Sauma S, Friedman E
Laboratory of Gastrointestinal Tumor Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
J Biol Chem. 1996 May 10;271(19):11422-6. doi: 10.1074/jbc.271.19.11422.
In a prior study, we have shown that stable transfection of expression plasmids for protein kinases C beta 1 (PKC beta 1) or PKC beta 2 into differentiated colon cancer cells led to elevated levels of PKC beta 1 or PKC beta 2 protein and PKC beta kinase activities in the transfectants, without altering PKC alpha levels. PKC gamma is not found in these cells, so the major modulation was in PKC beta. PKC beta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKC beta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKC beta 1 and each of two PKC beta 2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKC beta 1, PKC beta 2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a > 10-fold increase in ERK3 activity in each PKC beta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKC beta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKC beta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKC beta 1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKC beta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKC beta 1 or PKC beta 2.
在之前的一项研究中，我们已经表明，将蛋白激酶Cβ1（PKCβ1）或PKCβ2的表达质粒稳定转染到分化的结肠癌细胞中，会导致转染细胞中PKCβ1或PKCβ2蛋白水平及PKCβ激酶活性升高，而PKCα水平未发生改变。这些细胞中未发现PKCγ，因此主要的调节作用发生在PKCβ。PKCβ转染细胞表现出分化受阻、无胸腺小鼠中生长速率增加以及碱性成纤维细胞生长因子反应途径的恢复。在本研究中，我们将对这些PKCβ转染细胞的分析扩展至丝裂原活化蛋白激酶ERK3。通过凝胶内激酶分析对丝裂原活化蛋白激酶底物髓鞘碱性蛋白进行细胞裂解物分析，结果显示，与载体对照转染细胞相比，两种PKCβ1转染细胞及两种PKCβ2转染细胞中的每一种，在63 kDa（ERK3的大小）处均有活性增加。如蛋白质印迹法及用抗ERK3单克隆抗体进行免疫沉淀所证实，ERK3在PKCβ1、PKCβ2及对照转染细胞中的表达丰度相等。然而，用抗ERK3单克隆抗体进行免疫沉淀，随后进行免疫复合物激酶分析或凝胶内激酶分析，结果显示每种PKCβ转染细胞中ERK3活性增加超过10倍。因此，虽然转染的PKCβ过表达不会导致ERK3过表达，但确实会导致ERK3的组成性激活。由于12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯处理下调了两种PKCβ1转染细胞中的PKC和ERK3活性，因此建立了PKCβ过表达与ERK3激活之间存在因果关系。与对照相反，在每种PKCβ转染细胞的核及膜组分中均发现了ERK3活性，这可能解释了PKCβ1或PKCβ2水平升高细胞中ERK3的组成性激活。
