Endothelial cAMP-specific phosphodiesterase PDE3A is one of the major negative regulators of the endothelial barrier function in acute lung injury models. However, the mechanisms underlying its regulation still need to be fully resolved. We show here that the PDE3A is a newly described client of the molecular chaperone heat shock protein 90 (hsp90). In endothelial cells (ECs), hsp90 inhibition by geldanamycin (GA) led to a disruption of the hsp90/PDE3A complex, followed by a significant decrease in PDE3A protein levels. The decrease in PDE3A protein levels was ubiquitin-proteasome–dependent and required the activity of the E3 ubiquitin ligase C terminus of Hsc70-interacting protein. GA treatment also enhanced the association of PDE3A with hsp70, which partially prevented PDE3A degradation. GA-induced decreases in PDE3A protein levels correlated with decreased PDE3 activity and increased cAMP levels in EC. We also demonstrated that protein kinase G–dependent phosphorylation of PDE3A at Ser can signal the dissociation of PDE3A from hsp90 and PDE3A degradation. This was confirmed by endogenous PDE3A phosphorylation and degradation in 8-Br-cGMP– or 8-CPT-cGMP– and Bay 41-8543–stimulated EC and comparisons of WT- and phospho-mimic SD mutant PDE3A protein stability in transiently transfected HEK293 cells. In conclusion, we have identified a new mechanism of PDE3A regulation mediated by the ubiquitin-proteasome system. Further, the degradation of PDE3A is controlled by the phosphorylation of S and the interaction with hsp90. We speculate that targeting the PDE3A/hsp90 complex could be a therapeutic approach for acute lung injury.

中文翻译：

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Hsp90 客户蛋白磷酸二酯酶 3A PKG 依赖性蛋白酶体降解介导的环核苷酸串扰新机制


内皮 cAMP 特异性磷酸二酯酶 PDE3A 是急性肺损伤模型中内皮屏障功能的主要负调节因子之一。但其调控机制仍需彻底解决。我们在此表明​​ PDE3A 是分子伴侣热休克蛋白 90 (hsp90) 的新描述的客户。在内皮细胞 (EC) 中，格尔德霉素 (GA) 抑制 hsp90 会导致 hsp90/PDE3A 复合物破坏，随后 PDE3A 蛋白水平显着降低。 PDE3A 蛋白水平的降低依赖于泛素蛋白酶体，并且需要 Hsc70 相互作用蛋白的 E3 泛素连接酶 C 末端的活性。 GA 处理还增强了 PDE3A 与 hsp70 的关联，从而部分阻止了 PDE3A 降解。 GA 诱导的 PDE3A 蛋白水平降低与 EC 中 PDE3 活性降低和 cAMP 水平升高相关。我们还证明，PDE3A Ser 位点的蛋白激酶 G 依赖性磷酸化可以发出 PDE3A 从 hsp90 解离和 PDE3A 降解的信号。通过 8-Br-cGMP 或 8-CPT-cGMP 和 Bay 41-8543 刺激的 EC 中的内源性 PDE3A 磷酸化和降解以及瞬时转染 HEK293 细胞中 WT 和磷酸模拟 SD 突变体 PDE3A 蛋白稳定性的比较证实了这一点。总之，我们已经确定了泛素-蛋白酶体系统介导的 PDE3A 调节的新机制。此外，PDE3A 的降解是通过 S 的磷酸化以及与 hsp90 的相互作用来控制的。我们推测靶向 PDE3A/hsp90 复合物可能是治疗急性肺损伤的方法。