Ras proteins

Details Ras proteins Ras proteins

1998-05-08

2002-05-21

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...

C435S091200, C435S091400, C435S252300, C435S320100, C435S006120, C536S023100, C536S023500, C536S024310, C530S350000

Reexamination Certificate

active

06391580

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of seven Ras proteins and to the use of these sequences in the diagnosis, treatment, and prevention of disorders associated with cell proliferation, in particular, cancer and immune disorders.
BACKGROUND OF THE INVENTION
Guanine nucleotide-binding proteins (GTP-binding proteins, or G proteins) participate in a wide range of regulatory functions including metabolism, growth, differentiation, signal transduction, cytoskeletal organization, and intracellular vesicle transport and secretion. These proteins control a diverse sets of regulatory pathways in response to hormones, growth factors, neuromodulators, or other signaling molecules. When these molecules bind to transmembrane receptors, signals are propagated to effector molecules by intracellular signal transducing proteins. Many of these signal transducing proteins are members of GTP-binding proteins.
Low molecular weight (LMW) GTP-binding proteins are small proteins which consist of single polypeptides of 21-30 kDa. These proteins regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. In particular, the LMW GTP-binding proteins activate cellular proteins by transducing mitogenic signals involved in various cell functions in response to extracellular signals from receptors (Tavitian, A. (1995) C. R. Seances Soc. Biol. Fil. 189:7-12). During this process, the hydrolysis of GTP acts as an energy source as well as an on-off switch for the GTPase activity of the LMW GTP-binding proteins.
The LMW GTP-binding proteins can be classified into at least five subfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor. Despite their sequence variations, all five subfamilies share common conserved structural features. Four sequence regions, termed motifs I-IV, are conserved in the LMW GTP-binding proteins. Motif I is the most variable and has the signature, GXXXXGK. The lysine residue is essential in interacting with the &bgr;- and &ggr;-phosphates of GTP. Motif II, III, and IV are highly conserved, with DTAGQE, NKXD, and EXSAX as their respective signatures. These motifs regulate the binding of &ggr;-phosphate, GTP, and the guanine base of GTP, respectively. Most of the membrane-bound LMW GTP-binding proteins generally require a carboxy terminal isoprenyl group for membrane association and biological activity. The isoprenyl group is added posttranslationally by a mechanism which recognizes a terminal cysteine residue alone or a terminal cysteine-aliphatic amino acid-aliphatic amino acid-any amino acid (CAAX) motif. Additional membrane-binding energy is often provided by either internal palmitoylation or a carboxy terminal cluster of basic amino acids. The LMW GTP-binding proteins also have a variable effector region, located between motifs I and II, which is characterized as the interaction site for guanine nucleotide exchange factors (GEFs) or GTPase-activating proteins (GAPs). GEFs induce the release of GDP from the active form of the G protein, whereas GAPs interact with the inactive form by stimulating the GTPase activity of the G protein.
The Ras subfamily already indicated supra are essential in transducing signals from receptor tyrosine kinases (RTKs) to a series of serine/threonine kinases which control cell growth and differentiation. Activated Ras genes were initially found in human cancers and subsequent studies confirmed that Ras function is critical in the determination of whether cells continue to grow or become terminally differentiated. Stimulation of cell surface receptors activates Ras which, in turn, activates cytoplasmic kinases. The kinases translocate to the nucleus and activate key transcription factors that control gene expression and protein synthesis. (Barbacid, M. (1987)Ann. Rev Biochem. 56:779-827, Treisman, R. (1994) Curr. Opin. Genet. Dev. 4:96-98.) Mutant Ras proteins, which bind but can not hydrolyze GTP, are permanently activated, and cause continuous cell proliferation or cancer. TC2 1, a Ras-like protein, is found to be highly expressed in a human teratocarcinoma cell line. (Drivas, G. T. et al. (1990) Mol. Cell. Biol. 10: 1793-1798.) Rin and Rit are characterized as membrane-blinding, Ras-like proteins without the lipid-binding CAAX motif and carboxy terminal cysteine. (Lee, C.-H. J. et al. (1996) J. Neurosci. 16: 6784-6794.) Further, Rin is shown to localize in neurons and have calcium-dependant calmodulin-binding activity.
The other members of the LMW GTP-binding proteins have roles in signal transduction that vary with the function of the activated genes and the locations of the GTP-binding proteins that initiate the activity. The Rho GTP-binding proteins control signal transduction pathways that link growth factor receptors to actin polymerization which is necessary for normal cellular growth and division. The Rab proteins control the translocation of vesicles to and from membranes for protein localization, protein processing, and secretion. The ran GTP-binding proteins are located in the nucleus of cells and have a key role in nuclear protein import, the control of DNA synthesis, and cell-cycle progression. (Hall, A. (1990) Science 249:635-640, Scheffzek, K. et al. (1995) Nature 374:378-381.)
The discovery of seven human Ras proteins and the polynucleotides which encode them satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of disorders associated with cell proliferation, in particular, cancer and immune disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, Ras proteins, referred to collectively as “RASP” and individually as “RASP-1”, “RASP-2”, “RASP-3”, “RASP-4”, “RASP-5”, “RASP-6”, and “RASP-7”. In one aspect, the invention provides a substantially purified polypeptide, RASP, comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:7.
The invention further provides a substantially purified variant of RASP having at least 90% amino acid identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, or to a fragment of any of these sequences. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:7. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:7.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, a fragment of SEQ ID NO:1, a fragment of SEQ ID NO:2, a fragment of SEQ ID NO:3, a fragment of SEQ ID NO:4, a fragment of SEQ ID NO:5, a fragment of SEQ ID NO:6, and a fragment of SEQ ID NO:7, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising t

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