Mivechi N F, Trainor L D, Hahn G M
Stanford University School of Medicine, Department of Radiation Oncology, CBRL, CA 94305.
Biochem Biophys Res Commun. 1993 Apr 30;192(2):954-63. doi: 10.1006/bbrc.1993.1508.
We have examined protein phosphorylation in the presence of purified mammalian HSP-70 kDa using the phosphoproteins in the rabbit reticulocyte lysate system as a model. Purified HSP-70 added to the rabbit reticulocyte lysate decreased the general protein phosphorylation by 50-80% as measured by PAGE analysis of proteins labelled with gamma-(32P)-ATP. Reduction in protein phosphorylation was not due to the ATPase activity of HSP-70 as measured by thin layer chromatography. The reduction in protein phosphorylation was also not due to the reduced activities of the protein kinases. However, using (32P)-labelled phosphorylase-alpha as a substrate in the phosphatase assay system indicated increases in the activity of protein phosphatase 1(PP-1) and/or 2A (PP-2A) by 20-40% relative to control in the presence of increasing concentrations of HSP-70. Using a variety of activators and inhibitors of the two major protein phosphatases, PP-1 and PP-2A, we found that Mn2+ caused a similar pattern of dephosphorylation of proteins as measured by PAGE analysis. Both okadaic acid and microcystin, two protein phosphatase inhibitors, largely counteracted the HSP-70 effect as measured by gel electrophoresis or when (32P)-labelled phosphorylase-alpha was used as a substrate. We conclude that in this system HSP-70 activates specific protein phosphatases.
我们以兔网织红细胞裂解液系统中的磷蛋白为模型，研究了在纯化的哺乳动物70kDa热休克蛋白（HSP-70）存在下的蛋白质磷酸化情况。通过对用γ-(32P)-ATP标记的蛋白质进行聚丙烯酰胺凝胶电泳（PAGE）分析测定，添加到兔网织红细胞裂解液中的纯化HSP-70使总体蛋白质磷酸化降低了50%-80%。通过薄层色谱法测定，蛋白质磷酸化的降低并非由于HSP-70的ATP酶活性。蛋白质磷酸化的降低也不是由于蛋白激酶活性的降低。然而，在磷酸酶测定系统中使用(32P)标记的磷酸化酶-α作为底物表明，在HSP-70浓度增加的情况下，蛋白磷酸酶1（PP-1）和/或2A（PP-2A）的活性相对于对照增加了20%-40%。使用两种主要蛋白磷酸酶PP-1和PP-2A的多种激活剂和抑制剂，我们发现通过PAGE分析测定，Mn2+引起了类似的蛋白质去磷酸化模式。两种蛋白磷酸酶抑制剂冈田酸和微囊藻毒素，在通过凝胶电泳测定时或当使用(32P)标记的磷酸化酶-α作为底物时，很大程度上抵消了HSP-70的作用。我们得出结论，在该系统中HSP-70激活了特定的蛋白磷酸酶。
