Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester.
Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester.
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胰岛素和佛波酯激活 3T3-L1 细胞中的 S6 激酶活性。
DOI：
10.1073/pnas.82.13.4369
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发表时间：
1985
影响因子：
11.1
通讯作者：
Rosen,OM
中科院分区：
文献类型：
--
作者：
Tabarini,D;Heinrich,J;Rosen,OM
用0.1-1.0 nM胰岛素处理3 T3-L1细胞导致可溶性蛋白激酶的快速（5-15 min）活化，该蛋白激酶磷酸化核糖体蛋白S6中的丝氨酸残基。胰岛素刺激的激酶活性可在融合的、非生长的前脂肪细胞和脂肪细胞中检测到。在每毫升2微克放线菌酮的存在下，融合前的3 T3-L1细胞也通过获得S6激酶活性来响应胰岛素，该S6激酶活性的性质与生长抑制细胞中单独胰岛素引起的酶活性的性质相同。主要的胰岛素刺激的S6激酶具有约等于50，000 - 60，000的Mr;存在可变量的活性，其沉淀具有约80，000的Mr。可溶性酶在pH 8和pH 9之间表现出最佳活性，需要Mg 2+（10-20 mM），并且被Ca 2+（0.5 mM）、Mn 2+（0.05 mM）和NaF（30 mM）抑制。在磷酸转移酶反应中，GTP不能替代ATP; cAMP、cGMP、磷脂酰丝氨酸加二油酸甘油酯（cAMP依赖性蛋白激酶抑制剂）和肝素（0.7微克/毫升）没有作用。虽然用胰岛素处理3 T3-L1细胞不影响磷脂和Ca 2+依赖性蛋白激酶C的活性或亚细胞分布，暴露于佛波醇肿瘤促进剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）导致蛋白激酶C易位至膜，并激活可溶性磷脂和Ca 2 +-独立的S6蛋白激酶，具有与胰岛素诱导活性相同的活性和沉降行为。胰蛋白酶处理3 T3-L1胞质提取物或部分纯化的3 T3-L1蛋白激酶C产生少量的S6激酶活性Mr 50，000。这种活性，解决了蔗糖梯度离心，是较低的活性比引起的胰岛素或PMA，不像胰岛素和PMA产生的活动，是与组蛋白激酶活性。这些数据表明，无论是胰岛素或PMA引起的S6激酶既不是蛋白激酶C，它的磷脂，和Ca 2 +-独立的蛋白水解衍生物，也不是一个无活性的酶原，可以复制胰蛋白酶处理细胞提取物在体外的蛋白水解激活的结果。
Treatment of 3T3-L1 cells with 0.1-1.0 nM insulin results in rapid (5-15 min) activation of a soluble protein kinase that phosphorylates serine residues in ribosomal protein S6. The insulin-stimulated kinase activity is detectable in confluent, nongrowing preadipocytes and adipocytes. In the presence of 2 micrograms of cycloheximide per ml, preconfluent 3T3-L1 cells also respond to insulin by acquiring an S6 kinase activity whose properties are the same as those of the enzyme activity elicited by insulin alone in growth-inhibited cells. The principal insulin-stimulated S6 kinase has a Mr of approximately equal to 50,000-60,000; there is a variable amount of activity that sediments with a Mr of about 80,000. The soluble enzyme exhibits optimal activity between pH 8 and pH 9, requires Mg2+ (10-20 mM), and is inhibited by Ca2+ (0.5 mM), Mn2+ (0.05 mM), and NaF (30 mM). GTP cannot substitute for ATP in the phosphotransferase reaction; cAMP, cGMP, phosphatidylserine plus diolein, the cAMP-dependent protein kinase inhibitor, and heparin (0.7 micrograms/ml) are without effect. Although treatment of 3T3-L1 cells with insulin does not influence the activity or the subcellular distribution of the phospholipid and Ca2+-dependent protein kinase C, exposure to the phorbol tumor promoter phorbol 12-myristate 13-acetate (PMA) results in translocation of protein kinase C to the membrane and activation of a soluble phospholipid and Ca2+-independent S6 protein kinase that has the same magnitude of activity and sedimentation behavior as the insulin-induced activity. Trypsin treatment of either 3T3-L1 cytosolic extracts or partially purified 3T3-L1 protein kinase C generates a small amount of S6 kinase activity of Mr 50,000. This activity, resolved by sucrose gradient centrifugation, is less active than that elicited by either insulin or PMA and, unlike the activities generated by insulin and PMA, is associated with histone kinase activity. The data suggest that the S6 kinase elicited by either insulin or PMA is neither protein kinase C, its phospholipid, and Ca2+-independent proteolytic derivative nor the result of proteolytic activation of an inactive proenzyme that can be reproduced by trypsin treatment of cell extracts in vitro.