Regulation of the Cool/Pix Proteins

The Cool (cloned-outof library)/Pix (forPAK-interactive exchange factor) proteins directly bind to members of the PAK family of serine/threonine kinases and regulate their activity. Three members of the Cool/Pix family have shown distinct regulatory activities: (i) p50 Cool-1 inhibits Cdc42/Rac-stimulated PAK activity, (ii) p85 Cool-1 /β-Pix has a permissive effect on Cdc42/Rac-stimulated activity, and (iii) p90 Cool-2 /α-Pix strongly activates PAK. We initially suspected that these different functional effects were due to a binding interaction that occurs at the carboxyl-terminal ends of the larger Cool/Pix proteins, thus enabling them to stimulate (or at least permit) rather than inhibit PAK activity. This led to the identification of the Cat proteins (forCool-associated tyrosine phosphosubstrates). However, here we show that the Cat proteins bind to the carboxyl-terminal ends of p85 Cool-1 (residues 523–546) and Cool-2 (residues 647–670), and that the binding of Cat to Cool-2 in fact is not necessary for the Cool-2-mediated activation of PAK. Rather, an 18-amino acid region, designated T1, that is present in the Cool-1 proteins, but missing in Cool-2, is essential for controlling the regulation of PAK activity by Cool-1/β-Pix in vivo. Deletion of T1 yielded a p85 Cool-1 molecule that mimicked the Cool-2 protein and was capable of strongly stimulating PAK activity. However, when T1 was added to Cool-2, the ability of Cool-2 to directly activate PAK was lost. We conclude that T1 represents a novel regulatory domain that accounts for the specific functional effects on PAK activity exhibited by the different members of the Cool/Pix family.