Ludlow J W, Glendening C L, Livingston D M, DeCarprio J A
Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Mol Cell Biol. 1993 Jan;13(1):367-72. doi: 10.1128/mcb.13.1.367-372.1993.
The retinoblastoma gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. Pulse-chase experiments revealed that the change in RB gel electrophoretic migration which occurs near mitosis is due to enzymatic dephosphorylation (J. W. Ludlow, J. Shon, J. M. Pipas, D. M. Livingston, and J. A. DeCaprio, Cell 60:387-396, 1990). To determine the precise timing of RB dephosphorylation and whether a specific phosphatase is active in this process, we have utilized a nocodazole block and release protocol which allows a large population of cells to progress synchronously through mitosis. In such experiments, RB dephosphorylation began during anaphase and continued until complete dephosphorylation was apparent in the ensuing G1 period. In addition, late mitotic cell extracts were capable of dephosphorylating RB in vitro. This RB-specific mitotic phosphatase activity was more active in anaphase extracts than in pro- or metaphase extracts, which is consistent with the results obtained in vivo. Okadaic acid and protein phosphatase inhibitors 1 and 2 inhibited this specific RB phosphatase activity. These results suggest a role for serine and threonine phosphoprotein phosphatase type 1 in the late mitotic dephosphorylation of RB.
视网膜母细胞瘤基因产物（RB）经历细胞周期依赖性磷酸化和去磷酸化过程。脉冲追踪实验表明，在有丝分裂附近发生的RB凝胶电泳迁移变化是由于酶促去磷酸化所致（J. W. 勒德洛、J. 肖恩、J. M. 皮帕斯、D. M. 利文斯顿和J. A. 德卡普里奥，《细胞》60:387 - 396，1990）。为了确定RB去磷酸化的精确时间以及是否有特定的磷酸酶在此过程中起作用，我们采用了诺考达唑阻断和释放方案，该方案可使大量细胞同步进行有丝分裂。在这类实验中，RB去磷酸化在后期开始，并持续到随后的G1期明显完全去磷酸化。此外，有丝分裂后期细胞提取物能够在体外使RB去磷酸化。这种RB特异性有丝分裂磷酸酶活性在后期提取物中比在前期或中期提取物中更活跃，这与体内获得的结果一致。冈田酸以及蛋白磷酸酶抑制剂1和2抑制了这种特异性RB磷酸酶活性。这些结果表明1型丝氨酸和苏氨酸磷蛋白磷酸酶在RB有丝分裂后期去磷酸化过程中发挥作用。
