Lee R M, Rapp U R, Blackshear P J
Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1991 Jun 5;266(16):10351-7.
The protein product of the Raf-1 proto-oncogene is a protein serine/threonine kinase that is activated after stimulation of cells with insulin and other mitogens. To investigate the mechanism of this activation, we used purified Raf-1 expressed in E. coli as a substrate for a putative Raf-1 protein kinase kinase. In three different insulin-sensitive cell types, insulin activated Raf-1 kinase kinase activity in crude cytosolic cellular fractions. The insulin stimulation of this activity was evident as early as 2 min after exposure to insulin, maximal at 5-8 min, and inapparent at 15 min. Phosphoamino acid analysis of phosphorylated Raf-1 revealed that serine was the primary phosphate acceptor for the insulin-activated kinase or kinases; small amounts of phosphothreonine were also detected. The insulin effect occurred in cells depleted of protein kinase C, and in extracts depleted of endogenous Raf-1 kinase by immunodepletion; these data argue against protein kinase C or Raf-1 kinase itself being the insulin-stimulated activity. The insulin-activated kinase or kinases phosphorylated the Raf-1 protein on multiple sites in vitro, as evidenced by tryptic mapping; at least some of these appeared to overlap with sites phosphorylated in response to serum in intact cells. Several other mitogens and growth factors stimulated Raf-1 kinase kinase activity, including epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, serum, and phorbol 12-myristate 13-acetate. This insulin- and mitogen-stimulated Raf-1 kinase kinase activity may play a role in mediating the phosphorylation and possibly the activation of the Raf-1 kinase by insulin and other growth factors.
Raf-1原癌基因的蛋白质产物是一种蛋白质丝氨酸/苏氨酸激酶，在细胞受到胰岛素和其他有丝分裂原刺激后被激活。为了研究这种激活的机制，我们使用在大肠杆菌中表达的纯化Raf-1作为假定的Raf-1蛋白激酶激酶的底物。在三种不同的胰岛素敏感细胞类型中，胰岛素激活了粗制胞质细胞组分中的Raf-1激酶激酶活性。这种活性的胰岛素刺激早在暴露于胰岛素后2分钟就很明显，在5 - 8分钟时达到最大值，在15分钟时不明显。对磷酸化Raf-1的磷酸氨基酸分析表明，丝氨酸是胰岛素激活的一种或多种激酶的主要磷酸受体；也检测到少量的磷酸苏氨酸。胰岛素效应发生在蛋白激酶C缺失的细胞中，以及通过免疫去除内源性Raf-1激酶的提取物中；这些数据反对蛋白激酶C或Raf-1激酶本身是胰岛素刺激的活性。胰岛素激活的一种或多种激酶在体外使Raf-1蛋白在多个位点磷酸化，胰蛋白酶图谱证明了这一点；其中至少一些位点似乎与完整细胞中对血清反应而磷酸化的位点重叠。其他几种有丝分裂原和生长因子也刺激Raf-1激酶激酶活性，包括表皮生长因子、血小板衍生生长因子、成纤维细胞生长因子、血清和佛波酯12 - 肉豆蔻酸13 - 乙酸酯。这种胰岛素和有丝分裂原刺激的Raf-1激酶激酶活性可能在介导胰岛素和其他生长因子对Raf-1激酶的磷酸化以及可能的激活中起作用。
