Ser-660 Phosphorylation of Protein Kinase C beta II ( PKCβII) by mammalian target of rapamycin complex 2 (mTORC2) regulates High Glucose (HG)-induced Mesangial Cell hypertrophy

13^th Annual Conference on Nephrology & Renal Care

May 24-25, 2018 Tokyo, Japan

F Das, N Ghosh-Choudhury, M Mariappan, B S Kasinath and G Ghosh Choudhury

University of Texas Health Science Center, USA

Posters & Accepted Abstracts: J Nephrol Ther

Abstract :

Protein kinase C beta II (PKC�²II) has been implicated in Diabetic Nephropathy (DN). Mesangial cell (MC) hypertrophy is a pathologic feature of DN. PKC�²II undergoes phosphorylation at the hydrophobic motif site Ser-660 for its activity. We have shown that mTOR Complex 1 (C1) regulates MC hypertrophy. How activation of PKC�²II by Ser-660 phosphorylation fits into mTOR signaling to control MC hypertrophy is not known. HG significantly increased phosphorylation of PKC�²II at Ser-660 in a PI 3 kinase-dependent manner. siRNAs against PKC�²II, dominant negative PKC�²II and nonphosphorylatable mutant of PKC�²II, PKC�²IIS660A, blocked mTORC1 activity due to lack of PRAS40 phosphorylation, resulting in significant inhibition of HG-induced MC protein synthesis and hypertrophy. Also, PKC�²IIS660A attenuated phosphorylation of Akt at Ser-473, a putative mTOR complex 2 (C2) site. Specific inhibition of mTORC2 by shRNAs against rictor or Sin1, two exclusive and required components for its activity, suppressed HG-induced phosphorylation of PKC�²II Ser-660 and Akt Ser-473, resulting in attenuation of mTORC1 activity leading to inhibition of MC hypertrophy. Constitutively Active (CA) Akt or CA mTORC1 reversed shRictor- or shSin1-mediated inhibition of HG-induced MC hypertrophy. Furthermore, CA PKC�²II reversed the shRictor- or shSin1induced inhibition of HG-stimulated Akt Ser-473 phosphorylation and MC hypertrophy. Finally, we show increased phosphorylation of PKC�²II Ser660, PRAS40 and Akt Ser-473 in association with activation of mTORC1 in renal cortices of OVE26 mice with type 1 diabetes. These results provide the first evidence that HG-induced activation of mTORC2 phosphorylates and activates PKC�²II to increase the phosphorylation of Akt at Ser-473 to finally activate mTORC1 to induce MC hypertrophy. Thus, we uncover a specific role of mTORC2 for Akt/mTORC1 activation via PKC�²II Ser-660 phosphorylation. dasf@uthscsa.edu