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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 effectors 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 …