Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAF^V600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAF^V600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAF^V600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a ‘factory reset’ of the kinase prior to release.

HSP90 分子伴侣系统对客户蛋白激酶的激活受到 HSP90 多个位点、激酶特异性共伴侣 CDC37 和激酶客户本身磷酸化的影响。客户激酶调节性磷酸化的去除及其从 HSP90-CDC37 系统中的释放取决于 Ser/Thr 磷酸酶 PP5，PP5 通过其 N 端 TPR 结构域与 HSP90 结合。在这里，我们展示了与 HSP90-CDC37 结合的致癌蛋白激酶客户端 BRAF ^V600E的冷冻电镜结构，显示了 V600E 突变如何有利于 BRAF 与 HSP90-CDC37 的关联。 HSP90-CDC37-BRAF ^V600E与 PP5 处于自动抑制和激活构象的复合物的结构，连同其对 BRAF ^V600E和 CRAF 的磷酸酶活性的蛋白质组学分析，揭示了 PP5 如何通过招募到 HSP90 复合物而被激活。 PP5 全面使客户蛋白去磷酸化，去除调节伙伴（例如 14-3-3 蛋白）的相互作用位点，从而在释放之前对激酶进行“工厂重置”。