Direct Binding and In Vivo Regulation of the Fission Yeast p21-Activated Kinase Shk1 by the SH3 Domain Protein Scd2

Abstract

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 Nterminal 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.

Cold-induced PGC-1α expression modulates muscle glucose uptake through an insulin receptor/Akt-independent, AMPK-dependent pathway

“…As a transcriptional coactivator, PGC-1␣ possesses several functional domains that participate in protein-protein interaction (30). Motifs that allow for interaction with PPAR␥ (31), nuclear respiratory factor (48), cAMP response element binding protein-binding protein (16), steroid receptor coactivator-1 (5), and myocyte enhancer factor-2 (22), have been identified and are thought to participate in the PGC-1␣-dependent regulation of expression of genes involved in an array of specialized cell functions, such as the response to environmental thermal oscillations (31), mitochondrial biogenesis (31,48), energy and nutrient homeostasis (27), muscle fiber type differentiation (19), adipocyte differentiation (31), cellular response to cytokines (29), and control of insulin secretion (7,49).…”

The Cdc42 Binding and Scaffolding Activities of the Fission Yeast Adaptor Protein Scd2

“…The disruption of the cdc42 gene is lethal to fission yeast, and the cdc42 mutants exhibit abnormal morphological phenotypes (9). Genetic and molecular studies suggest that a Dbl homology domain-containing Scd1 protein functions as the guanine nucleotide exchange factor for the fission yeast Cdc42 protein and that the GTP-bound Cdc42 activates Ste20 homologous kinase 1 (Shk1) 1 (12)(13)(14)(15). The fission yeast Shk1 belongs to the family of the p21-activated kinase (PAK), which is a highly conserved serine/ threonine kinase activated by the Cdc42/Rac protein.…”

“…Recent studies indicate that the Cdc42/Rac protein relieves this autoinhibition, thereby allosterically inducing the activation of the kinase activity (18 -22). The fission yeast PAK family kinase, Shk1, is positively regulated by the adaptor protein, Scd2 (shape and conjugation deficiency 2, also called Ral3), as well as by Cdc42 (12,13). Co-expression of Scd2 enhances the binding of Shk1 to Cdc42 in a yeast two-hybrid system (12,13).…”

“…The fission yeast PAK family kinase, Shk1, is positively regulated by the adaptor protein, Scd2 (shape and conjugation deficiency 2, also called Ral3), as well as by Cdc42 (12,13). Co-expression of Scd2 enhances the binding of Shk1 to Cdc42 in a yeast two-hybrid system (12,13). Furthermore, the overexpression of Scd2 stimulates the autophosphorylation activity of Shk1 in vivo, whereas the recombinant Scd2 by itself is not able to stimulate it in vitro (12).…”

“…Co-expression of Scd2 enhances the binding of Shk1 to Cdc42 in a yeast two-hybrid system (12,13). Furthermore, the overexpression of Scd2 stimulates the autophosphorylation activity of Shk1 in vivo, whereas the recombinant Scd2 by itself is not able to stimulate it in vitro (12). These results indicate that Scd2 participates in the activation of Shk1 but is not sufficient for it.…”