Panasyuk Ganna, Nemazanyy Ivan, Zhyvoloup Alexander, Bretner Maria, Litchfield David W, Filonenko Valeriy, Gout Ivan T
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv 03143, Ukraine.
J Biol Chem. 2006 Oct 20;281(42):31188-201. doi: 10.1074/jbc.M602618200. Epub 2006 Aug 8.
Ribosomal S6 kinases (S6Ks) are principal players in the regulation of cell growth and energy metabolism. Signaling via phosphatidylinositol 3-kinase and mammalian target of rapamycin pathways mediates the activation of S6K in response to various mitogenic stimuli. The family of S6Ks consists of two forms, S6K1 and -2, that have cytoplasmic and nuclear splicing variants, S6K1 II and S6K1 I, respectively. Nuclear-cytoplasmic shuttling of both isoforms induced by mitogenic stimuli has been reported recently. Here we present the identification of protein kinase CK2 (CK2) as a novel binding and regulatory partner for S6K1 II. The interaction between S6K1 II and CK2beta regulatory subunit was initially identified in a yeast two-hybrid screen and further confirmed by co-immunoprecipitation of transiently expressed and endogenous proteins. The interaction between S6K1 II and CK2 was found to occur in serum-starved and serum-stimulated cells. In addition, we found that S6K1 II is a substrate for CK2. The localization of the CK2 phosphorylation site was narrowed down to Ser-17 in S6K1 II. Mutational analysis and the use of phosphospecific antibody indicate that Ser-17 is a major in vitro and in vivo phosphorylation site for CK2. Functional studies reveal that, in contrast to the wild type kinase, the phosphorylation-mimicking mutant of S6K1 II (S17E) retains its cytoplasmic localization in serum-stimulated cells. Treatment of cells with the nuclear export inhibitor leptomycin B revealed that the S17E mutant accumulates in the nucleus to the same extent as S6K1 II wild type. These results indicate that nuclear import of the S17E mutant is not affected, although the export is significantly enhanced. We also provide evidence that nuclear export of S6K1 is mediated by a CRM1-dependent mechanism. Taken together, this study establishes a functional link between S6K1 II and CK2 signaling, which involves the regulation of S6K1 II nuclear export by CK2-mediated phosphorylation of Ser-17.
核糖体S6激酶（S6Ks）是细胞生长和能量代谢调控的主要参与者。通过磷脂酰肌醇3激酶和雷帕霉素哺乳动物靶标途径的信号传导介导了S6K在各种促有丝分裂刺激下的激活。S6K家族由两种形式组成，即S6K1和 -2，它们分别具有细胞质和细胞核剪接变体，即S6K1 II和S6K1 I。最近有报道称，促有丝分裂刺激可诱导这两种亚型在细胞核与细胞质之间穿梭。在此，我们报告鉴定出蛋白激酶CK2（CK2）是S6K1 II的一种新型结合和调节伙伴。S6K1 II与CK2β调节亚基之间的相互作用最初是在酵母双杂交筛选中鉴定出来的，并通过瞬时表达和内源性蛋白的共免疫沉淀进一步得到证实。发现S6K1 II与CK2之间的相互作用发生在血清饥饿和血清刺激的细胞中。此外，我们发现S6K1 II是CK2的底物。CK2磷酸化位点的定位被缩小到S6K1 II中的Ser-17。突变分析和使用磷酸特异性抗体表明，Ser-17是CK2在体外和体内的主要磷酸化位点。功能研究表明，与野生型激酶相比，S6K1 II的磷酸化模拟突变体（S17E）在血清刺激的细胞中保留其细胞质定位。用核输出抑制剂雷帕霉素处理细胞表明，S17E突变体在细胞核中的积累程度与S6K1 II野生型相同。这些结果表明，S17E突变体的核输入不受影响，尽管其输出明显增强。我们还提供证据表明，S6K1的核输出是由一种依赖CRM1的机制介导的。综上所述，本研究建立了S6K1 II与CK2信号传导之间的功能联系，这涉及通过CK2介导的Ser-17磷酸化对S6K1 II核输出的调节。
