Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.
Bottom Line: By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly.As judged by biochemical and phenotypic criteria, a mutant (Fpk1(11A)), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine.Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function.
Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.Show MeSH
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Mentions: To assess whether phosphorylation of Fpk1 is Gin4 dependent in vivo, we first analyzed the migration pattern of Fpk1 fused to GFP using phosphate affinity (Phos-tag) gels (Kinoshita et al., 2009) and found a readily detectable level of a slower mobility Fpk1-GFP species whose appearance required Gin4 (Fig. 2 A, middle). This isoform was indeed due to phosphorylation because it was eliminated by treatment of the samples with calf intestinal phosphatase (CIP; Fig. 2 A, left), but persisted in the presence of CIP and the phosphatase inhibitor Na3VO4 (Fig. 2 A, right). To determine whether the Gin4-dependent phosphorylation of Fpk1-GFP observed in vivo may be direct, we tested whether Fpk1 serves as a substrate of Gin4 in vitro. To avoid any possibility of self-phosphorylation, catalytically inactive GST-Fpk1(D621A) (Roelants et al., 2010) purified from Escherichia coli was incubated with purified recombinant GST-Gin4 or an equivalent amount of a catalytically inactive mutant, GST-Gin4(K48A). We found that Fpk1 was readily phosphorylated by Gin4 (Fig. 2 B). Furthermore, when fused to GST, the N-terminal noncatalytic domain of Fpk1 was a much more robust substrate for Gin4 than its C-terminal (kinase) domain (Fig. 2 C). After exhaustive in vitro phosphorylation of Fpk1(1–472) by Gin4 in vitro, the sites of phosphorylation were mapped by mass spectrometry. This analysis revealed that 17 Ser or Thr residues were detectably modified, the majority of which fit the consensus (-R/KxxS-; Fig. S2 C), in accord with the phosphoacceptor site motif preference of Gin4 determined using synthetic peptide arrays (Mok et al., 2010). 11 of the most efficiently phosphorylated sites in Fpk1(1–472) were mutated to Ala, which decreased phosphorylation to a near-background level (Fig. 2 D), confirming that these sites were Gin4 targets.
Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.