Saville M K, Graham A, Malarkey K, Paterson A, Gould G W, Plevin R
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, U.K.
Biochem J. 1994 Jul 15;301 ( Pt 2)(Pt 2):407-14. doi: 10.1042/bj3010407.
The characteristics of protein tyrosine phosphorylation were examined in Rat-1 fibroblasts in response to endothelin-1 (ET-1) and 1-oleoyl-lysophosphatidic acid (LPA). Both agonists stimulated the biphasic tyrosine phosphorylation of at least three major proteins of approx. 120 kDa (pp116, pp120 and pp130) and two of 80 kDa (pp80 and pp70). Immunoprecipitation experiments indicated that the pp120 protein corresponded to the recently described focal adhesion protein kinase pp125fak. Phorbol 12-myristate 13-acetate, alone or in combination with the calcium ionophore A23187, also stimulated the phosphorylation of pp125fak but to a smaller extent than LPA or ET-1. Removal of both extracellular and intracellular Ca2+ did not significantly reduce LPA- and ET-1-stimulated tyrosine phosphorylation of pp125fak. In cells where protein kinase C activity was down-regulated or inhibited, ET-1-stimulated tyrosine phosphorylation of pp125fak was reduced to a greater extent than phosphorylation in response to LPA. In addition, ET-1-stimulated tyrosine phosphorylation of pp80 was decreased by 50-70% in response to protein kinase C inhibition at both 2 and 60 min whereas LPA-stimulated tyrosine phosphorylation of this protein was only reduced at 2 min. Pretreatment with pertussis toxin reduced the tyrosine phosphorylation of pp42 and pp44 forms of mitogen-activated protein kinase in response to both ET-1 and LPA but reduced the tyrosine phosphorylation of pp125fak only in response to LPA. These results indicate agonist-specific differences in the regulation of pathways mediating the tyrosine phosphorylation of pp125fak and other target proteins.
研究了大鼠1型成纤维细胞中蛋白质酪氨酸磷酸化的特征，以响应内皮素-1（ET-1）和1-油酰基-溶血磷脂酸（LPA）。两种激动剂均刺激了至少三种主要蛋白质的双相酪氨酸磷酸化，这些蛋白质分子量约为120 kDa（pp116、pp120和pp130）以及两种80 kDa的蛋白质（pp80和pp70）。免疫沉淀实验表明，pp120蛋白对应于最近描述的粘着斑蛋白激酶pp125fak。佛波醇12-肉豆蔻酸酯13-乙酸酯单独或与钙离子载体A23187联合使用，也刺激了pp125fak的磷酸化，但程度小于LPA或ET-1。去除细胞外和细胞内的Ca2+并没有显著降低LPA和ET-1刺激的pp125fak酪氨酸磷酸化。在蛋白激酶C活性下调或受抑制的细胞中，ET-1刺激的pp125fak酪氨酸磷酸化的降低程度大于对LPA反应的磷酸化降低程度。此外，在2分钟和60分钟时，蛋白激酶C抑制可使ET-1刺激的pp80酪氨酸磷酸化降低50 - 70%，而LPA刺激的该蛋白酪氨酸磷酸化仅在2分钟时降低。用百日咳毒素预处理可降低丝裂原活化蛋白激酶的pp42和pp44形式对ET-1和LPA反应的酪氨酸磷酸化，但仅降低对LPA反应的pp125fak酪氨酸磷酸化。这些结果表明，在介导pp125fak和其他靶蛋白酪氨酸磷酸化的信号通路调节中存在激动剂特异性差异。
