Two sterile-20 kinase-like proteins and methods of use thereof

Details Two sterile-20 kinase-like proteins and methods of use thereof Two sterile-20 kinase-like proteins and methods of use thereof

2000-10-10

2003-02-25

Monshipouri, M. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S252300, C435S320100, C435S325000, C435S006120, C536S023200

Reexamination Certificate

active

06524833

ABSTRACT:

BACKGROUND
Protein kinases play a key role in cell growth and differentiation. The p21-activated proteins kinases (PAKs) are related to a yeast serine/threonine protein kinase, Ste20. Ste20 is a member of a growing family of regulatory enzymes that may play roles in diverse phenomena such as cellular morphogenesis, the stress response and the pathogenesis of AIDS.
The growth, differentiation, maintenance and senescence of cells requires the transmission of signals. These signals can be derived from extracellular stimuli, such as hormone interaction with its receptor or physiological stress, or may be derived intracellularly from developmental programs.
The signals are transmitted via a signal cascade wherein proteins are phosphorylated or dephosphorylated in sequence. Protein kinases, the enzymes that phosphorylate, play a key role in many signaling pathways and therefore protein kinases play a key role in cell growth and differentiation. Protein kinases fall into two broad categories, tyrosine kinases (those that add phosphate to tyrosine residues) and serine/threonine kinases (those that add phosphate to either serine or threonine residues).
One group of signaling pathways involving protein kinases are the mitogen-activated protein (MAP) kinase cascades. The MAP kinase cascades are now recognized to participate in diverse signal transduction pathways rather than only mediating mitogenic signals from all surface receptors (Davis, R. J., 1994
, Trends in Biochem Science
, 19:470-473).
Genetic analyses in yeast have made it possible to identify some of the components of MAPK pathways, and to clarify the diversity of their functions and regulation (Herskowitz, I., 1995
, Cell
80:187-197). Mammalian cells have at least three MAPK pathway subtypes, the ERK (extracellular signal-regulated protein kinase) pathway, JNK (c-Jun NH
2
-terminal kinase)/SAPK (stress-activated protein kinase) pathway, and P38/Mpk2 pathway; together, these pathways mediate a wide variety of physiological responses (Davis, R. J., 1994,
Trends in Biochem. Science
19:470-473; Derijard, B. et al., 1994
Cell
76:1025-1037; Kyriakis, J. M. et al., 1994
Nature
369:156-160; Han, J. et al., 1994
Genes Dev
. 3:1336-1348; Lee, J. C. et al., 1994
Nature
372:739-746; Rouse, J. et al., 1994
Cell
78:1027-1037). MAPKs are activated by sequential protein phosphorylation reactions. The basic framework of the MAP kinase pathway, where MAPK is phosphorylated on Thr and Tyr residues and activated by MAPK kinase (MAPKK), before which MAPKK is itself phosphorylated and activated by MAPKK kinase (MAPKKK), is common from yeast to mammals (Nishida, E. and Gotoh, Y., 1993
Trends in Biochem. Science
18:128-131; Davis, 1994; Herskowitz, 1995; Marshall, C. J., 1994
Curr. Opin. Genet. Dev
. 4:82-89).
A new kinase group activated by G-protein and thought to act as MAPKKK kinase (MAPKKKK) has been identified in both yeast and mammals. In budding yeast, this new kinase group is known by its prototype member Sterile 20 (Ste20). Ste20 is activated by the &bgr;&agr; complex released from the heterotrimeric G protein complex upon pheromone receptor stimulation, and in turn activates Ste11 (a MAPKKK) (Leberer, E. et al., 1992
EMBO J
. 11:4815-4824; Ramer, S. W. and Davis, R. W., 1993
Proc. Natl. Acad. Sci. U.S.A
. 90:452-456). In mammals, this new kinase group is known by its prototype member p21-activated protein kinase (PAK). PAK (now called &agr;-PAK) has been identified as a protein kinase activated by the Rho family of small G-proteins, Rac1 and Cdc42 (Manser, E. et al., 1994
Nature
367:40-46); PAK also shows sequence similarity to yeast Ste20. Recently it has been clarified that PAK comprises a protein kinase family composed of several PAK isoforms, hPAK65 (Martin, G. A. et al., 1995
EMBO J
. 14:1970-1978), MPAK-3 (Bagrodia, S. et al., 1995
J. Biol. Chem
. 270:22731-22737 and &bgr;-PAK (Manser, E. et al., 1995
J. Biol. Chem
. 270:25070-25078), all of which are able to interact with Cdc42 and Rac1. Rac1 and Cdc42 have been implicated not only in cell motility (Ridley, A. J. et al., 1992
Cell
70:401-410; Kozma, R. et al., 1995
Mol. Cell. Biol
. 15:1842-1952; Nobes, C. D. and Hall, A., 1995
Cell
81:53-62), but also in the preferential activation of the JNK/SAPK and p38/Mpk2 pathways rather than the ERK pathway (Coso, O. A. et al., 1995
Cell
81:1137-1146; Minden, A. et al., 1995
Cell
81:1147-1157; Olson, M. F. et al., 1995
Science
269:1270-1272). This is in contrast to another small G-protein, Ras, which predominantly activates the ERK pathway through Raf activation (Minden, A. et al., 1994
Science
266:1719-1723). Although a direct interaction between PAKs and components of the JNK/SAPK and p38/Mpk2 pathways has not yet been demonstrated, these observations raise the intriguing possibility that PAK or PAK-related proteins mediate the signals from Rac1 and Cdc42 to the JNK/SAPK and p38/Mpk2 pathways, and, furthermore, that G-proteins differentially regulate MAPK pathways to achieve various physiological responses.
In contrast to the above-mentioned kinases, a group of Ste20-related kinases that lack the putative Cdc42/Rac1-binding domain has been identified in both yeast and mammals: Sps1, an upstream regulator of the MAPK pathways (Freisen et al.,
Genes and Dev
., 9:2162-2175 (1994) and MST-1 in Mammals; Creasy, C. L. and Chernoff, J., 1995
J. Biol. Chem
. 270:21695-21700). Although the specific activation of the SAPK pathway by GCK has recently been reported (Pombo, C. M. et al., 1995
Nature
377:750754), the upstream and downstream signaling pathways of this group of kinases remain to be clarified. Sterile 20-related kinases are regulatory molecules involved in mitogenic signaling as well as other cellular phenomena such as morphology and motility. These phenomena are important factors in development, cell differentiation, cancer and metastases. Therefore, the polynucleotides and polypeptides of the present invention allow manipulation of the signaling pathways involved and will allow the development of reagents to modulate the signaling pathways involved in these important cellular phenomena.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides encoding novel members of the Ste20 family of serine/threonine protein kinases, and the recombinantly produced polypeptides encoded by said polynucleotides.
The present invention is drawn to a purified nucleic acid comprising at least 45 continuous nucleotides of a nucleic acid sequence provided in SEQ ID NO: 3. The purified nucleic acid can comprise SEQ ID NO: 3, a complementary sequence or a sequence having greater about 600 bases in length, wherein said sequence hybridizes to SEQ ID NO: 3 under stringent conditions.
The present invention is also drawn to a purified nucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ:ID NO: 9, a sequence complementary to either SEQ ID NO: 1 or 9 and a sequence greater than 500 bases in length wherein said sequence hybridizes to SEQ ID NO: 1 or 9 under stringent conditions.
The present invention also encompasses expression vectors comprising the polynucleotides of the present invention and host cells harboring said vectors.
The present invention is also drawn to a purified nucleic acids encoding an amino acid sequence comprising SEQ ID NOs.: 2, 4, or 10. In one embodiment, the purified nucleic acid sequence comprising a nucleic acid sequence encoding at least 100 continuous amino acids of an amino acid sequence provided in SEQ ID NO: 2 or SEQ ID NO: 10. In another embodiment, the purified nucleic acid sequence comprising a nucleic acid sequence encoding at least 54 continuous amino acids of an amino acid sequence provided in SEQ ID NO: 4. The method of the present invention is also drawn to a method of making polypeptides encoded by SEQ ID NOs.:1, 3, or 9 comprising, transfecting a host cell with an expression vector comprising SEQ ID NOs.: 1, 3, or 9 and isolating the expressed protein.
The present invention is further drawn to polypeptides comprising SEQ ID NOs.: 2 or 10 and biol

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