The Chaperones Hsp90 and Cdc37 Mediate the Maturation and Stabilization of Protein Kinase C through a Conserved PXXP Motif in the C-terminal Tail
The Chaperones Hsp90 and Cdc37 Mediate the Maturation and Stabilization of Protein Kinase C through a Conserved PXXP Motif in the C-terminal Tail
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DOI：
10.1074/jbc.m808436200
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发表时间：
2009-02-20
影响因子：
4.8
通讯作者：
Newton, Alexandra C.
中科院分区：
文献类型：
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作者：
Gould, Christine M.;Kannan, Natarajan;Newton, Alexandra C.
蛋白激酶C（PKC）的生命周期受到酶成熟、维持活化活性酶和降解酶的机制的严格控制。在这里，我们表明，保守的PXXP基序（Kannan，N.，Haste，N.，Taylor，S.美国，和Neuwald，A. F.等人（2007）Proc.Natl. Acad. Sci.联合S. A. 104，1272-1277），在AGC（c-AMP依赖性蛋白激酶/蛋白激酶G/蛋白激酶C）激酶的C-末端尾部，控制常规和新型PKC同工酶的加工磷酸化，这是酶成熟为信号活性物质的必需步骤。在传统的PKC β II中，Pro-616和Pro-619突变为Ala，消除了激酶的磷酸化和活性。免疫共沉淀研究表明，传统的和新的，但不是非典型的，PKC同工酶结合的伴侣Hsp 90和Cdc 37通过PXXP依赖的机制。Hsp 90和Cdc 37的抑制剂显著降低PKC的磷酸化加工速率。在磷酸化处理的两个C-末端位点中，疏水基序而不是转向基序受Hsp 90调节。将纯化的Hsp 90覆盖到含有覆盖PKC β II催化结构域的肽的肽阵列上，确定了PXXP片段周围的区域，但不是PXXP基序本身，作为Hsp 90的主要结合决定簇。然而，这些Hsp 90结合区域通过在α E-螺旋中的PXXP基序和保守的Tyr（Tyr-446）之间形成的“分子钳”拴系到C-末端尾部。通过将Tyr突变为Ala来破坏钳，重现了突变PXXP基序的磷酸化缺陷。这些数据是一致的模型，其中的分子钳创建的PXXP基序在C-末端的尾巴和决定因素的α E-螺旋的催化结构域允许的伴侣蛋白Hsp 90和Cdc 37结合新合成的PKC，PKC的磷酸化处理所需的事件。
The life cycle of protein kinase C (PKC) is tightly controlled by mechanisms that mature the enzyme, sustain the activation-competent enzyme, and degrade the enzyme. Here we show that a conserved PXXP motif (Kannan, N., Haste, N., Taylor, S. S., and Neuwald, A. F. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 1272-1277), in the C-terminal tail of AGC (c-AMP-dependent protein kinase/protein kinase G/protein kinase C) kinases, controls the processing phosphorylation of conventional and novel PKC isozymes, a required step in the maturation of the enzyme into a signaling-competent species. Mutation of both Pro-616 and Pro-619 to Ala in the conventional PKC beta II abolishes the phosphorylation and activity of the kinase. Co-immunoprecipitation studies reveal that conventional and novel, but not atypical, PKC isozymes bind the chaperones Hsp90 and Cdc37 through a PXXP-dependent mechanism. Inhibitors of Hsp90 and Cdc37 significantly reduce the rate of processing phosphorylation of PKC. Of the two C-terminal sites processed by phosphorylation, the hydrophobic motif, but not the turn motif, is regulated by Hsp90. Overlay of purified Hsp90 onto a peptide array containing peptides covering the catalytic domain of PKC beta II identified regions surrounding the PXXP segment, but not the PXXP motif itself, as major binding determinants for Hsp90. These Hsp90-binding regions, however, are tethered to the C-terminal tail via a "molecular clamp" formed between the PXXP motif and a conserved Tyr (Tyr-446) in the alpha E-helix. Disruption of the clamp by mutation of the Tyr to Ala recapitulates the phosphorylation defect of mutating the PXXP motif. These data are consistent with a model in which a molecular clamp created by the PXXP motif in the C-terminal tail and determinants in the alpha E-helix of the catalytic domain allows the chaperones Hsp90 and Cdc37 to bind newly synthesized PKC, a required event in the processing of PKC by phosphorylation.