The Pleckstrin Homology Domain of Protein Kinase D Interacts Preferentially with the η Isoform of Protein Kinase C*
The Pleckstrin Homology Domain of Protein Kinase D Interacts Preferentially with the η Isoform of Protein Kinase C*
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蛋白激酶 D 的 Pleckstrin 同源结构域优先与蛋白激酶 C* 的 η 同工型相互作用
DOI：
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发表时间：
1999
影响因子：
4.8
通讯作者：
E. Rozengurt
中科院分区：
文献类型：
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作者：
R. Waldron;T. Iglesias;E. Rozengurt
本研究结果表明，蛋白激酶D（PKD）和PKCη在COS-7细胞中瞬时共表达形成复合物，该复合物可以用PKD或PKCη的特异性抗血清从细胞裂解物中免疫沉淀。PKD免疫复合物中PKCη的存在最初是通过体外激酶试验检测的，该试验显示除了对应于自磷酸化PKD的110-kDa条带外，还存在80-kDa磷酸化条带。通过Western印迹分析和肽磷酸化测定进一步验证了PKD和PKCη之间的关联，这些测定利用了PKC和PKD之间的不同底物特异性。根据相同的标准，PKD与PKCε仅形成非常弱的复合物，并且不结合PKC ε。当PKCη与含有PH结构域完全或部分缺失的PKD突变体共表达时，PKCη免疫反应性和PKD免疫沉淀物中的PKC活性均急剧降低。相反，缺失分子的氨基端部分，无论是富含半胱氨酸的区域，还是整个富含半胱氨酸的结构域，都不会干扰PKD与PKCη的结合。此外，谷胱甘肽S-转移酶-PKDPH融合蛋白优先结合PKCη。这些结果表明，PKD PH结构域可以区分单个酶家族的密切相关的结构，例如新型PKC ε和η，从而揭示了PH结构域介导的蛋白质-蛋白质相互作用之间先前未检测到的特异性程度。
The results presented here demonstrate that protein kinase D (PKD) and PKCη transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCη. The presence of PKCη in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCη was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCε, and did not bind PKCζ. When PKCη was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCη immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCη. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCη. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs ε and η, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.