Liebenhoff U, Brockmeier D, Presek P
Rudolf-Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, Giessen, Federal Republic of Germany.
Biochem J. 1993 Oct 1;295 ( Pt 1)(Pt 1):41-8. doi: 10.1042/bj2950041.
Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.
人血小板含有高水平的Src家族非受体蛋白酪氨酸激酶，尤其是pp60c-src，这表明这些酶在血小板生理学中起重要作用。事实上，响应各种血小板功能激动剂，许多蛋白质在酪氨酸残基处发生磷酸化。然而，尚未在血小板中显示Src相关酪氨酸激酶的酶促激活。在寻找负责的激酶时，我们发现所有测试的直接或间接激活血小板中蛋白激酶C的激动剂（佛波醇12-肉豆蔻酸酯、13-乙酸酯、凝血酶、血管加压素、胶原蛋白、钙离子载体A23187）在低ATP浓度下的免疫复合物测定中，通过IgG磷酸化测定增加了pp60c-src的总体活性。另一方面，通过福司可林直接或通过前列腺素E1间接升高环磷酸腺苷，或通过硝普钠升高环磷酸鸟苷，均未显著影响该酶的活性。为了证实酶活性的差异，我们测定了静息和凝血酶刺激血小板中pp60c-src的Km和Vmax值。凝血酶处理增加了pp60c-src的底物亲和力，ATP和外源性蛋白质底物酪蛋白蛋白的Km值降低2至3倍表明了这一点。在所使用的测定条件下，Vmax值仅略有改变。为了进一步排除pp60c-src磷酸化修饰作为底物亲和力改变的可能原因，我们分析了未刺激和刺激血小板中免疫沉淀的32P标记的pp60c-src的胰蛋白酶磷酸肽。上述血小板激动剂诱导Ser-12处pp60c-src磷酸化增加，Ser-12是被蛋白激酶C磷酸化的氨基酸。令人惊讶的是，我们发现环磷酸腺苷升高并不影响pp60c-src的32P标记。根据我们的数据，我们认为Ser-12处的磷酸化可能是导致pp60c-src底物亲和力增加的信号触发事件之一。
