The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C
The turn motif is a phosphorylation switch that regulates the binding of Hsp70 to protein kinase C
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DOI：
10.1074/jbc.m204335200
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发表时间：
2002-08-30
影响因子：
4.8
通讯作者：
Newton, AC
中科院分区：
文献类型：
--
作者：
Gao, TY;Newton, AC
热休克蛋白在确保细胞蛋白质的正确折叠和成熟方面发挥着核心作用。在这里，我们证明了热休克蛋白Hsp70在延长激活的蛋白激酶C的寿命中具有新的作用。我们在蛋白激酶C的羧基末端结合伙伴的筛选中鉴定了Hsp70。免疫共沉淀实验表明，Hsp70特异性地结合非磷酸化的转角基序(蛋白激酶C BetaII中的Thr(641))，这是蛋白激酶C家族成员成熟时被磷酸化的三个启动位点之一。在体内，Hsp70与蛋白激酶C的相互作用可以通过编码蛋白激酶C的羧基末端或Hsp70的羧基末端的融合蛋白的共表达而被取消。脉冲追逐实验表明，Hsp70不调节蛋白激酶C的成熟：通过磷酸化处理的速度。在存在或不存在破坏结构的情况下也是如此。相反，HSP70延长了成熟蛋白激酶Q的寿命，这种相互作用的中断促进了成熟的蛋白激酶C在细胞中不溶于洗涤剂的部分积累，然后去磷酸化。此外，对K562细胞的研究表明，中断与Hsp70的相互作用会减缓蛋白激酶CβII介导的细胞从PMA诱导的生长停滞中恢复的速度。最后，我们发现AGC超家族的其他成员(Akt/蛋白激酶B和蛋白激酶A)也通过其非磷酸化的转角基序与Hsp70结合。我们的数据与一个模型一致，在该模型中，Hsp70与成熟蛋白激酶C的去磷酸化羧基末端结合，从而稳定蛋白质并允许酶的重新磷酸化。这种相互作用的破坏会阻止重新磷酸化，并针对该酶进行下调。
Heat shock proteins play central roles in ensuring the correct folding and maturation of cellular proteins. Here we show that the heat shock protein Hsp70 has a novel role in prolonging the lifetime of activated protein kinase C. We identified Hsp70 in a screen for binding partners for the carboxyl terminus of protein kinase C. Co-immunoprecipitation experiments revealed that Hsp70 specifically binds the unphosphorylated turn motif (Thr(641) in protein kinase C betaII), one of three priming sites phosphorylated during the maturation of protein kinase C family members. The interaction of Hsp70 with protein kinase C can be abolished in vivo by co-expression of fusion proteins encoding the carboxyl terminus of protein kinase C or the carboxyl terminus of Hsp70. Pulse-chase experiments reveal that Hsp70 does not regulate the maturation of protein kinase C: the rate of processing by phosphorylation. is the same in the presence or absence of disrupting constructs. Rather, Hsp70 prolongs the lifetime of mature protein kinase Q disruption of the interaction promotes the accumulation of matured and then dephosphorylated protein kinase C in the detergent-insoluble fraction of cells. Furthermore, studies with K562 cells reveal that disruption of the interaction with Hsp70 slows the protein kinase C betaII-mediated recovery of cells from PMA-induced growth arrest. Last, we show that other members of the AGC superfamily (Akt/protein kinase B and protein kinase A) also bind Hsp70 via their unphosphorylated turn motifs. Our data are consistent with a model in which Hsp70 binds the dephosphorylated carboxyl terminus of mature protein kinase C, thus stabilizing the protein and allowing re-phosphorylation of the enzyme. Disruption of this interaction prevents re-phosphorylation and targets the enzyme for down-regulation.