Perdew G H, Wiegand H, Vanden Heuvel J P, Mitchell C, Singh S S
Department of Veterinary Science, The Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1997 Mar 25;36(12):3600-7. doi: 10.1021/bi9612529.
Several oncogenic protein kinases including c-raf-1 and pp60(v-src) are known to directly interact with the 90 kDa heat shock protein (hsp90)/p50 complexes. Using a monoclonal antibody to detect p50 during a purification scheme, p50 was purified to homogeneity. Internal amino acid sequence information was obtained and used to clone a partial cDNA. Comparison of the p50 sequence to other cloned proteins revealed 89% homology with a glycosaminoglycan-binding protein and 54% homology with Drosophila cell cycle control protein (cdc) 37. Monoclonal and polyclonal antibodies were produced against a cleaved fusion protein that recognizes p50 with a high level of specificity. These antibodies recognize the 50 kDa protein present in c-raf-1 and pp60(v-src) complexes. No other proteins were recognized with these antibodies suggesting that p50 is a unique protein. Immunocytochemical visualization of p50 in NIH 3T3 cells indicates a primarily cytoplasmic localization around the nuclear membrane. A survey of p50 expression in murine tissues on a protein blot revealed the following relative levels of expression; thymus > spleen > brain > heart > kidney > liver > lung > skeletal muscle. These results link studies demonstrating complexation of certain kinases with hsp90/p50 in mammalian cells and a number of reports in yeast and Drosophila, demonstrating the importance of cdc37 in cell cycle and kinase function.
已知包括c-raf-1和pp60(v-src)在内的几种致癌蛋白激酶可直接与90 kDa热休克蛋白（hsp90）/p50复合物相互作用。在纯化过程中使用单克隆抗体检测p50，将p50纯化至同质。获得了内部氨基酸序列信息并用于克隆部分cDNA。将p50序列与其他克隆蛋白进行比较，发现其与一种糖胺聚糖结合蛋白有89%的同源性，与果蝇细胞周期控制蛋白（cdc）37有54%的同源性。针对一种裂解融合蛋白制备了单克隆和多克隆抗体，该融合蛋白能高度特异性地识别p50。这些抗体识别存在于c-raf-1和pp60(v-src)复合物中的50 kDa蛋白。用这些抗体未识别出其他蛋白，这表明p50是一种独特的蛋白。在NIH 3T3细胞中对p50进行免疫细胞化学可视化显示，其主要定位于核膜周围的细胞质中。通过蛋白质印迹法对小鼠组织中p50表达的调查揭示了以下相对表达水平；胸腺＞脾脏＞脑＞心脏＞肾脏＞肝脏＞肺＞骨骼肌。这些结果将在哺乳动物细胞中证明某些激酶与hsp90/p50形成复合物的研究与酵母和果蝇中的一些报道联系起来，证明了cdc37在细胞周期和激酶功能中的重要性。
