PHLPP-Mediated Dephosphorylation of S6K1 Inhibits

PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Con-versely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phos-phorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1. Precise control of cell signaling is achieved by balancing