Crosstalk between protein kinases Pho85-Pho80 and Rim11 regulates Pah1 phosphatidate phosphatase function in lipid synthesis.

In Saccharomyces cerevisiae, the Pah1 phosphatidate phosphatase is essential for the synthesis of the storage lipid triacylglycerol. Through its utilization of the substrate phosphatidate, Pah1 also exerts a negative regulatory effect on phospholipid synthesis, a process required for cell growth and proliferation. The function of Pah1 is controlled by its membrane association, which is regulated by phosphorylation and dephosphorylation. Multiple protein kinases phosphorylate Pah1 to maintain it in the cytosol, whereas recruitment and dephosphorylation by the Nem1-Spo7 phosphatase complex promotes its translocation to the nuclear/endoplasmic reticulum membrane. Cyclin-dependent kinase Pho85-Pho80 and glycogen synthase kinase Rim11 both phosphorylate Pah1, thereby influencing its localization and inhibiting its phosphatidate phosphatase activity. In this study, we demonstrate that Rim11 is a bona fide substrate of the Pho85-Pho80 complex, with kinase activity dependent on Rim11, ATP, and reaction time. Pho85-Pho80 phosphorylates Rim11 at Ser-8, which enhances the ability of Rim11 to phosphorylate Pah1. A Rim11 peptide containing the Ser-8 site served as a substrate for Pho85-Pho80 and inhibited the phosphorylation of full-length Rim11 by the protein kinase complex. Mutational analysis revealed that the phosphorylation state of Pah1, as mediated by Pho85-Pho80 and Rim11, regulates the expression of the key phospholipid-synthesizing enzyme Cho1 phosphatidylserine synthase. Collectively, these findings deepen our understanding of how Pho85-Pho80 modulates Rim11 through phosphorylation and highlight the interconnected roles of these protein kinases in controlling Pah1 function in lipid synthesis.