Delayed Phosphorylation of Classical Protein Kinase C (PKC) Substrates Requires PKC Internalization and Formation of the Pericentrion in a Phospholipase D (PLD)-dependent Manner
Delayed Phosphorylation of Classical Protein Kinase C (PKC) Substrates Requires PKC Internalization and Formation of the Pericentrion in a Phospholipase D (PLD)-dependent Manner
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DOI：
10.1074/jbc.m110.152330
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发表时间：
2011-06-03
影响因子：
4.8
通讯作者：
Hannun, Yusuf A.
中科院分区：
文献类型：
--
作者：
El-Osta, Mohamad A.;Idkowiak-Baldys, Jola;Hannun, Yusuf A.
先前证明，蛋白激酶C（PKC）的持续活化（30-60分钟）导致PKC α和β II易位到中心周围，中心周围是循环区室的动态子集，其形成依赖于PKC和磷脂酶D（PLD）。在这里，我们研究了是否形成的中心区调节PKC磷酸化底物的能力，特别是如果它减少底物磷酸化通过螯合PKC。令人惊讶的是，使用检测经典PKC的磷酸化底物的抗体，结果显示，大多数PKC磷酸化底物以延迟动力学被磷酸化，与PKC易位到中心周围的时间框架相关。底物磷酸化被PLD抑制剂阻断，并且没有观察到对PKC β II突变体（F663 D）的激活的响应，该突变体在与PLD的相互作用和内化中有缺陷。通过阻断网格蛋白依赖性内吞作用也抑制了磷酸化，证明PKC依赖性主要磷酸化作用需要内吞作用。5-羟色胺受体激活5-羟色胺表现出类似的反应佛波酯12-肉豆蔻酸酯13-乙酸酯，暗示了G蛋白偶联受体信号传导的延迟动力学的潜在作用。候选底物的评价显示，PKC底物p70 S6 K激酶的磷酸化以类似的方式表现。基于毛细管电泳的分馏显示，这些PKC底物中的大多数驻留在中心周围富集的组分中，而不是在质膜中。最后，蛋白质组学分析的中心体富集馏分揭示了几种蛋白质作为已知的PKC底物和/或蛋白质参与内吞运输。这些结果揭示了一个重要的作用，PKC的内化和作为关键的决定因素/放大器的PKC行动的中心周围。
It was previously demonstrated that sustained activation (30-60 min) of protein kinase C (PKC) results in translocation of PKC alpha and beta II to the pericentrion, a dynamic subset of the recycling compartment whose formation is dependent on PKC and phospholipase D (PLD). Here we investigated whether the formation of the pericentrion modulates the ability of PKC to phosphorylate substrates, especially if it reduces substrate phosphorylation by sequestering PKC. Surprisingly, using an antibody that detects phosphosubstrates of classical PKCs, the results showed that the majority of PKC phosphosubstrates are phosphorylated with delayed kinetics, correlating with the time frame of PKC translocation to the pericentrion. Substrate phosphorylation was blocked by PLD inhibitors and was not observed in response to activation of a PKC beta II mutant (F663D) that is defective in interaction with PLD and in internalization. Phosphorylation was also inhibited by blocking clathrin-dependent endocytosis, demonstrating a requirement for endocytosis for the PKC-dependent major phosphorylation effects. Serotonin receptor activation by serotonin showed a similar response to phorbol 12-myristate 13-acetate, implicating a potential role of delayed kinetics in G protein-coupled receptor signaling. Evaluation of candidate substrates revealed that the phosphorylation of the PKC substrate p70S6K kinase behaved in a similar manner. Gradient-based fractionation revealed that the majority of these PKC substrates reside within the pericentrion-enriched fractions and not in the plasma membrane. Finally, proteomic analysis of the pericentrion-enriched fractions revealed several proteins as known PKC substrates and/or proteins involved in endocytic trafficking. These results reveal an important role for PKC internalization and for the pericentrion as key determinants/amplifiers of PKC action.