The p-21-activated kinase (PAK) family of serine-threonine kinases (reviewed by Bagrodia and Cerione, Trends in Cell Biology, vol. 9, pp. 350–355, 1999, and references contained therein) currently consists of four members, PAK1 (also known as PAKα), PAK2 (also known as PAKγ), PAK3 (also known as PAKβ), and PAK4. The kinase activity of PAKs is stimulated by binding to the GTP-bound forms of Cdc42 and p21Rac (hereafter referred to as Rac). The C-terminal region of PAK proteins contains the kinase catalytic domain, which shows the highest conservation of sequence homology between different members of the family. The N-terminal region contains a conserved motif thought to be responsible for binding to Cdc42 and Rac GTPases (the ‘GBD/CRIB’ motif, Burbelo et al., Journal of Biological Chemistry vol. 270, pp. 29071–29074, 1995). PAKs also contain in their N-terminal domains several copies of the PXXP protein motif that represents a binding site for SH3 protein domains. It has been shown that PAKs1–3 posses an N-terminal regulatory region (overlapping with the Cdc42/Rac binding domain) that is responsible for maintaining the kinase in a catalyically inactive form. PAK proteins have recently been shown to utilize sequences within the N-terminal domain for high-affinity binding to two SH3 domain-containing proteins, p85Cool-1/βPix and αPIX/Cool-2 (Manser et al., Mol. Cell vol. 1 pp.183–192, 1998, and Bagrodia et al., J. Biol. Chem. Vol 273, pp. 23633–23636, 1998). p85Cool-1/βPix localizes to peripheral focal complexes, and was found to recruit PAK1 from the cytoplasm to these complexes, while an alternatively spliced version of p85Cool-1/βPIX, p50Cool-1, appears to bind PAK3 and inhibits its kinase activity. Cool-2/αPIX stimulates PAK activity through an as yet unclear mechanism. Two tyrosine phosphorylated proteins, termed Cat-1 and Cat-2 (Cool-associated tyrosine phosphorylated proteins 1 and 2, Bagrodia et al., J Biol Chem vol. 274 pp. 22393–400, 1999) have recently been found to interact with p85Cool-1/βPIX and Cool-2/αPIX, but not with p50Cool-1. It therefore appears likely that Cat-1 and Cat-2 play crucial roles in PAK regulation, since they only interact with forms of Cool/Pix that promote PAK activity.
In addition to these interactions, PAK proteins have been shown to be recruited to activated tyrosine kinase receptors by the SH2/SH3 adapter protein Nck (Bokoch et al., J Biol Chem vol. 271 pp. 25746–9, 1996). This recruitment may provide a link between cell activation by growth factor receptors and PAK signaling pathways. It has also been shown that PAK kinase activity can be stimulated in the absence of Cdc42 or Rac binding by sphingosine and other membrane lipids (Bokoch et al., J Biol Chem vol. 273, pp. 8137–44, 1998), but repressed by products of sphingolipid metabolism (Lian et al., J Immunol vol. 161, pp. 4375–81, 1998).
The downstream consequences of PAK activity are also multifold and complex. PAK proteins have been found to affect assembly of focal contacts, cytosketal organization, neurite outgrowth, lamellipodia formation, membrane ruffling, regulation of cell motility and morphology. PAKs have also been found to activate nuclear mitogen-activated protein kinases (MAPKs), and importantly, to phosphorylate the kinase Raf1, a downstream effector of Ras proteins: in fact, kinase-defective PAK mutants revert the oncogenic activity of mutated Ras (Tang et al., Proc Natl Acad Sci USA vol. 95, pp. 5139–44, 1998). Additionally, PAKs become activated after stimulation of the T-cell receptor, and are required for activation of ERK2 and the NFAT transcription factor, and consequently gene expression by the T-cell receptor (Yablonski et al., EMBO J vol. 17 pp.5647–57, 1998).
In summary, PAK proteins are subject to diverse regulatory inputs, and transmit signals to diverse downstream effectors which are essential for many signaling pathways that are fundamental for cell morphology, motility/migration, proliferation, differentiation or cell death.
The present invention involves the surprising discovery of a novel polypeptide, herein designated p21-activated kinase 5 (henceforth to as “PAK5”) and its role as a key component, for example, in regulating cell proliferation, cell migration, cell differentiation, cytoskeletal organisation, gene expression, cell cycle progression, and cell death. PAK5 is, thus, useful in the search for novel agents that can modify and/or control these processes. These and other aspects of the invention are described below.
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