Department of Biology, Hope College, Holland, Michigan 49422-9000, USA.
J Biol Chem. 2010 Feb 12;285(7):4883-95. doi: 10.1074/jbc.M109.085225. Epub 2009 Nov 16.
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-1 cDNA 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-1 protein 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.
VACM-1/cul5 基因在体外的内皮细胞和癌细胞系中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-功能分析确定了特定的蛋白激酶 A 和 C（PKA 和 PKC）磷酸化和 Nedd8 蛋白修饰位点的共识位点。VACM-1/Cul5 序列中 PKA 特异性位点的突变（S730A）导致细胞生长增加和出现 Nedd8 修饰的 VACM-1/Cul5。本研究的目的是研究 PKA 依赖性 VACM-1/Cul5 磷酸化是否控制其 Neddylation 状态、PKC 的磷酸化，以及最终的生长。我们的结果表明，用抗 VACM-1 特异性小干扰 RNA 寡核苷酸转染大鼠肾上腺髓质内皮细胞会降低内源性 VACM-1 蛋白浓度并增加细胞生长。用针对 PKA 特异性磷酸化位点的抗体进行免疫沉淀，然后用抗 VACM-1 特异性抗体进行 Western blot 分析表明，与巨细胞病毒转染的细胞相比，用 (S730A)VACM-1 cDNA 转染的细胞中 VACM-1 蛋白的 PKA 依赖性磷酸化减少。这种变化与 VACM-1 蛋白被 Nedd8 修饰的增加有关。用 forskolin 诱导 PKA 活性会降低 VACM-1 蛋白被 Nedd8 修饰的程度。最后，用 (S730A)VACM-1/cul5 cDNA 转染大鼠肾上腺髓质内皮细胞并分别用佛波醇 12-肉豆蔻酸 13-乙酸酯（10 和 100nm）处理以诱导 PKC 活性，其生长速度明显快于对照细胞。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 途径的新型抗癌治疗方法。
