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Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

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Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

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Calcineurin and hyperosmotic stress do not affect Inp53 catalytic activity. (A) Coomassie-stained GST-Inp53 purified from cells treated with 1 μg/ml FK506 or vehicle and with or without 10 min of 1.25 M KCl. (B) The in vitro activity of each GST-Inp53 preparation shown in A was determined; the initial reaction rate is plotted as a function of PI(4,5)P2 concentration. GST purified equivalently displayed no phosphatase activity (Supplemental Figure S5).
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Figure 8: Calcineurin and hyperosmotic stress do not affect Inp53 catalytic activity. (A) Coomassie-stained GST-Inp53 purified from cells treated with 1 μg/ml FK506 or vehicle and with or without 10 min of 1.25 M KCl. (B) The in vitro activity of each GST-Inp53 preparation shown in A was determined; the initial reaction rate is plotted as a function of PI(4,5)P2 concentration. GST purified equivalently displayed no phosphatase activity (Supplemental Figure S5).

Mentions: To examine whether hyperphosphorylation induced by hyperosmotic stress or dephosphorylation by CN directly altered Inp53 catalytic activity, we purified Inp53 from yeast under conditions that maintained its phosphorylation state and SAC and IP5Pase domain activities (Figure 8A; Guo et al., 1999) and analyzed the kinetics of PI(4,5)P2 dephosphorylation by Inp53 in vitro. GST-Inp53 and GST were purified from cells treated either with vehicle or FK506 and grown in the presence or absence of hypertonic shock. GST-Inp53, but not GST, exhibited PI(4,5)P2 phosphatase activity in vitro (Figure 8B and Supplemental Figure S5) which was statistically equivalent in all preparations tested (Figure 8B). Thus we find no evidence that the intrinsic catalytic activity of Inp53 is altered either by the massive hyperphosphorylation that occurs after osmotic shock or by subsequent dephosphorylation via CN. However, changes in PI(4,5)P2 distribution in the membrane suggest that sites of Inp53 action are reorganized.


Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Calcineurin and hyperosmotic stress do not affect Inp53 catalytic activity. (A) Coomassie-stained GST-Inp53 purified from cells treated with 1 μg/ml FK506 or vehicle and with or without 10 min of 1.25 M KCl. (B) The in vitro activity of each GST-Inp53 preparation shown in A was determined; the initial reaction rate is plotted as a function of PI(4,5)P2 concentration. GST purified equivalently displayed no phosphatase activity (Supplemental Figure S5).
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Figure 8: Calcineurin and hyperosmotic stress do not affect Inp53 catalytic activity. (A) Coomassie-stained GST-Inp53 purified from cells treated with 1 μg/ml FK506 or vehicle and with or without 10 min of 1.25 M KCl. (B) The in vitro activity of each GST-Inp53 preparation shown in A was determined; the initial reaction rate is plotted as a function of PI(4,5)P2 concentration. GST purified equivalently displayed no phosphatase activity (Supplemental Figure S5).
Mentions: To examine whether hyperphosphorylation induced by hyperosmotic stress or dephosphorylation by CN directly altered Inp53 catalytic activity, we purified Inp53 from yeast under conditions that maintained its phosphorylation state and SAC and IP5Pase domain activities (Figure 8A; Guo et al., 1999) and analyzed the kinetics of PI(4,5)P2 dephosphorylation by Inp53 in vitro. GST-Inp53 and GST were purified from cells treated either with vehicle or FK506 and grown in the presence or absence of hypertonic shock. GST-Inp53, but not GST, exhibited PI(4,5)P2 phosphatase activity in vitro (Figure 8B and Supplemental Figure S5) which was statistically equivalent in all preparations tested (Figure 8B). Thus we find no evidence that the intrinsic catalytic activity of Inp53 is altered either by the massive hyperphosphorylation that occurs after osmotic shock or by subsequent dephosphorylation via CN. However, changes in PI(4,5)P2 distribution in the membrane suggest that sites of Inp53 action are reorganized.

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus