Specificity profiling of Pak kinases allows identification of novel phosphorylation sites.
The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.
Rennefahrt
Ulrike E E
UE
Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Deacon
Sean W
SW
Parker
Sirlester A
SA
Devarajan
Karthik
K
Beeser
Alexander
A
Chernoff
Jonathan
J
Knapp
Stefan
S
Turk
Benjamin E
BE
Peterson
Jeffrey R
JR
D020816:Amino Acid Motifs / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D064067:Rho Guanine Nucleotide Exchange Factors; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors; D017346:Protein Serine-Threonine Kinases; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins