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 fissi on 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 S cd2 (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 tao N-terminal SH3 domains of Scd2 is bath necessary and sufficient for int eraction with Shk1. While full-length Scd2 interacted with only the RI N-te rminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 i nteracted with both the R1 and R3 subdomains of Shk1, suggesting that the n on-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the auto phosphorylation 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 experim ents suggest that Scd2 may stimulate Shk1 catalytic function, at least in p art, 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 Cd c42 to positively regulate Shk1 function.