Hydrophobic motif site-phosphorylated protein kinase CßII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.

ABSTRACT

PKCßII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCßII contains the COOH-terminal hydrophobic motif site Ser-660. Whether this hydrophobic motif phosphorylation contributes to high glucose-induced mesangial cell hypertrophy has not been determined. Here we show that, in mesangial cells, high glucose increased phosphorylation of PKCßII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCßII, dominant negative PKCßII, and PKCßII hydrophobic motif phosphorylation-deficient mutant, we found that PKCßII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCßII via its phosphorylation at Ser-660 regulated phosphorylation of Akt at both catalytic loop and hydrophobic motif sites, resulting in phosphorylation and inactivation of its substrate PRAS40. Specific inhibition of mTORC2 increased mTORC1 activity and induced mesangial cell hypertrophy. In contrast, inhibition of mTORC2 decreased the phosphorylation of PKCßII and Akt, leading to inhibition of PRAS40 phosphorylation and mTORC1 activity and prevented mesangial cell hypertrophy in response to high glucose; expression of constitutively active Akt or mTORC1 restored mesangial cell hypertrophy. Moreover, constitutively active PKCßII reversed the inhibition of high glucose-stimulated Akt phosphorylation and mesangial cell hypertrophy induced by suppression of mTORC2. Finally, using renal cortexes from type 1 diabetic mice, we found that increased phosphorylation of PKCßII at Ser-660 was associated with enhanced Akt phosphorylation and mTORC1 activation. Collectively, our findings identify a signaling route connecting PI3-kinase to mTORC2 to phosphorylate PKCßII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.