Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase βII and Regulates Its Subcellular Localization

Abstract

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G 1 /S transition of the cell cycle. There are two forms of S6K, termed S6K␣ and S6K␤, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6K␣, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6K␤, but not S6K␣, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6K␤II by PKC-mediated phosphorylation.The ribosomal protein S6 (rpS6) kinase (S6K) belongs to the AGC family of Ser/Thr protein kinases which includes the protein kinase C's (PKCs), protein kinase B's, SGKs, and 90-kDa ribosomal S6 kinases (p90 RSKs). There are two forms of S6K, S6K␣ and S6K␤, which have cytoplasmic (S6K␣II and S6K␤II) and nuclear (S6K␣I and S6K␤I) variants derived from alternative splicings at the N terminus (2, 15). S6K␣ and S6K␤ have a very high level of overall sequence similarity, with the greatest homology in the kinase and kinase extension domains. However, both kinases differ significantly at their N-and Cterminal regulatory regions, sharing only 28 and 25% homology, respectively. The C terminus of S6K␤ contains a specific proline-rich region, which is absent in S6K␣ and might be involved in mediating protein-protein interactions with SH3 and WW domain-containing molecules. The presence of a PDZ domain-binding motif at the C terminus of S6K␣ may direct the kinase into distinct signaling complexes (8).The activity of S6K is regulated by phosphorylation and dephosphorylation events in cellular responses to various extracellular stimuli. The treatment of cells with growth factors, cytokines, and hormones leads to a rapid activation of S6K (10), while growth inhibitory agents, such as steroids and transforming growth factor ␤, suppress kinase activity (45, 52). The mechanism of activation of S6K␣ has been studied in detail by various laboratories and was shown to be a multistep phosphorylation process involving several Ser/Thr kinases (14,64).No direct, highly specific S6K inhibitor has yet been identified. Under these circumstances, the use of two indirect inhibitors, namely wortmannin (a phosphatidylinositol 3-kinase [PI3-K] inhibitor) and rapamycin (an mTOR inhibitor), has been instrumental in dissecting signaling even...

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