Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect
Phosphorylation of VACM-1/Cul5 by Protein Kinase A Regulates Its Neddylation and Antiproliferative Effect
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DOI：
10.1074/jbc.m109.085225
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发表时间：
2010-02-12
影响因子：
4.8
通讯作者：
Burnatowska-Hledin, Maria
中科院分区：
文献类型：
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作者：
Bradley, Shirley E.;Johnson, Alyssa E.;Burnatowska-Hledin, Maria
内皮细胞和癌细胞中VACM-1/cul5基因的表达抑制细胞增殖并降低MAPK的磷酸化。VACM-1蛋白序列的结构-功能分析确定了被蛋白激酶A和C （PKA和PKC）磷酸化的一致位点以及Nedd8蛋白修饰位点。VACM-1/Cul5 （(S730A)VACM-1）序列中pka特异性位点的突变导致细胞生长增加和nedd8修饰的VACM-1/Cul5的出现。本研究的目的是检验pka依赖的VACM-1/Cul5磷酸化是否控制其类化修饰状态、PKC磷酸化以及最终的生长。我们的研究结果表明，体外转染大鼠肾上腺髓质内皮细胞抗VACM-1特异性小干扰RNA寡核苷酸可降低内源性VACM-1蛋白浓度，促进细胞生长。用针对pka特异性磷酸化位点的抗体免疫沉淀细胞裂解液，并用抗VACM-1特异性抗体探测细胞裂解液，Western blot分析显示，与巨细胞病毒转染的细胞相比，转染（S730A）VACM-1cDNA的细胞中pka依赖性的VACM-1蛋白磷酸化水平降低。这种变化与Nedd8对VACM-1蛋白的修饰增加有关。用forskolin诱导PKA活性可以减少Nedd8对vacm -1蛋白的修饰。最后，转染（S730A）VACM-1/cul5 cDNA和用phorbol 12-肉豆酸酯13-乙酸酯（10 nM和100 nM）诱导PKC活性的大鼠肾上腺髓质内皮细胞生长明显快于对照细胞。这些结果表明，VACM-1/Cul5的抗增殖作用依赖于其翻译后修饰，并将有助于设计针对Nedd8途径的新抗癌疗法。
Expression of the VACM-1/cul5 gene in endothelial and in cancer cell lines in vitro inhibits cellular proliferation and decreases phosphorylation of MAPK. Structure-function analysis of the VACM-1 protein sequence identified consensus sites specific for phosphorylation by protein kinases A and C (PKA and PKC) and a Nedd8 protein modification site. Mutations at the PKA-specific site in VACM-1/Cul5 ((S730A)VACM-1) sequence resulted in increased cellular growth and the appearance of a Nedd8-modified VACM-1/Cul5. The aim of this study was to examine if PKA-dependent phosphorylation of VACM-1/Cul5 controls its neddylation status, phosphorylation by PKC, and ultimately growth. Our results indicate that in vitro transfection of rat adrenal medullary endothelial cells with anti-VACM-1-specific small interfering RNA oligonucleotides decreases endogenous VACM-1 protein concentration and increases cell growth. Western blot analysis of cell lysates immunoprecipitated with an antibody directed against a PKA-specific phosphorylation site and probed with anti-VACM-1-specific antibody showed that PKA-dependent phosphorylation of VACM-1 protein was decreased in cells transfected with (S730A)VACM-1cDNA when compared with the cytomegalovirus-transfected cells. This change was associated with increased modification of VACM-1 protein by Nedd8. Induction of PKA activity with forskolin reduced modification of VACM-1protein by Nedd8. Finally, rat adrenal medullary endothelial cells transfected with (S730A)VACM-1/cul5 cDNA and treated with phorbol 12-myristate 13-acetate (10 and 100 nM) to induce PKC activity grew significantly faster than the control cells. These results suggest that the antiproliferative effect of VACM-1/Cul5 is dependent on its posttranslational modifications and will help in the design of new anticancer therapeutics that target the Nedd8 pathway.