title: Hydrophobic motif site-phosphorylated protein kinase CβII between mTORC2 and Akt regulates high glucose-induced mesangial cell hypertrophy.
abstract: PKCβII controls the pathologic features of diabetic nephropathy, including glomerular mesangial cell hypertrophy. PKCβII 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 PKCβII at Ser-660 in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. Using siRNAs to downregulate PKCβII, dominant negative PKCβII, and PKCβII hydrophobic motif phosphorylation-deficient mutant, we found that PKCβII regulates activation of mechanistic target of rapamycin complex 1 (mTORC1) and mesangial cell hypertrophy by high glucose. PKCβII 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 PKCβII 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 PKCβII 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 PKCβII 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 PKCβII at the hydrophobic motif site necessary for Akt phosphorylation and mTORC1 activation, leading to mesangial cell hypertrophy.
authors.affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas;
authors.forename: Falguni
authors.initials: F
authors.lastname: Das
authors.affiliation: Veterans Affairs Research, South Texas Veterans Health Care System, San Antonio, Texas; Departments of Pathology, University of Texas Health Science Center at San Antonio, San Antonio, Texas choudhuryg@uthscsa.edu.
authors.forename: Nandini
authors.initials: N
authors.lastname: Ghosh-Choudhury
authors.affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas;
authors.forename: Meenalakshmi M
authors.initials: MM
authors.lastname: Mariappan
authors.affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Veterans Affairs Research, South Texas Veterans Health Care System, San Antonio, Texas;
authors.forename: Balakuntalam S
authors.initials: BS
authors.lastname: Kasinath
authors.affiliation: Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Veterans Affairs Research, South Texas Veterans Health Care System, San Antonio, Texas; Geriatric Research, Education and Clinical Research, South Texas Veterans Health Care System, San Antonio, Texas; and.
authors.forename: Goutam Ghosh
authors.initials: GG
authors.lastname: Choudhury
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D049108:Cell Enlargement; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism / Q000473:pathology; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism / Q000473:pathology; D003928:Diabetic Nephropathies / Q000378:metabolism* / Q000473:pathology; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000633:toxicity; D006801:Humans; D006984:Hypertrophy; D015151:Immunoblotting; D047468:Immunoprecipitation; D000076225:Mechanistic Target of Rapamycin Complex 2; D050527:Mesangial Cells / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D064546:Protein Kinase C beta / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014162:Transfection
chemical_list: D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D000076225:Mechanistic Target of Rapamycin Complex 2; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D064546:Protein Kinase C beta; D005947:Glucose
keywords: diabetic nephropathy; mTOR complex; protein kinase C; signal transduction