ArPIKfyve Regulates Sac3 Protein Abundance and Turnover DISRUPTION OF THE MECHANISM BY Sac3I41T MUTATION CAUSING CHARCOT-MARIE-TOOTH 4J DISORDER
ArPIKfyve Regulates Sac3 Protein Abundance and Turnover DISRUPTION OF THE MECHANISM BY Sac3I41T MUTATION CAUSING CHARCOT-MARIE-TOOTH 4J DISORDER
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DOI：
10.1074/jbc.c110.154658
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发表时间：
2010-08-27
影响因子：
4.8
通讯作者：
Shisheva, Assia
中科院分区：
文献类型：
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作者：
Ikonomov, Ognian C.;Sbrissa, Diego;Shisheva, Assia
哺乳动物磷脂酰肌醇（3，5）-二磷酸（PtdIns（3，5）P-2）磷酸酶Sac 3和PtdIns 3 P-5激酶PIKfyve的相关调节剂ArPIKfyve形成稳定的二元复合物，其在三元复合物中与PIKfyve缔合以增加PtdIns（3，5）P-2产生。ArPIKfyve-Sac 3子复合体是否在PIKfyve上下文之外起作用是未知的。在这里，我们表明，ArPIKfyve（WT）在哺乳动物细胞中的稳定或瞬时表达提高了稳态蛋白水平和Sac 3的PtdIns（3，5）P-2水解活性，而ArPIKfyve的敲低具有相反的效果。这些操作不改变Sac 3 mRNA水平，表明ArPIKfyve可能控制Sac 3蛋白降解。放线菌酮对COS细胞中蛋白质合成的抑制揭示了表达的Sac 3（WT）的显著快速周转（t（1/2）= 18.8 min），这是由蛋白酶体依赖性清除引起的，如通过抑制蛋白酶体活性后延长的Sac 3（WT）半衰期所证明的。ArPIKfyve（WT）而非N-或C-末端一半的共表达延长了Sac 3 WT半衰期，这与通过与全长ArPIKfyve缔合而增强的Sac 3蛋白稳定性一致。我们进一步证明，突变体Sac 3，窝藏致病Ile到Thr取代在41位发现的CMT 4J疾病患者，是类似于Sac 3（WT）的PtdIns（3，5）P2水解活性，与ArPIKfyve，或快速蛋白酶体依赖性清除。然而，值得注意的是，共表达的ArPIKfyve（WT）既没有升高稳态Sac 3（I41 T），也没有延长Sac 3（I41 T）半衰期，这表明与Sac 3（WT）不同，ArPIKfyve不能防止Sac 3（I41 T）快速丢失。总之，我们的数据确定了一种新的调节机制，ArPIKfyve通过减弱Sac 3蛋白酶体依赖性降解来增强Sac 3丰度，并表明这种机制的失败可能是CMT 4J发病机制中的主要分子缺陷。
The mammalian phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P-2) phosphatase Sac3 and ArPIKfyve, the associated regulator of the PtdIns3P-5 kinase PIKfyve, form a stable binary complex that associates with PIKfyve in a ternary complex to increase PtdIns(3,5)P-2 production. Whether the ArPIKfyve-Sac3 subcomplex functions outside the PIKfyve context is unknown. Here we show that stable or transient expression of ArPIKfyve(WT) in mammalian cells elevates steady-state protein levels and the PtdIns(3,5)P-2-hydrolyzing activity of Sac3, whereas knockdown of ArPIKfyve has the opposite effect. These manipulations do not alter the Sac3 mRNA levels, suggesting that ArPIKfyve might control Sac3 protein degradation. Inhibition of protein synthesis in COS cells by cycloheximide reveals remarkably rapid turnover of expressed Sac3(WT) (t(1/2) = 18.8 min), resulting from a proteasome-dependent clearance as evidenced by the extended Sac3(WT) half-life upon inhibiting proteasome activity. Coexpression of ArPIKfyve(WT), but not the N-or C-terminal halves, prolongs the Sac3WT half-life consistent with enhanced Sac3 protein stability through association with full-length ArPIKfyve. We further demonstrate that mutant Sac3, harboring the pathogenic Ile-to-Thr substitution at position 41 found in patients with CMT4J disorder, is similar to Sac3(WT) with regard to PtdIns(3,5)P2-hydrolyzing activity, association with ArPIKfyve, or rapid proteasome-dependent clearance. Remarkably, however, neither is the steady-state Sac3(I41T) elevated nor is the Sac3(I41T) half-life extended by coexpressed ArPIKfyve(WT), indicating that unlike with Sac3(WT), ArPIKfyve fails to prevent Sac3(I41T) rapid loss. Together, our data indentify a novel regulatory mechanism whereby ArPIKfyve enhances Sac3 abundance by attenuating Sac3 proteasome-dependent degradation and suggest that a failure of this mechanism could be the primary molecular defect in the pathogenesis of CMT4J.