Targeted Quantification of Phosphorylation Sites Identifies STRIPAK-Dependent Phosphorylation of the Hippo Pathway-Related Kinase SmKIN3

We showed recently that the erminal enter inase III (GCKIII) SmKIN3 from the fungus is involved in sexual development and hyphal septation. Our recent extensive global proteome and phosphoproteome analysis revealed that SmKIN3 is a target of the iatin- nteracting hosphatase nd inase (STRIPAK) multisubunit complex. Here, using protein samples from the wild type and three STRIPAK mutants, we applied absolute quantification by arallel- eaction onitoring (PRM) to analyze phosphorylation site occupancy in SmKIN3 and other eptation nitiation etwork (SIN) components, such as CDC7 and DBF2, as well as BUD4, acting downstream of SIN. For SmKIN3, we show that phosphorylation of S668 and S686 is decreased in mutants lacking distinct subunits of STRIPAK, while a third phosphorylation site, S589, was not affected. We constructed SmKIN3 mutants carrying phospho-mimetic and phospho-deficient codons for phosphorylation sites S589, S668, and S686. Investigation of hyphae in a Δ strain complemented by the S668 and S686 mutants showed a hyper-septation phenotype, which was absent in the wild type, the Δ strain complemented with the wild-type gene, and the S589 mutant. Furthermore, localization studies with SmKIN3 phosphorylation variants and STRIPAK mutants showed that SmKIN3 preferentially localizes at the terminal septa, which is distinctly different from the localization of the wild-type strains. We conclude that STRIPAK-dependent phosphorylation of SmKIN3 has an impact on controlled septum formation and on the time-dependent localization of SmKIN3 on septa at the hyphal tip. Thus, STRIPAK seems to regulate SmKIN3, as well as DBF2 and BUD4 phosphorylation, affecting septum formation. Phosphorylation and dephosphorylation of proteins are fundamental posttranslational modifications that determine the fine-tuning of their biological activity. Involved in this modification process is the recently identified iatin- nteracting hosphatase nd inase (STRIPAK) multisubunit complex, which is evolutionarily conserved from fungi to humans. STRIPAK functions as a macromolecular assembly communicating through physical interactions with other conserved signaling protein complexes to constitute larger dynamic protein networks. Its function is implied in many cellular processes, such as signal transduction pathways, growth, and cellular differentiation. We applied absolute quantification of protein phosphorylation by arallel- eaction onitoring (PRM) to analyze phosphorylation site occupanc