Phosphorylation of ribosomal protein S6 (RPS6) on ser235/236 and ser240/244 in the pancreatic beta cell increases cell size and insulin content and influences systemic glucose homeostasis. Our phosphoproteomic analysis identified the protein phosphatase, PPm1K, as a novel regulator of RPS6 phosphorylation. We find that beta cells account for most PPm1K expression within pancreatic islets and that exposure of INS1 832/13 beta cells to increasing concentrations of glucose results in suppression of PPm1K mRNA expression. siRNA mediated knockdown of PPm1K in INS1 832/13 cells, to levels achieved by glucose, increased insulin content and secretion in basal and glucose stimulated conditions that was accompanied by robust phosphorylation of RPS6 on ser235/236 and ser240/244. PPm1K knockdown did not increase phosphorylation of p70 S6 Kinase (S6K) or the mammalian target of rapamycin (mTOR) upstream of RPS6 suggesting that PPm1K regulates RPS6 phosphorylation in an mTOR-S6K independent manner. Accordingly, treatment with the mTORC1 inhibitor, Rapamycin, lowered S6K and RPS6 phosphorylation but did not override the effect of PPm1K knockdown to increase RPS6 phosphorylation compared to control siRNA treated cells. Lowering phosphorylation of PPm1K’s classical target, ser293 of the branched-chain keto acid dehydrogenase (BCKDH) with the small molecule, BT2, also did not impact the effect of PPm1K knockdown to increase RPS6 phosphorylation. Thus PPm1K exerts its effects on RPS6 phosphorylation in a BCKDH and mTOR/S6K independent manner. The effects of siRNA-mediated PPm1K knockdown on RPS6 phosphorylation were accompanied by a 1.5 fold increase in protein translation and the induction of an unfolded protein response as determined by an increase in XBP-1 splicing, and BiP expression. Together our data identify a new role for the phosphatase PPm1K as a glucose sensitive modulator of RPS6 phosphorylation and protein translation that impacts beta cell insulin content and secretion.
Y. Deleye: None. J. Herring: None. K. H. Hess: None. B. Leininger: None. R. W. Mcgarrah: None. J. S. Tessem: None. P. J. White: None.
American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)