Rho2 Is a Target of the Farnesyltransferase Cpp1 and Acts Upstream of Pmk1 Mitogen-activated Protein Kinase Signaling in Fission Yeast

Abstract

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.

The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

“…In fission yeast, Rho1p binds directly to the Pck1p and Pck2p protein kinases of the PKC family, and it functions as a positive regulator for these kinases (Arellano et al, 1999b;Sayers et al, 2000). Recently it has been shown that Pck2p interacts with Mkh1p (MAP-KKK) and activates the Pmk1p signaling pathway (Ma et al, 2006). Here, we show that Rgf1p, a Rho1p-specific GEF, acts upstream from the Pck2-Pmk1p MAPK cell integrity signaling pathway.…”

“…It has recently been shown that Rho2p and Pck2p act upstream from the Pmk1p MAPK signaling pathway, thereby resulting in the vic (viable in the presence of immunosuppressant FK506 and chloride ion) phenotype upon mutation (Ma et al, 2006). Because Rgf1p was linked to Pck2p, which in turn has been linked to the MAPK pathway, we decided to examine the functional relationship between Rgf1p and Pmk1p signaling by analyzing whether the rgf1⌬ mutants showed the vic phenotype.…”

“…The MAPK module is composed of MAPKKK Mkh1p (Sengar et al, 1997), MAPKK Pek1p/Shk1p (Sugiura et al, 1999;Loewith et al, 2000), and MAPK Pmk1p/Spm1p (Toda et al, 1996b;Zaitsevskaya-Carter and Cooper, 1997). Although this module was identified several years ago, little is known about its upstream components or the phosphorylation substrates activated by the cell integrity signaling pathway (Ma et al, 2006;Takada et al, 2007). The MAPKKKs at the head of the module are often activated through phosphorylation and/or as a result of their interaction with a small GTP-binding protein of the Ras/Rho family in response to extracellular stimuli (Roux and Blenis, 2004).…”

Eisosomes Regulate Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Cortical Clusters and Mitogen-activated Protein (MAP) Kinase Signaling upon Osmotic Stress

“…Rho GTPases represent molecular "switches" upstream of MAP kinases in the cell integrity pathway. In fission yeast, Rho2 is the specific activator of the pathway by acting through the PKC protein Pck2 in response to osmotic stress (20,45). The Rho-GEF Rgf1 is also required for Pmk1 activation upon osmotic stress and acts upstream of Pck2 (21).…”

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

“…Previous studies demonstrated that Pmk1 phosphorylation and activation in response to glucose starvation is mediated via Pck2, one of the two orthologs of PKC (8,18,19). Rho2, one of the six Rho GTPases, is a main positive regulator upstream of the Pmk1 in some stress conditions (26,42), although its role in activation of Pmk1 in response to glucose starvation is debatable (27). Under normal growth conditions, disruption of rho2 ϩ , pck2 ϩ , or pmk1 ϩ did not have any effect on Gad8 Ser-546 phosphorylation or Gad8 kinase activity (Fig.…”