Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2.
The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.
Chang
E
E
Department of Biology, New York University, New York, New York 10003-6688, USA.
Bartholomeusz
G
G
Pimental
R
R
Chen
J
J
Lai
H
H
Wang
L h
Lh
Yang
P
P
Marcus
S
S
D001665:Binding Sites; D018797:Cell Cycle Proteins; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000378:metabolism; D054462:p21-Activated Kinases; D018909:src Homology Domains
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; C089770:scd2 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C094341:shk1 protein, S pombe; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae