Regulation of the Cool/Pix proteins: key binding partners of the Cdc42/Rac targets, the p21-activated kinases.
The Cool (cloned-out of library)/Pix (for PAK-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)/beta-Pix has a permissive effect on Cdc42/Rac-stimulated activity, and (iii) p90(Cool-2)/alpha-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 (for Cool-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/beta-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.
Feng
Qiyu
Q
Department of Molecular Medicine, Cornell University, Ithaca, New York 14853, USA.
Albeck
John G
JG
Cerione
Richard A
RA
Yang
Wannian
W
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014162:Transfection; D014633:Valine / Q000737:chemistry; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D011993:Recombinant Fusion Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D014633:Valine