Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis.
Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis.
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DOI：
10.1177/2045893217753156
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发表时间：
2018-01
影响因子：
2.6
通讯作者：
Cioffi DL
中科院分区：
文献类型：
--
作者：
Hamilton CL;Abney KA;Vasauskas AA;Alexeyev M;Li N;Honkanen RE;Scammell JG;Cioffi DL
肺内皮细胞表达钙库操纵的钙进入电流（Isoc），这有助于内皮细胞间间隙的形成。Isoc由包括亲免素FKBP 51的蛋白质的异源复合物调节。FKBP 51通过尚未完全理解的机制抑制Isoc。在肺动脉内皮细胞（PAEC）中，我们已经表明FKBP 51增加微管聚合，这是FKBP 51抑制Isoc的关键事件。在神经元中，FKBP 51通过促进tau去磷酸化促进微管稳定性。然而，FKBP 51不具有磷酸酶活性。蛋白磷酸酶5（PP 5C/PPP 5C）可以使tau蛋白去磷酸化，并且与FKBP 51类似，PP 5C具有三肽重复序列（TPR），其介导与热休克蛋白90（HSP 90）伴侣/支架复合物的相互作用。因此，我们测试了PP 5C是否有助于FKBP 51介导的Isoc抑制。siRNA介导的PAEC中PP 5C表达的抑制和HEK 293细胞中PP 5C的遗传破坏都减弱了FKBP 51介导的Isoc抑制。重新引入有催化活性但无催化活性的PP 5C，恢复了FKBP 51介导的Isoc抑制。PAEC细胞分级分离研究在相同的膜组分中鉴定了PP 5C和ISOC异源复合物。此外，PP 5C与TRPC 4共沉淀，TRPC 4是ISOC通道的必需亚基。最后，为了确定PP 5C是否是FKBP 51介导的钙进入诱导的内皮细胞间间隙形成的抑制所必需的，我们通过宽视野显微镜测量间隙面积，并进行生物素间隙定量测定和电细胞底物阻抗传感（ECIS®）。总的来说，所呈现的数据表明PP 5C表达的抑制否定了FKBP 51的保护作用。这些观察结果将PP 5C鉴定为FKBP 51介导的Isoc抑制所需的ISOC异源复合物的新成员。
Pulmonary endothelial cells express a store-operated calcium entry current (Isoc), which contributes to inter-endothelial cell gap formation. Isoc is regulated by a heterocomplex of proteins that includes the immunophilin FKBP51. FKBP51 inhibits Isoc by mechanisms that are not fully understood. In pulmonary artery endothelial cells (PAECs) we have shown that FKBP51 increases microtubule polymerization, an event that is critical for Isoc inhibition by FKBP51. In neurons, FKBP51 promotes microtubule stability through facilitation of tau dephosphorylation. However, FKBP51 does not possess phosphatase activity. Protein phosphatase 5 (PP5C/PPP5C) can dephosphorylate tau, and similar to FKBP51, PP5C possesses tetratricopeptide repeats (TPR) that mediate interaction with heat shock protein-90 (HSP90) chaperone/scaffolding complexes. We therefore tested whether PP5C contributes to FKBP51-mediated inhibition of Isoc. Both siRNA-mediated suppression of PP5C expression in PAECs and genetic disruption of PP5C in HEK293 cells attenuate FKBP51-mediated inhibition of Isoc. Reintroduction of catalytically competent, but not catalytically inactive PP5C, restored FKBP51-mediated inhibition of Isoc. PAEC cell fractionation studies identified both PP5C and the ISOC heterocomplex in the same membrane fractions. Further, PP5C co-precipitates with TRPC4, an essential subunit of ISOC channel. Finally, to determine if PP5C is required for FKBP51-mediated inhibition of calcium entry-induced inter-endothelial cell gap formation, we measured gap area by wide-field microscopy and performed biotin gap quantification assay and electric cell-substrate impedance sensing (ECIS®). Collectively, the data presented indicate that suppression of PP5C expression negates the protective effect of FKBP51. These observations identify PP5C as a novel member of the ISOC heterocomplex that is required for FKBP51-mediated inhibition of Isoc.
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