Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain.
Protein kinase C-stimulated phosphorylation in vitro of a Mr 80,000 protein phosphorylated in response to phorbol esters and growth factors in intact fibroblasts. Distinction from protein kinase C and prominence in brain.
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发表时间：
1986-01
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通讯作者：
P. Blackshear;L. Wen;B. Glynn;L. Witters
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作者：
P. Blackshear;L. Wen;B. Glynn;L. Witters
在之前对完整 3T3-L1 成纤维细胞和脂肪细胞的研究中，我们证明酸性多组分 Mr 80,000 蛋白的磷酸化状态似乎是蛋白激酶 C 激活状态的特异性且有用的标记（Blackshear, P.J.、Witters, L.A.、Girard, P.R.、Kuo, J.F. 和 Quamo, S.N. (1985) J. Biol.化学，260，13304-13315）。在本研究中，我们证明来自大鼠脂肪组织的 Mr 80,000 蛋白是体外蛋白激酶 C 的底物，并在二维凝胶上与来自小鼠 3T3-L1 脂肪细胞的类似蛋白（通过将完整细胞暴露于 32Pi 和佛波醇 12-肉豆蔻酸 13-乙酸酯进行标记）共同迁移。两种 32P 蛋白的部分蛋白水解图几乎相同，支持这样的假设：体外由蛋白激酶 C 磷酸化的位点以及体内对佛波醇 12-肉豆蔻酸酯 13-乙酸酯的反应的位点是相似或相同的。尽管它们的表观分子量相似，但我们能够通过几个物理标准区分 Mr 80,000 蛋白和蛋白激酶 C。 Mr 80,000 蛋白激酶 C 底物存在于所有受检大鼠组织的部分中，但在大鼠大脑中最为突出。佛波醇 12-肉豆蔻酸酯 13-乙酸酯还刺激几种类型的培养神经元细胞中 Mr 80,000 蛋白的磷酸化，表明该蛋白在胆碱能神经传递中可能发挥作用。 Mr 80,000 蛋白似乎是多种细胞类型中蛋白激酶 C 激活的有用标记。
In previous studies in intact 3T3-L1 fibroblasts and adipocytes, we demonstrated that the phosphorylation state of an acidic, multicomponent Mr 80,000 protein appeared to be a specific and useful marker for the activation state of protein kinase C (Blackshear, P.J., Witters, L.A., Girard, P.R., Kuo, J.F., and Quamo, S.N. (1985) J. Biol. Chem. 260, 13304-13315). In the present studies, we demonstrate that the Mr 80,000 protein from rat adipose tissue was a substrate for protein kinase C in vitro, and co-migrated on two-dimensional gels with the analogous protein from murine 3T3-L1 adipocytes labeled by exposure of intact cells to 32Pi and phorbol 12-myristate 13-acetate. Partial proteolytic maps of the two 32P-proteins were nearly identical, supporting the postulate that the sites phosphorylated by protein kinase C in vitro, and in response to phorbol 12-myristate 13-acetate in vivo, were similar or identical. Despite their similar apparent molecular weights, we were able to distinguish between the Mr 80,000 protein and protein kinase C by several physical criteria. The Mr 80,000 protein kinase C substrate was found in fractions of all rat tissues examined, but was most prominent in rat brain. Phorbol 12-myristate 13-acetate also stimulated phosphorylation of the Mr 80,000 protein in several types of cultured neuronal cells, suggesting a possible role for this protein in cholinergic neurotransmission. The Mr 80,000 protein appears to be a useful marker for protein kinase C activation in a variety of cell types.