35H, A SEQUENCE ISOLATED AS A PROTEIN-KINASE-C BINDING-PROTEIN, IS A NOVEL MEMBER OF THE ADDUCIN FAMILY
35H, A SEQUENCE ISOLATED AS A PROTEIN-KINASE-C BINDING-PROTEIN, IS A NOVEL MEMBER OF THE ADDUCIN FAMILY
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DOI：
10.1074/jbc.270.43.25534
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发表时间：
1995-10-27
影响因子：
4.8
通讯作者：
JAKEN, S
中科院分区：
文献类型：
--
作者：
DONG, LQ;CHAPLINE, C;JAKEN, S
我们最近从大鼠肾cDNA文库中克隆了蛋白激酶C（PKC）结合蛋白的部分cDNA（35 H），并证明它是体外PKC底物（Chapline，C.，Ramsay，K.，Klauck，T.，和Jaken，S.（1993）J.Biol.Chem.268，6858-6861）。采用额外的文库筛选和cDNA末端的5'快速扩增来获得完整的开放阅读框架。氨基酸序列分析、DNA序列分析和北方分析表明35 H是与α-和β-内收蛋白相关的独特cDNA。制备的35 H细菌融合蛋白的抗血清识别两个多肽的80和90 kDa的肾匀浆和培养的肾近端小管上皮细胞提取物的免疫印迹。35 H-相关的蛋白质是类似的α-和β-内收蛋白，因为它们优先回收的Triton X-100不溶性（细胞骨架，CSK）部分的细胞提取物，并主要定位于细胞的边界。佛波酯刺激CSK 35 H蛋白的磷酸化，从而强调根据PKC结合活性分离的序列在体外也是PKC底物。磷酸化形式的35 H蛋白优先回收的可溶性部分，从而证明磷酸化调节它们的CSK协会，从而，他们在调节细胞骨架组装的功能，我们已经分离出另一个PKC结合蛋白的部分cDNA（克隆45）从大鼠成纤维细胞库与α-内收蛋白的实质同源性。抗血清对这个表达的序列识别蛋白质的120 kDa，报告的α-内收蛋白的大小，对肾近端小管上皮细胞提取物的免疫印迹。与克隆45（α-内收蛋白）抗血清交叉反应的120-kDa蛋白与35 H免疫复合物共沉淀，表明α-内收蛋白在体内与35 H蛋白相关。总之，这些结果表明，35 H是一种新的，广泛表达的内收蛋白形式，能够与α-内收蛋白形成异源二聚体。我们建议将这种内收蛋白同系物命名为γ-内收蛋白。
We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha- and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies, We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha-adducin. We propose naming this adducin homologue gamma-adducin.