Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20.
The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules.
Winters
Matthew J
MJ
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 377 Plantation St., Four Biotech, Rm. 330, Worcester, MA 01605, USA.
Pryciak
Peter M
PM
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D019143:Evolution, Molecular; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D020930:MAP Kinase Kinase Kinases; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C487575:NBP2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C073129:BEM1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D020930:MAP Kinase Kinase Kinases; C078754:STE20 protein, S cerevisiae; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
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