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首页> 外文期刊>World Journal of Gastroenterology >Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase
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Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase

机译：G蛋白偶联受体激酶2的Pleckstrin同源域与PKC结合并影响PKC激酶的活性

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AIM: To study the detail mechanism of interaction between PKC and GRK_2 and the effect of GRK_2 on activity of PKC. METHODS: The cDNA of pleckstrin homology (PH) domain located in GRK_2 residue 548 to 660 was amplified by PCR with the mRNA of human GRK_2 (β1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK_2 PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK_2 PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK_2 in culture mammalian cells (6 cell lines: PC-3,MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK_2 PH domain, GST-GRK_2 PH domain fusion protein and BTK PH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK_2 to PKC was also detected by western blot and Co-immunoprecipitation. RESULTS: The binding of GRK_2 PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK_2 PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK_2 PH domain. CONCLUSION: GRK_2 can bind with PKC and down-regulated PKC activity.
机译：目的：研究PKC与GRK_2相互作用的详细机制以及GRK_2对PKC活性的影响。方法：以人新鲜胎盘中人源GRK_2（β1-肾上腺素能受体激酶）的mRNA为模板，通过PCR扩增出位于人RKK_2残基548至660之间的PH蛋白结构域的cDNA，插入表达载体pGEX-PH。用上述GRK_2 PH结构域蛋白的cDNA序列和GST（谷胱甘肽-S-转移酶）-GRK_2 PH结构域融合蛋白的表达载体，构建了BTK（Bruton酪氨酸激酶）PH结构域和GST蛋白。通过SDS-PAGE和共免疫沉淀法测定培养哺乳动物细胞（PC-3，MDCK，SGC7901，Jurkat细胞等6种细胞系）中GRK_2的表达。通过SDS-PAGE和Western blot检测体外GRK_2 PH结构域，GST-GRK_2 PH结构域融合蛋白和BTK PH结构域与PKC的结合，长时间刺激肾上腺素后，Western blot检测GRK_2与PKC的结合和免疫共沉淀。结果：Western blot证实了GRK_2 PH结构域与PKC的体外结合，以及长时间刺激肾上腺素的结合以及BTK PH结构域与PKC的结合也得到了证实。在本研究中，GRK_2 PH结构域与PKC和下调PKC活性相关，但Btk PH结构域与GRK_2 PH结构域相比上调PKC活性。结论：GRK_2可以与PKC结合并下调PKC活性。

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