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: We have demonstrated previously (Roelants et al., 2010) that the flippase-activating protein kinase Fpk1 (and its paralogue Fpk2) is subject to two physiologically important levels of control: via inhibitory phosphorylation by the membrane-stress activated protein kinase Ypk1 (and its paralogue Ypk2); and via stimulation specifically by the complex sphingolipid MIPC (by a mechanism not yet understood). As we show here, an equally important new component of this regulatory network is inhibitory phosphorylation by the septin-associated and cell cycle–regulated protein kinase Gin4 (Fig. 7). This specific function is a previously unrecognized role for Gin4, which was thought to be involved only in proper septin collar formation via direct phosphorylation of septin Shs1 (Mortensen et al., 2002; Asano et al., 2006). It has been suggested that this modification promotes formation of the septin collar that demarcates the bud neck during bud emergence, thereby generating a stable scaffold that recruits other proteins needed for bud growth and then cytokinesis (McMurray and Thorner, 2009; Oh and Bi, 2011). Consistent with such a function, after mitosis, Shs1 is dephosphorylated by the Rts1-bound isoform of phosphoprotein phosphatase 2A, concomitant with splitting of the septin collar into two discrete rings at cytokinesis (Dobbelaere et al., 2003). However, aberrant septin structure at the bud neck is displayed by only a very small fraction of the cells in a culture of a gin4Δ mutant (Longtine et al., 1998). Moreover, time-lapse microscopy of gin4Δ cells expressing a GFP-tagged septin revealed that, in the majority of these rare anomalous cells, the abnormal septin morphology eventually resolves into a uniform collar and the cell then divides (McMurray and Thorner, 2009). The findings we have presented here demonstrate that Gin4 control of Fpk1 governs the level of PtdEth in the inner leaflet of the PM and argue that a fundamentally important function of Gin4 is its role in regulation of PM bilayer asymmetry.
Affiliation: Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.