Pilkinton Mark, Sandoval Raudel, Barrett Kelly, Tian Xinyong, Colamonici Oscar R
University of Illinois, Department of Pharmacology, 835 S. Wolcott, Room E403 (M/C 868), Chicago, IL 60612, USA.
Blood Cells Mol Dis. 2007 Nov-Dec;39(3):272-7. doi: 10.1016/j.bcmd.2007.05.006. Epub 2007 Jul 6.
Progression through the G1-phase of the cell cycle requires that cyclin D and CDK4 phosphorylate pRB and the other pocket proteins, p107 and p130. Cyclin E and CDK2 further phosphorylate pRB to complete its inactivation and allow the cell to enter S-phase. These phosphorylation events lead to the inactivation of the antiproliferative effect of the pocket proteins. The pocket proteins are the main targets of CDK4, and its unregulated activity can contribute to carcinogenesis. Mip/LIN9 is a recently described protein with growth suppressor, as well as growth promoting effects due to its ability to stabilize B-Myb and induce genes required for S phase and mitosis. The finding that a mutation that deletes the first 84 amino acids of Mip/LIN-9 corrects the defects of the CDK4 knockout mouse suggests that it should have a growth repressor effect that is blocked by CDK4. However, overexpression of cyclin D only partially blocks the inhibitory effect of Mip/LIN-9 on cell proliferation. Here, we performed experiments to further understand the antiproliferative effect of Mip/LIN-9 within the context of the pocket proteins. Our results suggest that there is a pocket protein-independent mechanism of the Mip/LIN-9 antiproliferative effect since it can be observed in cells with ablation of the three members of the family, and in NIH3T3 cells expressing the adenovirus E1A-12S protein. Altogether, the independence from the pocket proteins and the partial blockade of the antiproliferative effect produced by expression of cyclin D suggest that the role of Mip/LIN-9 downstream of CDK4 may be more closely related to the activation of B-Myb and the induction of S/M genes. Importantly, the regulatory effect of CDK4 is not due to direct phosphorylation of Mip/LIN-9 by this kinase or even CDK2, suggesting an indirect mechanism such as phosphorylation of the pocket proteins.
细胞周期G1期的进展需要细胞周期蛋白D和CDK4使pRB以及其他口袋蛋白p107和p130磷酸化。细胞周期蛋白E和CDK2进一步使pRB磷酸化以完成其失活，并使细胞进入S期。这些磷酸化事件导致口袋蛋白的抗增殖作用失活。口袋蛋白是CDK4的主要靶标，其不受调控的活性可能导致癌症发生。Mip/LIN9是最近描述的一种蛋白质，具有生长抑制作用，同时由于其能够稳定B-Myb并诱导S期和有丝分裂所需的基因，也具有生长促进作用。删除Mip/LIN-9前84个氨基酸的突变能够纠正CDK4基因敲除小鼠的缺陷，这一发现表明它应该具有被CDK4阻断的生长抑制作用。然而，细胞周期蛋白D的过表达仅部分阻断了Mip/LIN-9对细胞增殖的抑制作用。在这里，我们进行了实验，以在口袋蛋白的背景下进一步了解Mip/LIN-9的抗增殖作用。我们的结果表明，存在一种不依赖口袋蛋白的Mip/LIN-9抗增殖作用机制，因为在该家族三个成员缺失的细胞以及表达腺病毒E1A-12S蛋白的NIH3T3细胞中都能观察到这种作用。总之，不依赖口袋蛋白以及细胞周期蛋白D表达对其抗增殖作用的部分阻断表明，CDK4下游的Mip/LIN-9的作用可能与B-Myb的激活和S/M基因的诱导更密切相关。重要的是，CDK4的调节作用不是由于该激酶甚至CDK2对Mip/LIN-9的直接磷酸化，这表明存在一种间接机制，如口袋蛋白的磷酸化。
