The novel Rho GTPase-activating protein family protein, Rga8, provides a potential link between Cdc42/p21-activated kinase and Rho signaling pathways in the fission yeast, Schizosaccharomyces pombe.
The PAK family kinase, Shk1, is an essential regulator of polarized growth in the fission yeast, Schizosaccharomyces pombe. Here we describe the characterization of a novel member of the RhoGAP family, Rga8, identified from a two-hybrid screen for proteins that interact with the Shk1 kinase domain. Although deletion of the rga8 gene in wild type S. pombe cells results in no obvious phenotypic defects under normal growth conditions, it partially suppresses the cold-sensitive growth and morphological defects of S. pombe cells carrying a hypomorphic allele of the shk1 gene. By contrast, overexpression of rga8 is lethal to shk1-defective cells and causes morphological and cytokinesis defects in wild type S. pombe cells. Consistent with a role for Rga8 as a downstream target of Shk1, we show that the Rga8 protein is directly phosphorylated by Shk1 in vitro and phosphorylated in a Shk1-dependent fashion in S. pombe cells. Fluorescence photomicroscopy of the GFP-Rga8 fusion protein indicates that Rga8 is localized to the cell ends during interphase and to the septum-forming region during cytokinesis. In S. pombe cells carrying the orb2-34 allele of shk1, Rga8 exhibits a monopolar pattern of localization, providing evidence that Shk1 contributes to the regulation of Rga8 localization. Although molecular analyses suggest that Rga8 functions as a GAP for the S. pombe Rho1 GTPase, genetic experiments suggest that Rga8 and Rho1 have a positive functional interaction and that gain of Rho1 function, like gain of Rga8 function, is lethal to Shk1-defective cells. Our results suggest that Rga8 is a Shk1 substrate that negatively regulates Shk1-dependent growth control pathway(s) in S. pombe, potentially through interaction with the Rho1 GTPase.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D017353:Gene Deletion; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D054462:p21-Activated Kinases; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D029702:Schizosaccharomyces pombe Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C094341:shk1 protein, S pombe; C515914:shk2 protein, S pombe; D020741:rho GTP-Binding Proteins