Heidenreich K A, Toledo S P
Department of Medicine, University of California-San Diego, La Jolla 92093.
Endocrinology. 1989 Sep;125(3):1458-63. doi: 10.1210/endo-125-3-1458.
As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.
作为确定胰岛素作用早期步骤（可能参与神经元对胰岛素生长反应）的初步尝试，我们研究了胰岛素受体激活是否会增加培养的胎儿神经元中核糖体蛋白S6的磷酸化，以及蛋白激酶的激活是否参与此过程。当神经元与32Pi孵育2小时后，在最后30分钟加入胰岛素（100 ng/ml）可增加32Pi掺入一种32K微粒体蛋白中。在30分钟的胰岛素暴露下，大多数其他神经元蛋白中32Pi的掺入未发生改变。在存在外源性大鼠肝脏40S核糖体和[γ-32P]ATP的情况下，胰岛素处理的神经元的胞质提取物显示核糖体蛋白S6的磷酸化比未处理细胞的提取物增加了3至8倍。在神经元暴露于胰岛素期间加入环己酰亚胺并不抑制胞质激酶活性的增加。胰岛素对S6激酶活性的激活呈剂量依赖性（在低至0.1 ng/ml的胰岛素浓度下即可观察到），孵育20分钟后达到最大值。向体外激酶反应中添加磷脂酰丝氨酸、二油精和Ca2+对核糖体蛋白S6的磷酸化没有影响。同样，用（Bu）2cAMP处理神经元不会改变神经元胞质提取物对核糖体蛋白S6的磷酸化。我们得出结论，胰岛素激活一种胞质蛋白激酶，该激酶使神经元中的核糖体S6磷酸化，且不同于蛋白激酶C和cAMP依赖性蛋白激酶。这种激酶的刺激可能在神经元的胰岛素信号转导中起作用。
