Nelson D A, Krucher N A, Ludlow J W
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
J Biol Chem. 1997 Feb 14;272(7):4528-35. doi: 10.1074/jbc.272.7.4528.
pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.
pRb通过限制细胞不恰当地进入细胞分裂周期来控制细胞增殖。由于有丝分裂退出过程中pRb的去磷酸化激活了其生长抑制功能，因此鉴定使pRb去磷酸化的蛋白磷酸酶并表征其调控机制，对于阐明细胞生长控制机制至关重要。通过对有丝分裂CV-1P细胞提取物进行分级分离，我们鉴定出使pRb去磷酸化的蛋白磷酸酶为1型丝氨酸/苏氨酸磷酸蛋白磷酸酶（PP1）。分子大小分析表明，催化酶（PP1c）存在于高分子量复合物中，预测分子量为166 kDa。鉴定了有丝分裂细胞提取物中与PP1相互作用的蛋白。两种与PP1相互作用的蛋白（41 kDa和110 kDa）显示与来自含有磷酸化酶磷酸酶活性的分离有丝分裂细胞提取物级分中的PP1c形成不同的复合物。然而，只有110 kDa与PP1相互作用的蛋白存在于含有pRb定向磷酸酶活性的级分中，将该蛋白鉴定为有丝分裂期间PP1对pRb功能的假定激活剂。
