Grosso Stefano, Volta Viviana, Sala Leonardo A, Vietri Marina, Marchisio Pier Carlo, Ron Dorit, Biffo Stefano
Molecular Histology and Cell Growth, HSR, 20132 Milan, Italy.
Biochem J. 2008 Oct 1;415(1):77-85. doi: 10.1042/BJ20080463.
RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.
活化C激酶1受体（RACK1）是一种丰富的支架蛋白，它能结合活性蛋白激酶CβII（PKCβII），在体外增强其活性。RACK1也被描述为小核糖体亚基的一个组成部分，靠近mRNA出口通道。在本研究中，我们检验了PKCβII在翻译控制中发挥特定作用的假说，并验证了它是否可能与核糖体机制相关联。我们发现，特异性抑制PKCβI/II会降低翻译水平，与全局抑制PKC的效果相同，但不影响雷帕霉素靶蛋白（mTOR）靶点的磷酸化。这些结果表明，PKCβII作为一种特定的PKC同工型，以mTOR非依赖的方式影响翻译，可能靠近核糖体机制。通过远缘Western分析，我们发现PKCβII在体外能结合核糖体。免疫共沉淀研究表明，一小部分但可重复的PKCβII与含有核糖体的膜相关联，这表明在体内PKCβII也可能与核糖体机制发生物理相互作用。多核糖体图谱显示，PKC的激活导致多核糖体/80S比值增加，同时PKCβII向梯度较重的部分移动。一种抑制活性PKCβII与RACK1结合的RACK1衍生肽会降低多核糖体/80S比值和甲硫氨酸掺入，这表明PKCβII与RACK1的结合对于PKC介导的翻译控制很重要。最后，通过小干扰RNA（siRNA）下调RACK1会损害PKC介导的翻译增加。本研究结果综合表明，PKCβII可作为一种特定的PKC同工型，以mTOR非依赖的方式调节翻译，可能靠近核糖体机制。
