Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-19
2002-04-09
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S252300, C435S325000, C536S023100
Reexamination Certificate
active
06368829
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acids and encoded polypeptides which interact with the TGF-&bgr; receptor complex and which is a negative regulator of TGF-&bgr; 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
During mammalian embryogenesis and adult tissue homeostasis transforming growth factor &bgr; (TGF-&bgr;) 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&bgr;R-I and T&bgr;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 etal.,
Curr. Opin. Cell Biol
., 8:139-145, 1996). The two receptors, which both are required for signaling, act in sequence; T&bgr;R-I is a substrate for the constitutively active T&bgr;R-II kinase (Wrana et al.,
Nature
, 370:341-347, 1994; Wieser et al.,
EMBO J
., 14:2199-2208, 1995). TGF-&bgr; 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).
Genetic studies of TGF-&bgr;-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 etal.,
Proc. Natl. Acad. Sci. USA
, 93:790-794, 1996) genes, respectively. The products of these related genes perform essential functions downstream of TGF-&bgr;-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 a.,
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).
Different Smads specify different responses using functional assays in Xenopus. Whereas Smadl induces ventral mesoderm, a BMP-like response, Smad2 induces dorsal mesoderm, an activin/TGF-&bgr;-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 Smadl phosphorylation, whereas TGF-&bgr; 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; Macias-Silva et al.,
Cell
, 87:1215-1224, 1996; Nakao et al.,
J. Biol. Chem
., 272:2896-2900, 1996).
Smad4 is a common component of TGF-&bgr;, 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-&bgr; or activin Smad4 interacts with Smad2 or Smad3, and upon BMP challenge a heteromeric complex of Smad4 and Smad I 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; Macias-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
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-&bgr; 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.
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.
In other embodiments, the foregoing isolated polypeptide consists of a fragment or variant of the foregoing which retains the activity of the foregoing.
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.
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.
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 20amino 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/
Dijke Peter Bon
Engotröm Ulla
Heldin Carl-Henrik
Piek Ester
Souchelnytskyi Serhly
Carlson Karen Cochrane
Ludwig Institute for Cancer Research
Wolf Greenfield & Sacks P.C.
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