Begum N, Robinson L J, Draznin B, Heidenreich K A
Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501.
Endocrinology. 1993 Nov;133(5):2085-90. doi: 10.1210/endo.133.5.8404657.
In this study, we examined the developmental expression and regulation by insulin and insulin-like growth factor-I (IGF-I) of protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) in cultured fetal chick neurons. Protein phosphatase activities were measured using 32P-labeled phosphorylase-a or 32P-labeled S6 kinase substrate peptide. In cell extracts from day 1-5 cultures, 40-45% of spontaneous protein phosphatase activity was due to PP-1. PP-2A accounted for the remaining 55-60% of enzyme activity. Spontaneous PP-1 activity increased by 100% in day 2 cultures and remained constant thereafter. PP-2A activity increased by 48% in day 2 cultures, with minimal increases in enzyme activity in later cultures. Under the assay conditions employed, at all times in culture a significant proportion (45-50%) of PP-1 was in an inactive form that could be reactivated by trypsin. PP-2A activity was not influenced by trypsin. Insulin stimulated neuronal PP-1 activity in day 4 and 5 cultures, but had no effect in earlier cultures. The activation of PP-1 by insulin was rapid, with a maximal effect (30-40% increase over basal levels) at 5 min with 10 ng/ml insulin. Insulin did not alter total (trypsin-released) PP-1 activity, the content of PP-1 catalytic subunit, or PP-2A activity at any time in culture. In contrast to insulin, IGF-I had no effect on PP-1 activity at any time in culture, but significantly increased PP-2A activity in day 5 cultures. Maximal stimulation of PP-2A activity by IGF-I was observed at 10 min, with an EC50 of 5 ng/ml. These results indicate that chick forebrain neurons contain both PP-1 and PP-2A activities and that neuronal PP-1 and PP-2A activities are differentially regulated by insulin and IGF-I. We conclude that although insulin and IGF-I share many steps in signal transduction, these growth factors have distinct actions on neuronal phosphatase activity that may impact on differences in their neurotropic actions.
在本研究中，我们检测了培养的鸡胚神经元中蛋白磷酸酶-1（PP-1）和蛋白磷酸酶-2A（PP-2A）的发育表达以及胰岛素和胰岛素样生长因子-I（IGF-I）对它们的调节作用。使用32P标记的磷酸化酶-a或32P标记的S6激酶底物肽来测量蛋白磷酸酶活性。在第1 - 5天培养的细胞提取物中，40 - 45%的自发蛋白磷酸酶活性归因于PP-1。PP-2A占其余55 - 60%的酶活性。自发的PP-1活性在第2天培养物中增加了100%，此后保持恒定。PP-2A活性在第2天培养物中增加了48%，在后期培养物中酶活性增加 minimal。在所采用的测定条件下，在培养的所有时间里，相当大比例（45 - 50%）的PP-1处于无活性形式，可被胰蛋白酶重新激活。PP-2A活性不受胰蛋白酶影响。胰岛素在第4天和第5天培养物中刺激神经元PP-1活性，但在早期培养物中没有作用。胰岛素对PP-1的激活迅速，在5分钟时，10 ng/ml胰岛素可产生最大效应（比基础水平增加30 - 40%）。胰岛素在培养的任何时间都不会改变总（胰蛋白酶释放的）PP-1活性、PP-1催化亚基的含量或PP-2A活性。与胰岛素相反，IGF-I在培养的任何时间对PP-1活性都没有影响，但在第5天培养物中显著增加PP-2A活性。IGF-I对PP-2A活性的最大刺激在10分钟时观察到，EC50为5 ng/ml。这些结果表明鸡胚前脑神经元同时含有PP-1和PP-2A活性，并且神经元PP-1和PP-2A活性受到胰岛素和IGF-I的差异调节。我们得出结论，尽管胰岛素和IGF-I在信号转导中有许多共同步骤，但这些生长因子对神经元磷酸酶活性有不同的作用，这可能影响它们的神经营养作用差异。
