Kazlauskas A, Cooper J A
Department of Cell Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
J Cell Biol. 1988 Apr;106(4):1395-402. doi: 10.1083/jcb.106.4.1395.
One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.
用血小板衍生生长因子（PDGF）、12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）、二酰基甘油或其他几种促细胞分裂剂刺激瑞士3T3细胞后，早期事件之一是一种稀少的细胞质蛋白（p42）在酪氨酸和丝氨酸位点发生近乎化学计量的磷酸化。已知TPA和二酰基甘油可直接刺激蛋白丝氨酸/苏氨酸激酶——蛋白激酶C（PKC）的活性。PDGF和其他几种促细胞分裂剂直接刺激酪氨酸激酶，间接刺激PKC。因此，我们研究了PKC在瑞士3T3细胞p42酪氨酸磷酸化过程中的作用。首先，六种刺激p42磷酸化的试剂也刺激了一种已知的PKC底物——一种80,000道尔顿的蛋白（p80）的磷酸化。其次，在PKC缺陷细胞（通过长时间暴露于TPA使PKC活性降低到无法检测水平的细胞）中，PDGF诱导的p42磷酸化降低了三到四倍。对来自PDGF刺激的PKC缺陷细胞的磷酸化p42进行磷酸氨基酸分析，结果显示主要是磷酸丝氨酸，只有微量的磷酸酪氨酸，这表明PKC缺陷导致的PDGF刺激的p42酪氨酸磷酸化减少幅度大于三到四倍。最后，比较PKC缺陷细胞与未处理细胞的抗磷酸酪氨酸免疫沉淀物发现，大多数其他PDGF诱导的酪氨酸磷酸化事件非常相似。这些数据表明，像PDGF这样直接刺激某些蛋白酪氨酸磷酸化的促细胞分裂剂，通过涉及PKC的一系列事件诱导p42酪氨酸磷酸化。
