Omary M B, Baxter G T, Chou C F, Riopel C L, Lin W Y, Strulovici B
Stanford University School of Medicine, Gastroenterology Division, California 94305.
J Cell Biol. 1992 May;117(3):583-93. doi: 10.1083/jcb.117.3.583.
A 40-kD protein kinase C (PKC)epsilon related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKC epsilon specific antibody showed that the 40-kD species, and not native PKC epsilon (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKC epsilon, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. A peptide pseudosubstrate specific for PKC epsilon inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates. Tryptic peptide map analysis of the cytokeratins that were phosphorylated by purified rat brain PKC epsilon or as immunoprecipitates by the associated kinase showed similar phosphopeptides. Furthermore, PKC epsilon immunoreactive species and CK8/18 colocalized using immunofluorescent double staining. We propose that a kinase related to the catalytic fragment of PKC epsilon physically associates with and phosphorylates cytokeratins 8 and 18.
发现一种40-kD蛋白激酶C（PKC）ε相关活性与人类上皮特异性细胞角蛋白（CK）多肽8和18相关联。该激酶活性与CK8和18共免疫沉淀，并使CK的免疫沉淀物磷酸化。使用抗PKCε特异性抗体对CK8/18免疫沉淀物进行免疫印迹分析表明，与细胞角蛋白相关的是40-kD蛋白，而非天然的PKCε（90 kD）。重组实验表明，纯化的CK8或CK18与纯化的PKCε的40-kD胰蛋白酶片段相关联，或与从组成性表达该片段但不表达CK8/18的细胞中获得的类似蛋白相关联。一种对PKCε特异的肽类假底物可抑制完整细胞中CK8/18的磷酸化，或在对CK8/18免疫沉淀物进行的激酶分析中起抑制作用。对经纯化的大鼠脑PKCε磷酸化或经相关激酶免疫沉淀的细胞角蛋白进行胰蛋白酶肽图谱分析，显示出相似的磷酸肽。此外，使用免疫荧光双重染色法发现PKCε免疫反应性蛋白与CK8/18共定位。我们提出，一种与PKCε催化片段相关的激酶在物理上与细胞角蛋白8和18相关联并使其磷酸化。
