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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 controls inner leaflet PtdEth and Cdc42-GTP level. During progression through the cell cycle, Gin4 localizes at the incipient bud site, and phosphorylates and inhibits Fpk1 at the same location, preventing flippase activation and keeping PtdEth level on the inner leaflet low. Because inner leaflet PtdEth promotes Cdc42-GTP inactivation (Saito et al., 2007; Das et al., 2012), Gin4-imposed reduction in PtdEth allows for buildup of Cdc42-GTP to stimulate recruitment and function of effectors, like formin Bni1, that promote bud emergence. Further accumulation of Gin4 at the septin collar will continue to suppress the inner leaflet PtdEth level, allowing for localized protection of Cdc42-GTP, perhaps facilitating recruitment and activation of effectors required for efficient cytokinesis. Despite a normally beneficial role, flippase function also permits entry of toxic xenobiotics. To help prevent an influx of such noxious agents when the PM barrier is already compromised by heat stress or other membrane-perturbing or -damaging agents, an independent stress response mediated by protein kinase Ypk1 also down-regulates flippase function by inhibiting Fpk1. Full Ypk1 activity requires its stimulation by two PM-associated protein kinases: eisosome-bound Pkh1 and the TORC2 complex.
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fig7: Gin4 controls inner leaflet PtdEth and Cdc42-GTP level. During progression through the cell cycle, Gin4 localizes at the incipient bud site, and phosphorylates and inhibits Fpk1 at the same location, preventing flippase activation and keeping PtdEth level on the inner leaflet low. Because inner leaflet PtdEth promotes Cdc42-GTP inactivation (Saito et al., 2007; Das et al., 2012), Gin4-imposed reduction in PtdEth allows for buildup of Cdc42-GTP to stimulate recruitment and function of effectors, like formin Bni1, that promote bud emergence. Further accumulation of Gin4 at the septin collar will continue to suppress the inner leaflet PtdEth level, allowing for localized protection of Cdc42-GTP, perhaps facilitating recruitment and activation of effectors required for efficient cytokinesis. Despite a normally beneficial role, flippase function also permits entry of toxic xenobiotics. To help prevent an influx of such noxious agents when the PM barrier is already compromised by heat stress or other membrane-perturbing or -damaging agents, an independent stress response mediated by protein kinase Ypk1 also down-regulates flippase function by inhibiting Fpk1. Full Ypk1 activity requires its stimulation by two PM-associated protein kinases: eisosome-bound Pkh1 and the TORC2 complex.

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.


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 controls inner leaflet PtdEth and Cdc42-GTP level. During progression through the cell cycle, Gin4 localizes at the incipient bud site, and phosphorylates and inhibits Fpk1 at the same location, preventing flippase activation and keeping PtdEth level on the inner leaflet low. Because inner leaflet PtdEth promotes Cdc42-GTP inactivation (Saito et al., 2007; Das et al., 2012), Gin4-imposed reduction in PtdEth allows for buildup of Cdc42-GTP to stimulate recruitment and function of effectors, like formin Bni1, that promote bud emergence. Further accumulation of Gin4 at the septin collar will continue to suppress the inner leaflet PtdEth level, allowing for localized protection of Cdc42-GTP, perhaps facilitating recruitment and activation of effectors required for efficient cytokinesis. Despite a normally beneficial role, flippase function also permits entry of toxic xenobiotics. To help prevent an influx of such noxious agents when the PM barrier is already compromised by heat stress or other membrane-perturbing or -damaging agents, an independent stress response mediated by protein kinase Ypk1 also down-regulates flippase function by inhibiting Fpk1. Full Ypk1 activity requires its stimulation by two PM-associated protein kinases: eisosome-bound Pkh1 and the TORC2 complex.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4315245&req=5

fig7: Gin4 controls inner leaflet PtdEth and Cdc42-GTP level. During progression through the cell cycle, Gin4 localizes at the incipient bud site, and phosphorylates and inhibits Fpk1 at the same location, preventing flippase activation and keeping PtdEth level on the inner leaflet low. Because inner leaflet PtdEth promotes Cdc42-GTP inactivation (Saito et al., 2007; Das et al., 2012), Gin4-imposed reduction in PtdEth allows for buildup of Cdc42-GTP to stimulate recruitment and function of effectors, like formin Bni1, that promote bud emergence. Further accumulation of Gin4 at the septin collar will continue to suppress the inner leaflet PtdEth level, allowing for localized protection of Cdc42-GTP, perhaps facilitating recruitment and activation of effectors required for efficient cytokinesis. Despite a normally beneficial role, flippase function also permits entry of toxic xenobiotics. To help prevent an influx of such noxious agents when the PM barrier is already compromised by heat stress or other membrane-perturbing or -damaging agents, an independent stress response mediated by protein kinase Ypk1 also down-regulates flippase function by inhibiting Fpk1. Full Ypk1 activity requires its stimulation by two PM-associated protein kinases: eisosome-bound Pkh1 and the TORC2 complex.
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.

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