Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity

Abstract

Schizosaccharomyces pombe rho1+ is required for maintenance of cell integrity and polarization of the actin cytoskeleton. However, no other effector besides the (1,3)β-D-glucan synthase enzyme has been identified in S. pombe. We have further investigated if rho1+ signalling could be also mediated by the two protein kinase C homologues, pck1p and pck2p. We show in this study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effecters do. Interestingly, the interaction was mapped in a different part of the proteins than in Saccharomyces cerevisiae Pkc1p. Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabilizes the kinases. Detailed biochemical analysis suggests that pck2p is more important in the regulation of the enzyme (1-3)β-D-glucan synthase. Thus, overexpression of pck2+, but not pck1+, caused a general increase in cell wall biosynthesis, mainly in β-glucan, and (1-3)β-D-glucan synthase activity was considerably augmented. When this activity was separated into soluble and membrane fractions and reconstituted, the increase caused by pck2+ overexpression was exclusively detected in the membrane component. We also show that both protein kinase C homologues are required for the maintenance of cell integrity. pck1Δ and pck2Δ strains present a number of defects related to the cell wall, indicating that this structure might be co-ordinately regulated by both kinases. In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence indicates that both pck1+ and pck2+ interact with cps1+ and gls2+, two genes similar to S. cerevisiae FKS1 and FKS2 that encode membrane subunits of the (1-3)β-D-glucan synthase, pck1+ also showed a genetic interaction with ras1+ and ral1+ suggesting the existence of a functional link between both signalling pathways.