrature.) stimulation with 10 ng/ml of
TGF-.beta.1. The values were normalized for transfection efficiency
using the .beta.-gal reporter gene under transcriptional control of
cytomegalovirus promoter. Representative results of three
independent experiments are shown. Consistent with previous
observations (Zhang et al., 1996), no difference in signaling in
the absence or presence of transfected wild-type Smad2 was found,
indicating that level of endogenous Smad2 is sufficient for full
response. Expression of any Smad2 with Ser465 or Ser467 replaced
with alanine residues(s) led to a decrease of the luciferase
signal. Introduction of aspartic acid residues, in order to mimic
the negative charge of the phosphate group, did not rescue the
stimulation of luciferase expression. Notably, Smad2/Ser464A also
acted as a dominant negative inhibitor. However, in some
experiments, the inhibitory effect of this mutant on TGF-.beta.
signaling was less pronounced than those of Smad2/S465A and
Smad2/S467A mutants. Thus, Ser464 and in particular the
phosphorylatable residues Ser465 and Ser467 in Smad2, are required
for TGF-.beta.-induced p3TP-Lux transcriptional response.
Example 4
Association of Wild-Type Smad2 and Smad2 Mutants with
T.beta.R-I
[0147] The interaction of Smad2 with the TGF-.beta. receptor
complex was investigated by co-expression of wild-type Smad2 and
Smad2 mutants with TGF-.beta. receptor sin COS-1 cells (FIG. 5).
COS-1 cells were transfected with wild-type Smad2 (W.T.) or Smad2
mutants in combination with T.beta.R-II and wild-type (W.T.) or
kinase-inactive (K.R.) forms of T.beta.R-I. Constructs for WT and
KR T.beta.R-I tagged at C-terminus with HA epitope were used. The
receptors were affinity cross-linked with .sup.125I-TGF-.beta.1.
Cell lysates were subjected to immunoprecipitation with Smad2
antiserum and analyzed by SDS-PAGE and autoradiography. Migration
position of T.beta.R-I and T.beta.R-II are shown. Smad2-receptor
interaction was determined by the ability of an antiserum against
Smad2 to co-immunoprecipitate the receptors, cross-linked with
.sup.125I-TGF-.beta.1. In accordance with previous results
(Macas-Silva et al., 1996), it was determined that wild-type Smad2
interacted with T.beta.R-I, provided that T.beta.R-I was
kinase-inactive and phosphorylated by T.beta.R-II kinase; in
contrast, a Smad2 mutant with the three C-terminal serines altered
to alanines, was able to bind with high affinity also to activated
wild-type T.beta.R-I. The single and double serine mutants of Smad2
were analyzed in a similar experimental setup for their abilities
to interact with wild-type T.beta.R-I or a kinase-inactive
T.beta.R-I mutant in complex with T.beta.R-II. The double mutant
interacted with T.beta.R-I in a similar manner as the triple
mutant. In addition, the single mutants were also found to interact
with wild-type T.beta.R-I. Interestingly, although Ser464 is not an
in vivo phosphorylation site, Smad2/S464A interacted with
T.beta.R-I as efficiently as the Smad2/S465A and Smad2/S467A
mutants (FIG. 5). T.beta.R-I and T.beta.R-II were expressed at
equal levels, as confirmed by immunoprecipitation of the
crosslinked .sup.125I-TGF-.beta.-receptor complex, followed by
SDS-PAGE and autoradiography; also Smad2 was expressed at equal
levels as determined by immunoblotting of cell lysates, resolved by
SDS-PAGE, with Smad2 antiserum (data not shown). These data are in
concordance with the dominant negative effects of Smad2 and mutated
at Ser464, Ser465 and/or Ser467 on TGF-.beta.-mediated stimulation
of gene expression (FIG. 4), and suggests that the mechanism
involves competition of the Smad2 mutants and endogenous Smad2 for
binding to T.beta.R-I.
Example 5
GST-Smad4 Binds with Higher Affinity to Phosphorylated Peptides
Derived from the C-terminus of Smad2 than to the Corresponding
Non-Phosphorylated Peptides
[0148] To investigate whether phosphorylated Ser465 and Ser467 of
Smad2 are directly involved in heteromeric interaction with Smad4,
phosphorylated and non-phosphorylated peptides corresponding to the
C-terminus of Smad2 were made and tested for their ability to bind
GST-Smad4 fusion proteins. Short C-terminal peptide of Smad2 SSMS
(SNp; SEQ ID NO: 3) or its phosphorylated counterpart (SPp), as
well as long C-terminal-peptide TQMGSPSVRCSSMS (LNp; SEQ ID NO: 4)
or its phosphorylated counterpart (LPp), coupled to CNBr-Sepharose
(peptide beads), were incubated with GST or GST-Smad4 fusion
protein. For competition large excesses (200 .mu.M) of
non-phosphorylated short (SNp) or long (LNp) peptides or their
phosphorylated counterpart (SPp and LPp, respectively) were used
(block). Proteins bound to the beads, were resolved by SDS-PAGE,
transferred to nitrocellulose membrane and immunoblotted with
anti-GST antibodies. Migration positions of GST and GST-Smad4 are
shown by double arrows. Molecular masses of standard proteins are
indicated on the left side on gel in which protein-stained GST and
GST-Smad4 are shown. It was found that Smad4 bound to a doubly
phosphorylated peptide containing the four C-terminal amino acid
residues of Smad2 (SSpMSp SEQ ID NO: 3), but weakly (or not) to the
corresponding non-phosphorylated counterpart (SSMS) (FIG. 6). We
also found that the phosphorylated form of a longer peptide
containing the 14 C-terminal amino acid residues of Smad2
(TQMGSPSVRCSSpMSp SEQ ID NO: 4) bound GST-Smad4 even more
efficiently than the SSpMSp peptide. The phosphorylated long
peptide also bound GST-Smad4 more efficiently, than the
non-phosphorylated counterpart, however, a considerable binding was
observed also to the long non-phosphorylated peptide. This
indicates that interaction between Smad2 and Smad4 is not only
dependent on phosphorylation of Ser465 and Ser467 in Smad2, but
involves also interaction with the sequence upstream of the
phosphorylation sites. The interaction was shown to be specific,
since it was blocked by an excess of short or long phosphorylated
peptides, but not by an excess of an irrelevant phosphopeptide,
phosphoserine, or peptides derived from Smad2; the GST protein
showed no binding to phosphorylated peptides (FIG. 6; and data not
shown).
Example 6
Preparation of Smad2 Phospho Tail Antibody
[0149] Rabbit antisera to TGF-.beta.-receptor-mediated
phosphorylated Smad2 was made against the phosphorylated tail of
Smad2. A three lysine peptide (KKK) was added to the N-terminus of
the amino acids of SEQ ID NO:3 to facilitate coupling to carrier
protein. The resulting peptide, KKKSSMS (SEQ ID NO:6), in which
underlined serines (amino acids 5 and 7, equivalent to Ser465 and
Ser467 of Smad2) are phosphoserines, was coupled to keyhole limpet
hemocyanin with glutaraldehyde, mixed with Freund's adjuvant, and
used to immunize rabbits according to standard protocols for
preparation of polyclonal antibodies. Blood was drawn from
immunized rabbits and antisera prepared according to standard
procedures. Characterization of the phosphoserine specific antisera
(referred to as Smad2 phospho tail antibody) is described in the
following examples. Additional Smad2 phosphoserine specific
antisera are prepared as described above, using longer Smad2
peptides such as a 14 mer peptide (SEQ ID NO:4), 8 mer peptides
corresponding to amino acids 4-11, 5-12, 6-13 and 7-14 of SEQ ID
NO:4, 4 mer peptides corresponding to amino acids 8-11, 9-12, and
10-13 of SEQ ID NO:4. Other peptides can be chosen as desired to
prepare still other Smad2 phosphoserine specific antisera. One or
more of the serine residues in the foregoing peptides are
phosphorylated.
Example 7
Smad2 Phospho Tail Antibody Recognizes TGF-.beta. Type I Receptor
Mediated Phosphorylated Smad2 by Western Blot Analysis
[0150] COS cells were transfected with TGF-.beta. type I and type
II receptors and Smad2 expression plasmids in the absence or
presence of different amounts of Smad7 expression plasmid. Cells
were treated without or with TGF-.beta., and cell lysates were
prepared and used for Western blotting using the Smad2 phospho tail
antibody prepared in Example 6. The results of these experiments
are shown in FIG. 7. In the absence of Smad7 and TGF-.beta., Smad2
is weakly phosphorylated (lane 1), which is increased after ligand
addition (lane 4). The phosphorylation in the absence of ligand is
caused by ligand independent heteromeric complex formation of
receptors in COS cells. In the presence of increasing amounts of
Smad7, which inhibits receptor dependent Smad2 phosphorylation on
Ser465 and Ser467 in C-tail, a dose dependent decrease in Smad2
phosphorylation was observed. Thus the Smad2 phospho tail antibody
recognized TGF-.beta. type I receptor mediated phosphorylated
Smad2. The same blot was reprobed with Smad2 specific antibody; the
equal expression of Smad2 in each transfection was shown.
Example 8
Smad2 Phospho Tail Antibody Recognizes Phosphorylated Smad2 in
Extracts from Metabolically Labeled and TGF-.beta. Stimulated
Cells
[0151] To determine whether the Smad2 phospho tail antibody binds
Smad2 phosphorylated following TGF-.beta. stimulation, the
glioblastoma cell line U-343 MGa 35L was metabolically labeled with
.sup.35S-methionine and .sup.35S-cysteine for 4 hours. During the
last hour of labeling cells were incubated with or without 10 ng/ml
TGF-.beta.1. Cell lysates were precleaned by incubation with
protein A-Sepharose beads. After centrifugation to remove the
protein A-Sepharose beads, the cell extracts were subjected to
immunoprecipitation using Smad-specific antisera. Antiserum `543`
recognizes Smad2, `542` is specific for Smad3, `DPC` is raised
towards Smad 4 and `597` is the Smad2 phospho tail antibody
prepared in Example 6. To show specificity of the antisera,
controls were included containing the respective peptides to which
the antisera were raised. Incubations were done for 2 hours after
which the immunocomplexes were collected on protein-A Sepharose
beads. The beads were washed three times with lysis buffer (125 mM
NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM PMSF, 1.5% trasylol,
and 1% Triton X-100), three times with RIPA buffer (150 mM NaCl,
0.1% SDS, 0.5% deoxycholate, 0.5% Triton X-100, 50 mM Tris-HCl pH
8.0), and three times with high salt buffer (20 mM Tris-HCl pH 7.5,
500 mM NaCl, 1% Triton X-100). Immunocomplexes were separated on a
8.5% SDS-PAGE gel and visualized using a Fuji-X BioImager. As shown
in FIG. 8, U-343 MGa 35L clearly expressed Smad2 protein as
specifically recognized by the 543 antiserum (lanes 1, 2, 9 and
10), while Smad4 was brought down by the DPC antibody (lanes 5, 6,
13, 14). Smad3 protein was not detected in these cells. In the
absence of TGF-.beta. l, the Smad2 phospho tail antiserum (597) did
not detect any Smad protein (lanes 7, 8). Upon treatment of the
cells for one hour with 10 ng/ml of TGF-.beta.1, Smad2 phospho tail
antibody clearly and specifically recognized phosphorylated Smad2
(lanes 11, 12). These data indicate that the Smad2 phospho tail
antiserum can be successfully applied for the detection of
TGF-.beta. induced phosphorylated Smad2 present in extracts from
metabolically labeled cells.
Example 9
Preparation of Phospho Tail Antibodies which Recognize Pathway
Restricted Smad1, Smad3, Smad5, and Smad9
[0152] Rabbit antisera to TGF-.beta.-receptor-mediated
phosphorylated Smad1, Smad3, Smad5, and Smad9 are made against
peptides representing the phosphorylated tail of these Smad
proteins. A three lysine peptide (KKK) is added to the N-terminus
of the amino acids of SEQ ID NO:8 (SSVS) to facilitate coupling to
carrier protein. The resulting peptides, e.g. KKKSSVS (SEQ ID
NO:9), in which one or more of the serines are phosphoserines is
coupled to keyhole limpet hemocyanin with glutaraldehyde. Longer
peptides are used as needed to prepare Smad-specific antibodies.
For example, additional peptides are prepared by the addition of
the KKK linker to longer peptides derived from the C-terminus of
Smad1, Smad3, Smad5 and Smad9, e.g. 14 mers analogous to SEQ ID
NO:4 including SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID
NO:13, or intermediate length peptides such as 8 mers corresponding
to amino acids 7-14 of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
and SEQ ID NO:13. Still other peptides are used to prepare
antibodies to pathway-restricted Smad proteins, including Smad
C-terminal peptides which lack the two most C-terminal amino acids
(e.g., 4 mers corresponding to amino acids 9-12 of SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, or 8 mers
corresponding to amino acids 5-12 of SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, and SEQ ID NO:13). For all of the foregoing peptides,
one or more of the C-terminal serine residues are
phosphorylated.
[0153] The coupled peptides are mixed with Freund's adjuvant and is
used to immunize rabbits according to standard methods for
preparing polyclonal antibodies. Blood is drawn from immunized
rabbits and antisera prepared according to standard procedures.
Characterization of the phosphoserine specific antisera is
described in the following examples. The specificity of the
polyclonal antibodies for phosphorylated pathway-restricted Smad
proteins and not nonphosphorylated pathway-restricted Smad proteins
can be tested by methods well known to one of ordinary skill in the
art, including immunoprecipitation and Western blotting. Antisera
to phosphorylated pathway-restricted Smad proteins which crossreact
with nonphosphorylated pathway-restricted Smad proteins can be
affinity purified, if desired, to obtain polyclonal antibodies
which recognize only phosphorylated pathway-restricted Smad
proteins. Alternatively, monoclonal antibodies to phosphorylated
pathway-restricted Smad proteins can be prepared as described below
in Example 12. Each hybridoma clone producing monoclonal antibodies
can be tested for expression of antibodies which recognize
selectively the phosphorylated form of pathway-restricted Smad
proteins according to standard methods.
Example 10
Pathway-Restricted Smad Phospho Tail Antibodies Recognize
TGF-.beta. Superfamily Type I Receptor-Mediated Phosphorylated
Pathway-Restricted Smads by Western Blot Analysis
[0154] COS cells are transfected as described in Example 7 with
TGF-.beta. superfamily type I and type II receptors and a
pathway-restricted Smad expression plasmid (Smad1, Smad3, Smad5, or
Smad9), in the absence or the presence of different amounts of
Smad7 expression plasmid. For testing antibodies which recognize
phosphorylated Smad1, Smad5 or Smad9, cells are transfected with
BMP type I and type II receptors and a Smad1, Smad5 or Smad9
expression plasmid. For testing antibodies which recognize
phosphorylated Smad3, cells are transfected with TGF-.beta. or
activin type I and type II receptors and a Smad3 expression
plasmid. Cells then are treated with or without TGF-.beta., activin
or BMP molecules according to the receptors transfected, and cell
lysates are prepared and are used for Western blotting using
pathway specific Smad phospho tail antisera according to standard
procedures. In the presence of TGF-.beta., activin or BMP
molecules, the pathway specific Smads are phosphorylated and
recognized by the phospho tail antibodies. Antibodies which are
selective for individual phosphorylated Smad proteins can be
identified by blocking experiments using the various peptides
against which the antibodies are raised (and non-phosphorylated
peptides). For example, an antibody raised against a Smad1
phosphopeptide which does not bind the cognate antigen in the
presence of excess Smad1 phosphopeptide, but does bind the cognate
antigen in the presence of nonphosphorylated Smad1 peptide or
Smad3, Smad5 and/or Smad9 phosphopeptides is an antibody which
selectively binds Smad1 phosphorylated at the C-terminus. Therefore
the pathway restricted Smad phospho tail antibodies recognize
TGF-.beta. type I receptor-mediated phosphorylated pathway specific
Smads.
Example 11
Pathway Specific Smad Phospho Tail Antibody Recognizes
Phosphorylated Pathway Specific Smads in Extracts from
Metabolically Labeled and TGF-.beta. Stimulated Cells
[0155] Cells lines which overexpress pathway restricted Smads
(e.g., naturally overexpress, or which are transfected with
expression plasmids) are metabolically labeled with 35S-methionine
and .sup.35S-cysteine for 4 hours. During the last hour of
labeling, cells are incubated with or without 10 ng/ml TGF-.beta.1.
Cells are lysed according to standard procedures. Cell lysates are
precleaned by incubation with protein A-Sepharose beads. After
centrifugation to remove the beads, the cell extracts are subjected
to immunoprecipitation using the pathway specific Smad phospho tail
antisera. To show specificity of the antisera, controls are
included containing the respective pathway specific Smad peptides
to which the antisera have been raised. Incubations are done for 2
hours, after which the immunocomplexes are collected on protein-A
Sepharose beads. The beads are washed three times with lysis buffer
(125 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM PMSF, 1.5%
trasylol, and 1% Triton X-100), three times with RIPA buffer (150
mM NaCl, 0.1% SDS, 0.5% deoxycholate, 0.5% Triton X-100, 50 mM
Tris-HCl pH 8.0), and three times with high salt buffer (20 mM
Tris-HCl pH 7.5, 500 mM NaCl, 1% Triton X-100). Immunocomplexes are
separated on a 8.5% SDS-PAGE gel and visualized using a Fuji-X
Biolmager. Upon treatment of the cells for one hour with 10 ng/ml
of TGF-.beta.1, pathway specific Smad phospho tail antibodies
clearly and specifically recognize phosphorylated pathway specific
Smad proteins. Therefore the antibodies effectively
immunoprecipitate the respective pathway specific Smad proteins
present in extracts from metabolically labeled cells.
Example 12
Preparation of Monoclonal Antibodies
[0156] 1. Immunization of Mice
[0157] Mice (e.g., Balb/c female; Jackson Laboratories, Bar Harbor,
Me.) are immunized by subcutaneous and/or intraperitoneal injection
with a pathway-restricted Smad peptide having one or more
C-terminal phosphorylated serine residues suspended in Dulbecco's
phosphate buffered saline, then emulsified with an equal volume of
complete Freund's adjuvant (Sigma Chemical Co., St. Louis, Mo.).
The mice are given two intraperitoneal booster immunizations of
phosphorylated Smad peptide suspended in Dulbecco's phosphate
buffered saline (GIBCO, Grand Island, N.Y.), then emulsified with
an equal volume of incomplete Freund's adjuvant (Sigma Chemical
Co., St. Louis, Mo.) at 14 day intervals following the initial
immunization.
[0158] 2. Screening of Mice for Antibody Production
[0159] The presence of antibodies to phosphorylated Smad peptide is
tested by immunoprecipitation or Western blotting according to
standard procedures. For example, ten days following the third and
final immunization, a small amount of blood is collected by
retro-orbital bleed from each mouse and is clotted. A 1:1000
dilution of each of the serum samples collected from the immunized
mice (50%1) is added to a sample of labeled phosphorylated Smad
peptide (labeling can be, e.g., .sup.32P phosphorylation, biotin
conjugation, inclusion of a Flag antibody tag, etc.), mixed well
and incubated for 60 minutes at 4.degree. C. Protein A-Sepharose is
added to the reaction and incubated for 30 min at 4.degree. C. The
bound antibody is recovered by centrifugation. Pellets are washed,
and SDS-PAGE sample buffer added followed by boiling to disrupt
immune complexes. The samples are then analyzed by SDS-PAGE and
autoradiography (for radioactive labeling) or other detection
method such as chemiluminescence. Alternatively, radioactivity in
the washed pellets is measured by scintillation counting. The mouse
sera exhibiting the highest degree of binding to phosphorylated
Smad peptide is selected for cell fusion to create the monoclonal
antibodies.
[0160] 3. Preparation of Hybridomas
[0161] Hybridoma cell lines are prepared according to standard
protocols; one example is provided below. Briefly, after the mouse
with the best antibody titre to the phosphorylated Smad peptide is
selected, it is rested for a total of 4 weeks after its last
immunization. The mouse is then boosted with phosphorylated Smad
peptide by intraperitoneal injection in Dulbecco's phosphate
buffered saline. Four days later, the mouse is euthanized by
cervical dislocation and the spleen is removed and teased apart
into a cell suspension and washed in Dulbecco's phosphate buffered
saline. The spleen cells are counted and mixed with SP 2/0 myeloma
cells (ATCC Accession No. CRL8006, Rockville, Md.) that are
incapable of secreting either heavy or light immunoglobulin chains
(Kearney et al., J. Immunology, 123:1548, 1979) at a ratio of 2:1
(spleen cell:myeloma cells) and then fused using polyethylene
glycol 1450 (ATCC, Rockville, Md.) according to the standard
procedure developed by Kohler and Milstein (Nature, 256:495, 1975)
in eight 96-well tissue culture plates in selective HAT medium.
[0162] Between 10 and 21 days after fusion, hybridoma colonies
become visible and are screened by immunprecipitation, as described
above. Alternatively, ELISA assays or other immunobinding assay can
be used to determine which hybridomas produce antibodies that bind
phosphorylated Smad peptide. All hybridoma colonies that give a
positive response are expanded to 24-well cultures and subcloned by
limiting dilution to produce monoclonal cell lines. At this point,
additional screening is done with the hybridomas to identify which
hybridoma produces an anti-Smad protein antibody. Culture media
harvested from hybridoma cultures (supernatants) are screened as
described above, e.g. by ELISA (enzyme-linked immuno-adsorbent
assay) to identify positive clones.
[0163] The monoclonal antibodies optionally are examined to
determine the subclass of the antibody using an typing kit such as
an ISOstrip Kit (Boehringer Mannheim, Indianapolis, Ind.). A small
aliquot (e.g., 5 .mu.l) of hybridoma supernatant is diluted in PBS
and added to a test tube containing blue latex beads bound to
anti-mouse Ig antibodies. An isotyping strip is then placed in each
tube and the bead/antibody solution moves up the strip by capillary
action until the solution passes an antibody bound line containing
antibodies specific for the different isotopes. A blue line appears
in the area of the strip for each isotype detected in the hybridoma
supernatant.
Example 13
Separation and Sequencing of the Heavy and Light Chains of
Anti-Smad Monoclonal Antibody
[0164] The antibody may be isolated from the hybridomas and
purified by any method known in the art. At least two methods may
be used to separate the heavy and light chains of the purified
antibody for sequence determination. The first method employs a
semi-preparative SDS-PAGE followed by electroblotting onto a
polyvinyldifluoride (PVDF) membrane. Briefly, the purified antibody
is subjected to slab gel electrophoresis in SDS after reduction
with 2-merecaptoethanol. The resolved heavy and light chains are
then transferred onto a membrane such as an IMMOBILON.RTM. membrane
(a PVDF membrane from Millipore, Bedford, Mass.) using the
electroblotting method of Matsudaira (J. Biol. Chem. 261:10035,
1987). Bands corresponding to the heavy and light chains which are
identified with Coomassie Brilliant Blue staining may then be
excised from the membrane and processed for N-terminal
sequencing.
[0165] A second more complicated method permits larger amounts of
the heavy and light chains to be isolated in solution. This method
involves a dialysis step in which the purified antibody sample is
dialyzed against 0.1M Tris-HCl, 1 mM EDTA, pH 8.0, at 4.degree. C.
and then subjected to oxidative sulfitolysis in
NaSO.sub.3Na.sub.2S.sub.2O.sub.0, essentially as described by
Morehead at al. (Biochemistry 23:2500, 1984). Following
sulfitolysis, the antibody preparation is dialyzed against 1M
acetic acid, lyophilized to dryness, reconstituted in 1M acetic
acid, and subjected to gel filtration in a SEPHADEX G-75 column in
1M acetic acid. The purity of the heavy and light chains following
this step can then be assessed by analytical SDS-PAGE and then
concentrated for sequencing.
[0166] N-terminal amino acid sequencing may be performed using any
commercial amino acid sequencer such as an Applied Biosystems Model
477A protein-peptide sequencer. Analysis of the isolated chains is
performed following the instructions of the manufacturer of the
sequencer.
Example 14
Oligonucleotide Primer Design and Cloning of Smad Phospho Tail
Monoclonal Antibody Nucleic Acids
[0167] 1. Preparation of Oligonucleotides
[0168] Based upon the information which is obtained from the
foregoing amino acid sequence analyses, degenerate oligonucleotide
primers can be designed for use in PCR. Other non-degenerate
primers may be designed based upon nucleotide sequence information
obtained following PCR amplification of cDNA encoding the complete
heavy and light chains.
[0169] Oligonucleotide primers are synthesized by standard methods
using a commercially available sequencer such as an Applied
Biosystems Model 380B Synthesizer.
[0170] Alternatively, PCR amplification of the IgG, Fd heavy chain
fragments and light chains may be performed using the individual
heavy and light chain variable region gene families, and 3'
constant region primers for IgG.sub.1, k or 1 as previously
described (Kang et al., in Methods, A Companion to Methods in
Enzymology: Vol. 2, R. A. Lemer and D. R. Burton, ed. Academic
Press, NY, pp 111-118, 1991). Primers may contain restriction
enzyme sites to allow the sequential ligation of Fd and light chain
libraries for various other recombinant uses into a phage display
vector.
[0171] 2. PCR Amplification and DNA Sequencing of Heavy and Light
Chains
[0172] Total cytoplasmic RNA is isolated from the hybridoma cell
lines by any method known in the art. First strand cDNA is
synthesized directly from total cytoplasmic RNA using reverse
transcriptase. Polymerase chain reaction (PCR) amplifications then
are carried out according to standard protocols using a thermal
cycler or similar equipment. After amplification aliquots of the
PCR mixtures are subjected to electrophoresis in agarose gels
containing ethidium bromide. The PCR fragments of interest are
excised from the gels and purified by e.g. electroelution. The
gel-purified PCR fragments are digested with appropriate
restriction enzymes and ligated to a cloning vector such as
pBluescript. Competent bacterial cells are transformed with the
ligation mixture, grown, and lysed for preparation of DNA. Plasmid
DNA is purified by any technique known in the art for purifying
DNA, such as the Qiagen plasmid maxiprep kit (Qiagen, Chatsworth,
Calif.). Sequencing is then performed on an automated DNA
sequencer. Derived sequences for heavy chain Fd fragments and light
chains can then be aligned using various commercially available
software packages and the Genbank database.
[0173] Equivalents
[0174] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0175] All references disclosed herein are incorporated by
reference in their entirety.
[0176] A Sequence Listing is presented followed by what is
claimed:
Sequence Listing
[0177] (1) GENERAL INFORMATION
[0178] (i) APPLICANT: Souchelnytskyi, Serheyi
[0179] Tamaki, Kiyoshi
[0180] Engstrom, Ulla
[0181] Wernstedt, Christer
[0182] Piek, Esther
[0183] ten Dijke, Peter
[0184] Heldin, Carl-Henrik
[0185] (ii) TITLE OF THE INVENTION: SMAD2 PHOSPHORYLATION AND
INTERACTION WITH SMAD4
[0186] (iii) NUMBER OF SEQUENCES: 15
[0187] (iv) CORRESPONDENCE ADDRESS:
[0188] (A) ADDRESSEE: Wolf, Greenfield & Sacks, P.C.
[0189] (B) STREET: 600 Atlantic Avenue
[0190] (C) CITY: Boston
[0191] (D) STATE: MA
[0192] (E) COUNTRY: USA
[0193] (F) ZIP: 02210
[0194] (v) COMPUTER READABLE FORM:
[0195] (A) MEDIUM TYPE: Diskette
[0196] (B) COMPUTER: IBM Compatible
[0197] (C) OPERATING SYSTEM: DOS
[0198] (D) SOFTWARE: FastSEQ for Windows Version 2.0
Sequence CWU 1
1
* * * * *