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Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.

Roelants FM, Su BM, von Wulffen J, Ramachandran S, Sartorel E, Trott AE, Thorner J - J. Cell Biol. (2015)

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.Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function.Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.

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Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

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Gin4 phosphorylates Fpk1 in vivo and in vitro. (A) Wild-type strain (YFR221, WT) or an isogenic gin4Δ (YFR224) expressing Fpk1-GFP were grown to mid-exponential phase and lysed. The resulting extracts were either not treated or treated with CIP or CIP + Na3VO4, resolved on a Phos-tag gel, and analyzed by immunoblotting with anti-GFP antibody. (B) GST-Gin4 (pAB1, WT) or catalytically inactive (“kinase-dead,” KD) mutant, GST-Gin4(K48A) (pAT103), were purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1 (pFR143, WT) or catalytically inactive (KD) mutant GST-Fpk1(D621A) (pFR144), also purified from E. coli. Resulting products were resolved by SDS-PAGE and analyzed by autoradiography (left) and staining with Coomassie dye (right). (C) GST-Gin4 (pAB1, WT) was purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1(1–472) (pBS1, N) or catalytically inactive GST-Fpk1(473–893; D621A) (pBS2, C), which were also purified from E. coli, and the products were analyzed as in B. (D) As in C, except that either GST-Gin4 (pAB1, WT) or catalytically inactive (KD) GST-Gin4(K48A) (pAT103) were incubated with either GST-Fpk1(1–472) (pBS1, WT) or GST-Fpk1(1–472; 11A) in which 11 Gin4 phosphorylation sites were mutated to Ala (pJW2).
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fig2: Gin4 phosphorylates Fpk1 in vivo and in vitro. (A) Wild-type strain (YFR221, WT) or an isogenic gin4Δ (YFR224) expressing Fpk1-GFP were grown to mid-exponential phase and lysed. The resulting extracts were either not treated or treated with CIP or CIP + Na3VO4, resolved on a Phos-tag gel, and analyzed by immunoblotting with anti-GFP antibody. (B) GST-Gin4 (pAB1, WT) or catalytically inactive (“kinase-dead,” KD) mutant, GST-Gin4(K48A) (pAT103), were purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1 (pFR143, WT) or catalytically inactive (KD) mutant GST-Fpk1(D621A) (pFR144), also purified from E. coli. Resulting products were resolved by SDS-PAGE and analyzed by autoradiography (left) and staining with Coomassie dye (right). (C) GST-Gin4 (pAB1, WT) was purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1(1–472) (pBS1, N) or catalytically inactive GST-Fpk1(473–893; D621A) (pBS2, C), which were also purified from E. coli, and the products were analyzed as in B. (D) As in C, except that either GST-Gin4 (pAB1, WT) or catalytically inactive (KD) GST-Gin4(K48A) (pAT103) were incubated with either GST-Fpk1(1–472) (pBS1, WT) or GST-Fpk1(1–472; 11A) in which 11 Gin4 phosphorylation sites were mutated to Ala (pJW2).

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.


Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.

Roelants FM, Su BM, von Wulffen J, Ramachandran S, Sartorel E, Trott AE, Thorner J - J. Cell Biol. (2015)

Gin4 phosphorylates Fpk1 in vivo and in vitro. (A) Wild-type strain (YFR221, WT) or an isogenic gin4Δ (YFR224) expressing Fpk1-GFP were grown to mid-exponential phase and lysed. The resulting extracts were either not treated or treated with CIP or CIP + Na3VO4, resolved on a Phos-tag gel, and analyzed by immunoblotting with anti-GFP antibody. (B) GST-Gin4 (pAB1, WT) or catalytically inactive (“kinase-dead,” KD) mutant, GST-Gin4(K48A) (pAT103), were purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1 (pFR143, WT) or catalytically inactive (KD) mutant GST-Fpk1(D621A) (pFR144), also purified from E. coli. Resulting products were resolved by SDS-PAGE and analyzed by autoradiography (left) and staining with Coomassie dye (right). (C) GST-Gin4 (pAB1, WT) was purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1(1–472) (pBS1, N) or catalytically inactive GST-Fpk1(473–893; D621A) (pBS2, C), which were also purified from E. coli, and the products were analyzed as in B. (D) As in C, except that either GST-Gin4 (pAB1, WT) or catalytically inactive (KD) GST-Gin4(K48A) (pAT103) were incubated with either GST-Fpk1(1–472) (pBS1, WT) or GST-Fpk1(1–472; 11A) in which 11 Gin4 phosphorylation sites were mutated to Ala (pJW2).
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fig2: Gin4 phosphorylates Fpk1 in vivo and in vitro. (A) Wild-type strain (YFR221, WT) or an isogenic gin4Δ (YFR224) expressing Fpk1-GFP were grown to mid-exponential phase and lysed. The resulting extracts were either not treated or treated with CIP or CIP + Na3VO4, resolved on a Phos-tag gel, and analyzed by immunoblotting with anti-GFP antibody. (B) GST-Gin4 (pAB1, WT) or catalytically inactive (“kinase-dead,” KD) mutant, GST-Gin4(K48A) (pAT103), were purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1 (pFR143, WT) or catalytically inactive (KD) mutant GST-Fpk1(D621A) (pFR144), also purified from E. coli. Resulting products were resolved by SDS-PAGE and analyzed by autoradiography (left) and staining with Coomassie dye (right). (C) GST-Gin4 (pAB1, WT) was purified from E. coli, incubated with γ-[32P]ATP and either GST-Fpk1(1–472) (pBS1, N) or catalytically inactive GST-Fpk1(473–893; D621A) (pBS2, C), which were also purified from E. coli, and the products were analyzed as in B. (D) As in C, except that either GST-Gin4 (pAB1, WT) or catalytically inactive (KD) GST-Gin4(K48A) (pAT103) were incubated with either GST-Fpk1(1–472) (pBS1, WT) or GST-Fpk1(1–472; 11A) in which 11 Gin4 phosphorylation sites were mutated to Ala (pJW2).
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.

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.Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function.Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.

Show MeSH
Related in: MedlinePlus