Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2000-05-08
2002-11-05
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S328000, C435S325000, C435S326000, C435S327000, C435S328000, C435S329000, C435S330000, C435S350000
Reexamination Certificate
active
06475775
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acids and encoded polypeptides which interact with the PTPL1 phosphatase and which are GTPase activating proteins. 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
The Rho family of Ras-like GTPases, which includes Rho, Rac and Cdc42, control actin-based cytoskeletal rearrangements (reviewed in Hall,
Annu. Rev. Cell Biol.
10:31-54, 1994; Zigmond,
Curr. Opin. Cell Biol.
8:66-73, 1996). Rho regulates receptor-mediated assembly of focal adhesions and stress fibers (Ridley and Hall,
Cell
70:389-399, 1992), while Rac regulates the formation of membrane ruffles (Ridley et al.,
Cell
70:401-410, 1992) and Cdc42 controls the formation of filopodia (Nobes and Hall,
Cell
81:53-62, 1995). Rho proteins have also been shown to be important in the regulation of cell proliferation (reviewed in Symons,
Trends Biochem. Sci.
21:178-181, 1996). As members of the Ras superfamily, Rho proteins function as molecular switches, having an active, GTP-bound form, and an inactive, GDP-bound form. The active, GTP-bound form, is negatively regulated by GTPase activating proteins (GAPs) which enhance the intrinsic GTPase activity of Rho proteins. A number of GAPs that are active on proteins of the Rho family have been identified (reviewed in Lamarche and Hall,
TIG
10:436-440, 1994). These include p50RhoGAP (Lancaster et al.,
J. Biol. Chem.
269:1137-1142, 1994), Myr5 (Reinhard et al.,
EMBO J.
14:697-704, 1995), and p190 (Settleman et al.,
Nature
359:153-154, 1992) which are also active on Rac and Cdc42. Another GAP, p122-RhoGAP (Homma and Emori,
EMBO J.
14:286-291, 1995) appears to be specific for Rho.
Intracellular protein tyrosine phosphatases (PTPs) are a diverse group of proteins involved in signal transduction (reviewed in Streuli,
Curr. Opin. Cell Biol.
8:182-188, 1996). They contain a conserved PTP domain which specifically dephosphorylates tyrosine residues and, in addition, domains that regulate their subcellular localization and activity (reviewed in Mauro and Dixon,
Trends Biochem. Sci.
19:151-155, 1994). For example, the SH2 domains of SHP-1 and SHP-2 enables these PTPs to localize to and interact with activated growth factor receptors (Mauro and Dixon, 1994). Correct localization of PTPs is of importance, since the PTP domains usually have broad substrate specificity.
PTPL1 (Saras et al.,
J. Biol. Chem.
269:24082-24089, 1994) also called PTP-BAS (Maekawa et al.,
FEBS Lett.
337:200-206, 1994), hPTP1E (Banville et al.,
J. Biol. Chem.
269:22320-22327, 1994) and FAP-1 (Sato et al.,
Science
268:411-415, 1995), is a 250 kDa protein expressed in many tissues and cell lines. PTPL1 is fully described in PCT published application WO95/06735. It contains an N-terminal leucine zipper motif followed by a domain with homology to the Band 4.1 superfamily. Band 4.1-like domains are found in proteins involved in the linkage of actin filaments to the plasma membrane (Arpin et al.,
Curr. Opin. Cell Biol.
6:136-141, 1994). Five PDZ domains [PDZ is derived from PSD-95 (Cho et al.,
Neuron
9:929-942, 1992), Dlg-A (Woods and Bryant,
Cell
66:451-464, 1991) and ZO-1 (Itoh et al.,
J. Cell. Biol.
121:491-502, 1993), each of which contains three such domains] are present between the Band 4.1-like domain and the C-terminal PTP domain. These domain structures of about 90 amino acid residues have also been called GLGF repeats or DHRs and are identified in a variety of proteins (Ponting and Phillips,
Trends Biochem. Sci.
20:102-103, 1995). A PDZ domain of PTPL1 has been shown to interact with the C-terminal tail of the membrane receptor Fas (Sato et al., 1995) and PDZ domains of PSD-95 bind to the C-terminals of the NMDA-receptor and Shaker-type K
+
channels (Kim et al.,
Nature
378:85-88, 1995; Kornau et al.,
Science
269:1737-1740, 1995). The crystal structures of two PDZ domains have recently been published (Doyle et al.,
Cell
85:1067-1076, 1996; Morais Cabral et al.,
Nature
382:649-652, 1996).
There exists a need to influence the receptor-mediated intracellular signal transduction pathways to treat disease. There also exists a need to identify the gene(s) responsible for increased or decreased signal transduction and to provide a genetic therapy for treating diseases resulting from aberrant signal transduction.
An object of the invention is to provide compounds that desirably influence the signal transduction by the Rho family of Ras-like GTPases.
Another object of the invention is to provide therapeutics for treating diseases resulting from aberrant signal transduction by the Rho family of Ras-like GTPases.
Still another object of the invention is to provide diagnostics and research tools relating to PARG, PTPL1 and the Rho family of Ras-like GTPases. These and other objects will be described in greater detail below.
SUMMARY OF THE INVENTION
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and agents which bind such polypeptides, including antibodies. The foregoing can be used in the diagnosis or treatment of conditions characterized by the expression of a PARG nucleic acid or polypeptide. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, we present the cDNA cloning of a PTPL1-associated RhoGAP, PARG, a 150 kDa protein that contains a GAP domain that displays strong activity towards Rho. Furthermore, the C-terminal tail of PARG specifically interacts with the fourth PDZ domain (PDZ4) of PTPL1.
According to one aspect of the invention, an isolated nucleic acid molecule is provided. The molecule hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NO:1. The isolated nucleic acid molecule codes for a GTPase activating polypeptide. The invention further embraces nucleic acid molecules that differ from the foregoing isolated nucleic acid molecules in codon sequence due to the degeneracy of the genetic code. The invention also embraces complements of the foregoing nucleic acids.
In preferred embodiments, the isolated nucleic acid molecule comprises a molecule consisting of the nucleic acid sequence of SEQ ID NO:1. More preferably, the isolated nucleic acid molecule comprises a molecule consisting of nucleotides 184-3966 of SEQ ID NO:1. Preferably the isolated nucleic acid comprises a molecule having a sequence which encodes amino acids 666-853 of SEQ ID NO:2, amino acids 613-652 of SEQ ID NO:2, and/or amino acids 193-509 of SEQ ID NO:2.
According to another aspect of the invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule comprises a molecule consisting of a unique fragment of nucleotides 184-3966 of SEQ ID NO:1 between 12 and 3781 nucleotides in length and complements thereof, provided that the isolated nucleic acid molecule excludes molecules consisting solely of nucleotide sequences selected from the group consisting of accession numbers T32345 (SEQ ID NO:3), Z28937 (SEQ ID NO:4), Z28520 (SEQ ID NO:5), AA431926 (SEQ ID NO:14), AA326126 (SEQ ID NO:15), AA342471 (SEQ ID NO:16), AA716829 (SEQ ID NO:17), L49573, Z43348 (SEQ ID NO:18), AA303722 (SEQ ID NO:19), T32495 (SEQ ID NO:20), AA330162 (SEQ ID NO:21), Z25350 (SEQ ID NO:22), AA794256 (SEQ ID NO:23), T32506 (SEQ ID NO:24), T32263 (SEQ ID NO:25), F06673 (SEQ ID NO:26), AA462548 (SEQ ID NO:27), X85558 (SEQ ID NO:28), R14952 (SEQ ID NO:29), AA870705 (SEQ ID NO:30), AA120493 (SEQ ID NO:3 1), AA415591 (SEQ ID NO:32), AA1 31400 (SEQ ID NO:33), C76597 (SEQ ID NO:34), C76601 (SEQ ID NO:35), AA870475 (SEQ ID NO:36), AA234871 (SEQ ID NO:37), C77518 (SEQ ID NO:38), and AA672012 (SEQ ID NO:39). In one embodiment, the
Aspenström Pontus
Franzen Petra
Gonez Leonel Jorge
Heldin Carl-Henrik
Hellman Ulf
Ludwig Institute for Cancer Research
McKelvey Terry
Wolf Greenfield & Sacks P.C.
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