Guan Lingjie, Song Kyung, Pysz Marybeth A, Curry Kathryn J, Hizli A Asli, Danielpour David, Black Adrian R, Black Jennifer D
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
J Biol Chem. 2007 May 11;282(19):14213-25. doi: 10.1074/jbc.M610513200. Epub 2007 Mar 13.
We reported previously that protein kinase Calpha (PKCalpha), a negative regulator of cell growth in the intestinal epithelium, inhibits cyclin D1 translation by inducing hypophosphorylation/activation of the translational repressor 4E-BP1. The current study explores the molecular mechanisms underlying PKC/PKCalpha-induced activation of 4E-BP1 in IEC-18 nontransformed rat ileal crypt cells. PKC signaling is shown to promote dephosphorylation of Thr(45) and Ser(64) on 4E-BP1, residues directly involved in its association with eIF4E. Consistent with the known role of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway in regulation of 4E-BP1, PKC signaling transiently inhibited PI3K activity and Akt phosphorylation in IEC-18 cells. However, PKC/PKCalpha-induced activation of 4E-BP1 was not prevented by constitutively active mutants of PI3K or Akt, indicating that blockade of PI3K/Akt signaling is not the primary effector of 4E-BP1 activation. This idea is supported by the fact that PKC activation did not alter S6 kinase activity in these cells. Further analysis indicated that PKC-mediated 4E-BP1 hypophosphorylation is dependent on the activity of protein phosphatase 2A (PP2A). PKC signaling induced an approximately 2-fold increase in PP2A activity, and phosphatase inhibition blocked the effects of PKC agonists on 4E-BP1 phosphorylation and cyclin D1 expression. H(2)O(2) and ceramide, two naturally occurring PKCalpha agonists that promote growth arrest in intestinal cells, activate 4E-BP1 in PKC/PKCalpha-dependent manner, supporting the physiological significance of the findings. Together, our studies indicate that activation of PP2A is an important mechanism underlying PKC/PKCalpha-induced inhibition of cap-dependent translation and growth suppression in intestinal epithelial cells.
我们之前报道过，蛋白激酶Cα（PKCα）作为肠上皮细胞生长的负调节因子，通过诱导翻译阻遏物4E-BP1的低磷酸化/激活来抑制细胞周期蛋白D1的翻译。当前研究探讨了PKC/PKCα诱导IEC-18非转化大鼠回肠隐窝细胞中4E-BP1激活的分子机制。结果显示PKC信号传导促进4E-BP1上Thr(45)和Ser(64)的去磷酸化，这两个残基直接参与其与eIF4E的结合。与磷酸肌醇3激酶（PI3K）/Akt/雷帕霉素靶蛋白（mTOR）途径在4E-BP1调节中的已知作用一致，PKC信号传导短暂抑制了IEC-18细胞中的PI3K活性和Akt磷酸化。然而，PI3K或Akt的组成型活性突变体并不能阻止PKC/PKCα诱导的4E-BP1激活，这表明PI3K/Akt信号传导的阻断不是4E-BP1激活的主要效应器。PKC激活未改变这些细胞中S6激酶活性这一事实支持了这一观点。进一步分析表明，PKC介导的4E-BP1低磷酸化依赖于蛋白磷酸酶2A（PP2A）的活性。PKC信号传导使PP2A活性增加约2倍，磷酸酶抑制阻断了PKC激动剂对4E-BP1磷酸化和细胞周期蛋白D1表达的影响。H₂O₂和神经酰胺是两种天然存在的PKCα激动剂，可促进肠细胞生长停滞，它们以PKC/PKCα依赖的方式激活4E-BP1，支持了这些发现的生理意义。总之，我们的研究表明，PP2A的激活是PKC/PKCα诱导肠上皮细胞中帽依赖性翻译抑制和生长抑制的重要机制。
