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

Schizosaccharomyces pombe rho1(+) is required for maintenance of cell integ rity and polarization of the actin cytoskeleton, However, no other effector besides the (1,3)beta-D-glucan synthase enzyme has been identified in S, p ombe. We have further investigated if rho1(+) signalling could be also medi ated by the two protein kinase C homologues, pck1p and pck2p, We show in th is study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effecters do. Interestingly, the interaction was mappe d in a different part of the proteins than in Saccharomyces cerevisiae Pkc1 p, Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabiliz es the kinases, Detailed biochemical analysis suggests that pck2p is more i mportant in the regulation of the enzyme (1-3)beta-D-glucan synthase, Thus, overexpression of pck2(+), but not pck1(+), caused a general increase in c ell wall biosynthesis, mainly in beta-glucan, and (1-3)beta-D-glucan syntha se activity was considerably augmented. When this activity was separated in to 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 ma intenance of cell integrity. pck1 Delta and pck2 Delta strains present a nu mber of defects related to the cell wall, indicating that this structure mi ght be co-ordinately regulated by both kinases, In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence ind icates 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)beta-D-glucan synthase, pck1(+) also showed a genetic interact ion with ras1(+) and ral1(+) suggesting the existence of a functional link between both signalling pathways.