Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1
Human carbon catabolite repressor protein (CCR4)-associative factor 1: cloning, expression and characterization of its interaction with the B-cell translocation protein BTG1
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DOI：
10.1042/bj3360471
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发表时间：
1998-12-01
影响因子：
4.1
通讯作者：
Dicker, IB
中科院分区：
文献类型：
--
作者：
Bogdan, JA;Adams-Burton, C;Dicker, IB
人BTG 1蛋白被认为是一种潜在的肿瘤抑制因子，因为其过表达抑制NIH 3 T3细胞增殖。然而，人们对BTG 1如何发挥其抗增殖活性知之甚少。在这项研究中，我们使用的酵母'双杂交'系统，以筛选相互作用的蛋白质合作伙伴，并确定了人类碳分解代谢物阻遏蛋白（CCR 4）-相关因子1（hCAF-1），小鼠CAF-1（mCAF-1）和酿酒酵母yCAF-1/POP 2的同源物。在体外，hCAF-1/BTG 1复合物的形成依赖于BTG 1上推定的p34（cdc 2）激酶位点（Ser-159）的磷酸化。在酵母中，Ala-159突变体不与hCAF-1相互作用。另外，Ser-159的磷酸化对细胞周期激酶p34（CDK 2）/cyclin E和p34（CDK 2）/cyclin A有特异性，而对p34（CDK 4）/cyclin D1和P34（cdc 2）/cyclin B无特异性。用大鼠主动脉平滑肌细胞（RSMC）的原代培养物进行的细胞同步性实验证明，大鼠CAF-I（rCAF-1）的信使和蛋白水平在细胞接触条件下上调，如先前对BTG 1所报道的[Wilcox，Scott，Subramanian，Ross，Adams-Burton，Stoltenborg和Corjay（1995）Circulation 92，I34-I35]。免疫印迹和免疫组化分析表明，rCAF-1定位于细胞核的接触抑制RSMCs，在那里它是物理上与BTG 1，确定与抗hCAF-1抗血清免疫共沉淀。在NIH 3 T3和骨肉瘤（U-2-OS）细胞中过表达hCAF-1本身具有抗增殖性，集落形成分别减少67%和90%。总之，这些结果表明，hCAF-1/BTG 1复合物的形成是由BTG 1（Ser-159）的磷酸化驱动的，并暗示该复合物参与细胞分裂的信号传导事件，导致与细胞-细胞接触相关的细胞增殖变化。
The human BTG1 protein is thought to be a potential tumour suppressor because its overexpression inhibits NIH 3T3 cell proliferation. However, little is known about how BTG1 exerts its anti-proliferative activity. In this study, we used the yeast 'two-hybrid' system to screen for interacting protein partners and identified human carbon catabolite repressor protein (CCR4)-associative factor 1 (hCAF-1), a homologue of mouse CAF-I (mCAF-1) and Saccharomyces cerevisiae yCAF-1/POP2. In vitro the hCAF-1/BTG1 complex formation was dependent on the phosphorylation of a putative p34(cdc2) kinase site on BTG1 (Ser-159). In yeast, the Ala-159 mutant did not interact with hCAF-1. In addition, phosphorylation of Ser-159 in vitro showed specificity for the cell cycle kinases p34(CDK2)/cyclin E and p34(CDK2)/cyclin A, but not for p34(CDK4)/cyclin D1 Or P34(cdc2)/cyclin B. Cell synchrony experiments with primary cultures of rat aortic smooth-muscle cells (RSMCs) demonstrated that message and protein levels of rat CAF-I (rCAF-1) were up-regulated under conditions of cell contact, as previously reported for BTG1 [Wilcox, Scott, Subramanian, Ross, Adams-Burton, Stoltenborg and Corjay (1995) Circulation 92, I34-I35]. Western blot and immunohistochemical analysis showed that rCAF-1 localizes to the nucleus of contact-inhibited RSMCs, where it was physically associated with BTG1, as determined by co-immunoprecipitation with anti-hCAF-1 antisera. Overexpression of hCAF-1 in NIH 3T3 and osteosarcoma (U-2-OS) cells was itself anti-proliferative with colony formation reduced by 67 % and 90 % respectively. Taken together, these results indicate that formation of the hCAF-1/BTG1 complex is driven by phosphorylation at BTG1 (Ser-159) and implicates this complex in the signalling events of cell division that lead to changes in cellular proliferation associated with cell-cell contact.