A tyrosine-phosphorylated protein that binds to an important regulatory region on the cool family of p21-activated kinase-binding proteins

The pal-activated kinases (Pak) are major targets of the small GTPases Cdc4 2 and Rac, We, and others, recently identified a family of proteins termed Cool/Pix, which interact with Pak3, In cells, p50(Cool-1) suppresses Pak ac tivation by upstream activators; p85(Cool-1) has a permissive effect on Pak activation, and we now show that the closely related Cool-2 stimulates Pak kinase activity. To understand the differential regulation of Pak by Cool proteins, we screened for Cool-interacting proteins by affinity purificatio n and microsequencing, This has led to the identification of two closely re lated proteins called Cat (Cool-associated, tyrosine phosphorylated), which contain a zinc finger followed by three ankyrin repeats. Cat-1 is identica l to the recently identified binding partner for the beta-adrenergic recept or kinase (beta ARK or GRK-2), which was shown to have Arf-GAP activity. Ca t-1 and Cat-2 both bind to the COOH-terminal region of p85(Cool-1) and p85( Cool-2) but not bind to p50(Cool-1), Cat-1 is tyrosine-phosphorylated in gr owing NIH 3T3 fibroblasts, and its tyrosine phosphorylation is increased fo llowing cell spreading on fibronectin, decreased in cells arrested in mitos is, and increased in the ensuing G(1) phase. Cat proteins are tyrosine-phos phorylated when co-expressed in cells with the focal adhesion kinase Fak an d Src, These findings suggest that in addition to playing a role in Cool/Pa k interactions, Cat proteins may serve as points of convergence between G p rotein-coupled receptors, integrins, Arf GTPases, cell cycle regulators, an d Cdc42/Rac/Pak signaling pathways.