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

Reagents.

d-glucose, d-mannitol, Nonidet P-40, PMSF, Na₃VO₄, protease inhibitor cocktail, and actin antibody were purchased from Sigma. Phospho-PKCβII (Ser-660), phospho-Akt (Ser-473), phospho-Akt (Thr-308), Akt, phospho-4EBP-1 (Thr-37/46), 4EBP-1, phospho-S6 kinase (Thr-389), S6 kinase, phospho-PRAS40 (Thr-246), PRAS40, mTOR, and rictor antibodies were purchased from Cell Signaling Technology. HA antibody was purchased from Covance. PKCβII, p85 regulatory subunit of PI3-kinase, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), and GFP antibodies and pooled siRNAs against PKCβII were purchased from Santa Cruz Biotechnology. shRNA expression vectors against human rictor (shRictor 1 and shRictor 2) and scramble vector were obtained from Addgene and described previously (10). GFP-tagged PKCβII660A expression vector was a gift from Dr. Y. Hannnun, Medical University of South Carolina. Dominant negative PKCβII K371R and constitutively active PKCβII CAT plasmids were obtained from Addgene. Constitutively active Myr Akt, constitutively active mTOR, which acts as mTORC1, and adenovirus vectors expressing the deletion mutant of p85 regulator subunit of PI3-kinase and PTEN were described previously (2, 10, 11, 45).

Cell culture and adenovirus infection.

Previously, we and Abboud and coworkers (9, 68) have described the preparation of normal human mesangial cells from renal cortex of human kidneys unsuitable for transplantation. These cells have been extensively used by us and were cultured in DMEM in the presence of 10% fetal bovine serum as described previously (6, 9). Cells were incubated in serum free medium for 24 h before incubation with 25 mM glucose (high glucose). As osmotic control 5 mM glucose plus 20 mM mannitol (low glucose) was used. Where necessary, mesangial cells were infected with adenovirus vectors in serum free medium at a multiplicity of infection of 50 for 24 h as described previously (11). As control, infection with an adenovirus vector expressing green fluorescence protein (Ad GFP) was used.

Animals.

OVE26 mice expressing pancreatic β-cell-specific calmodulin transgene and their control littermate FVB mice were purchased from The Jackson Laboratories. OVE26 mice are hyperglycemic at 3 days of age and develop significant renal hypertrophy, glomerular hypertrophy, and albuminuria at 2 mo of age (15, 84). The mice were kept in the University of Texas Health Science Center animal facility. They had free access to food and water. After the control FVB and OVE26 mice were euthanized at 3 mo of age, both kidneys were removed. Renal cortical tissues from each mouse were isolated and frozen as described previously (13). The University of Texas Health Science Center Institutional Animal Care and Use Committee approved the animal protocol.

Cell lysis, immunoprecipitation, and immunoblotting.

After high-glucose treatment, the cells were washed with PBS. The monolayer was harvested in radioimmune precipitation buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1 mM Na₃VO₄, 1% NP-40, and 0.1% protease inhibitor cocktail). Similarly, renal cortexes from control and diabetic mice were harvested in the same RIPA buffer. The cells and renal cortexes were lysed for 30 min at 4°C. The cell extracts were centrifuged at 12,000 g for 30 min. The supernatant was collected and the protein concentration was determined. Equal amounts of proteins were separated by SDS-PAGE. Separated proteins were transferred to membrane. Immunoblotting was performed using indicated antibodies. The protein bands were developed with horseradish peroxidase-conjugated secondary antibodies (10, 12). For immunoprecipitation, equal amounts of protein were incubated with PKCβII antibody and immunoprecipitated using protein G agarose beads. The immunebeads were suspended in sample buffer followed by electrophoresis and immunoblotting using phospho-PKCβII (Ser-660) antibody as described above.

mTORC2 immunecomplex kinase assay.

Renal cortical lysates were immunoprecipitated with rictor antibody. The immunoprecipitates were washed three times with the RIPA buffer at 4°C. Then, the immunoprecipitates were washed twice with the mTORC2 immunecomplex kinase assay buffer (25 mM HEPES pH 7.4, 100 mM potassium acetate, and 1 mM MgCl₂). The immunoprecipitates were resuspended in 20 μl immunecomplex kinase assay buffer. One-hundred and twenty-five nanograms of recombinant PKCβII and 500 μM ATP were added. The reaction mixture was incubated at 37°C for 30 min and was terminated by adding 4× SDS sample buffer. The protein was analyzed by immunoblotting with phospho-PKCβII (Ser-660) antibody. For control, 25 ng of recombinant PKCβII were immunoblotted separately with PKCβII antibody.

Protein synthesis.

After incubation, the mesangial cells were labeled with ³⁵S-methionine. Protein synthesis was determined as ³⁵S-methionine incorporation as described previously (11, 14).

Measurement of cellular hypertrophy.

The cells were trypsinized and counted in a hemocytometer. Cells were then centrifuged at 4,000 g at 4°C. The cell pellet was washed with PBS and lysed in RIPA buffer as described above. The protein content in the total number of cells was determined. Hypertrophy was determined as a ratio of total protein to number of cell, as described previously (14).

Statistics.

The data were analyzed by ANOVA followed by Student-Newman-Keuls analysis or by paired Student's t-test. A P < 0.05 was considered significant (10, 11).

High glucose-induced increase in hydrophobic motif site phosphorylation of PKCβII is PI3-kinase dependent and regulates mesangial cell hypertrophy.

Glomeruli from diabetic rodents and high glucose-treated glomerular mesangial cells in culture show increased activity of different PKC isoforms (28). However, activation of PKCβII is involved in diabetic complications of kidney. Similar to other PKC isotypes, PKCβII requires phosphorylation for activation. All members of the AGC kinase family including PKCβII contain a hydrophobic motif site, Ser-660, which undergoes phosphorylation. Although Ser-660-unphosphorylated PKCβII can undergo lipid-dependent activation, this phosphorylation is necessary for its dissociation from membrane (19, 34). To systematically investigate the role of PKCβII hydrophobic motif phosphorylation in mesangial cell function, we evaluated its status in response to high glucose. The phospho-PKCβII (Ser-660) antibody predominantly recognizes phosphorylated PKCβII at Ser-660. Due to high conservation around the hydrophobic motif sites among other PKC isotypes, the antibody cross reacts weakly with other isotypes of PKC when they are phosphorylated. Therefore, PKCβII immunoprecipitates were used from lysates of mesangial cells incubated with high glucose. High glucose increased phosphorylation of PKCβII at Ser-660 in a time-dependent and sustained manner for up to 48 h (Fig. 1, A and B). Many PKC isotypes have been shown to be activated by PI3-kinase-dependent PDK1-mediated activation loop phosphorylation (40). However, the role of PI3-kinase in phosphorylation of PKCβII hydrophobic motif site has not been characterized. The PI3-kinase inhibitor Ly294002 significantly blocked high glucose-stimulated Ser-660 phosphorylation of PKCβII (Fig. 1C). To confirm this observation, we used a deletion mutant of the p85 regulatory subunit of PI3-kinase, which confers dominant negative function to this enzyme. Expression of dominant negative PI3-kinase inhibited high glucose-induced PKCβII phosphorylation (Fig. 1D). Similarly, expression of the tumor suppressor protein PTEN, which is an endogenous inhibitor of PI3-kinase signaling, abolished phosphorylation of PKCβII induced by high glucose (Fig. 1E).

We have shown previously that high glucose-induced PI3-kinase activity contributes to mesangial cell hypertrophy (14, 45). Our results above show that PKCβII is a downstream target of PI3-kinase signaling. Also, PKCβ knockout mouse showed protection from diabetic renal, especially glomerular hypertrophy (49). Since this model does not discriminate between the roles of PKCβ1 and PKCβII, and PKCβII activity is increased in diabetic renal glomeruli, we tested the specific role of PKCβII in mesangial cell hypertrophy. We used a pool of siRNAs specifically targeting PKCβII. The results showed that downregulation of PKCβII significantly inhibited high glucose-induced protein synthesis (Fig. 1F). Consequently, mesangial cell hypertrophy as determined by the ratio of total protein to cell number in response to high glucose was also inhibited (Fig. 1G). To determine the role of PKCβII kinase activity in mesangial cell hypertrophy, we employed a kinase dead mutant with a mutation at the Lys-371 to Arg (70). Expression of this mutant markedly inhibited the high glucose-stimulated protein synthesis and hypertrophy of mesangial cells (Fig. 1, H and I). The hydrophobic motif site phosphorylation is associated with the activities of various PKC isotypes. However, its role in a specific biological activity such as protein synthesis is poorly understood. Expression of PKCβII nonphosphorylatable mutant significantly prevented both protein synthesis and hypertrophy of mesangial cells in response to high glucose (Fig. 1, J and K). These results suggest that the hydrophobic motif site phosphorylation of PKCβII is essential for high glucose-induced mesangial cell hypertrophy.

PKCβII increases mTORC1 signaling.

We and others have shown that mTORC1 activation is necessary for renal hypertrophy in rodents (61, 65). Also, we showed the requirement of mTORC1 in high glucose-induced mesangial cell hypertrophy (14). To examine the role of PKCβII in mTORC1 activation, we determined the phosphorylation of its two endogenous substrates, S6 kinase and 4EBP-1. As expected, high glucose increased phosphorylation of these two proteins (Fig. 2). siRNAs targeting PKCβII blocked high glucose-stimulated phosphorylation of S6 kinase and 4EBP-1 (Fig. 2, A and B). Expression of dominant negative PKCβII inhibited high glucose-stimulated phosphorylation of S6 kinase and 4EBP-1 (Fig. 2, C and D). Similarly, nonphosphorylatable PKCβII S660A abrogated the phosphorylation of both mTORC1 substrates (Fig. 2, E and F).

Ser-660 phosphorylation of PKCβII is necessary for Akt activation.

Our previous studies demonstrated that high glucose-induced mesangial cell hypertrophy is dependent on phosphorylation/inactivation of PRAS40, an Akt substrate and a negative regulator of mTORC1 activity (14). We determined the role of PKCβII in phosphorylation of PRAS40. Both siRNAs against PKCβII and dominant negative PKCβII inhibited high glucose-induced phosphorylation of PRAS40 at Thr-246 (Fig. 3, A and B). Similarly, the PKCβII Ser-660A phosphorylation-deficient mutant blocked the phosphorylation of PRAS40 in response to high glucose (Fig. 3C). We and others previously reported that Akt kinase activation is associated with renal hypertrophy in rodents (33, 45). It is known that activated Akt kinase phosphorylates PRAS40 at Thr-246 (62). Thus inhibition of its phosphorylation by PKCβII mutant indicates a role of this kinase in activation of Akt. Indeed high glucose increased the phosphorylation of Akt at both the hydrophobic motif (Ser-473) and catalytic loop (Thr-308) sites in a time-dependent manner similar to that observed with PKCβII Ser-660 phosphorylation (Figs. 3, D and E, and 1, A and B). Note that the hydrophobic motif site phosphorylation of Akt at Ser-473 is mediated by mTORC2 (64). However, downregulation of PKCβII by siRNAs or dominant negative PKCβII expression abrogated high glucose-induced phosphorylation of Akt at both sites (Fig. 3, F and G). Moreover, the expression of PKCβII Ser-S660A inhibited the phosphorylation of Akt at these sites (Fig. 3H). These results indicate that Ser-660 phosphorylated PKCβII regulates high glucose-stimulated Akt activation necessary for the phosphorylation of PRAS40, which results in activation of mTORC1 leading to mesangial cell hypertrophy.

mTORC2 regulates high glucose-induced PKCβII hydrophobic motif phosphorylation and mTORC1 activation.

It was shown previously that kinase activity of mTORC2 phosphorylates the hydrophobic motif site of conventional PKCα isotype (63). We tested whether mTORC2 could phosphorylate the hydrophobic motif site of PKCβII. A classical way of studying the role of mTORC2 is to downregulate one of its exclusive components rictor (10, 63). With the use of two independent shRNAs to downregulate endogenous rictor, which is necessary for mTORC2 activity (63), our results showed inhibition of high glucose-induced phosphorylation of PKCβII at Ser-660 (Fig. 4A). Importantly, high glucose-induced phosphorylation of PKCβII at this site was inhibited by the PI3-kinase inhibitor Ly294002 (Fig. 1C). To directly determine the involvement of PI3-kinase in mTORC2-mediated phosphorylation of PKCβII, we immunoprecipitated rictor, the specific and required component of mTORC2, from lysates of mesangial cells treated with Ly294002 before incubation with high glucose. The immunoprecipitates were used in an immunecomplex kinase assay to determine mTORC2 activity using recombinant PKCβII protein as substrate in the presence of ATP. The reaction product was immunoblotted with phospho-PKCβII (Ser-660). High glucose significantly increased the mTORC2 activity, which was inhibited by Ly 294002 (Fig. 4B). These results indicate that high glucose activates mTORC2 in a PI3-kinase-sensitive manner.

We have shown above that hydrophobic motif site phosphorylated PKCβII regulates mTORC1 activation by high glucose (Fig. 2, E and F). Since mTORC2 regulates phosphorylation of PKCβII, we systematically examined the involvement of mTORC2 in mTORC1 activation in high glucose-treated mesangial cells. Since phosphorylation of PRAS40 is necessary for mTORC1 activation by high glucose, we first tested the effect of inhibition of mTORC2 activity on its phosphorylation. Inhibition of mTORC2 by downregulation of rictor with two independent shRNAs showed suppression of PRAS40 phosphorylation in response to high glucose (Fig. 4C). Also, downregulation of rictor resulted in the inhibition of phosphorylation of Akt at both sites (Fig. 4D), which is necessary for its activation and PRAS40 phosphorylation (62, 64). Therefore, we tested the role of mTORC2 in high glucose-stimulated mTORC1 activity. Downregulation of rictor by two individual shRNAs inhibited high glucose-increased mTORC1 activity as judged by phosphorylation of S6 kinase (Fig. 4E). To confirm mTORC2 regulation of mTORC1, we examined phosphorylation of 4EBP-1, another direct substrate of mTORC1. Inhibition of expression of rictor abrogated high glucose-stimulated 4EBP-1 phosphorylation (Fig. 4F). Note that inhibition of mTORC2 by shRNA against rictor increased basal phosphorylation of both S6 kinase and 4EBP-1 (Fig. 4, E and F; see discussion). These results conclusively show that mTORC2 contributes to the activation of mTORC1 in response to high glucose in mesangial cells; in contrast, mTORC2 appears to constitutively inhibit mTORC1 in the basal state.

mTORC2 regulates mesangial cell hypertrophy via Akt-mTORC1.

We have previously shown that both Akt and mTORC1 regulate mesangial cell hypertrophy (14, 45). Since we have shown above that mTORC2 contributes to the activation of mTORC1 in response to high glucose (Fig. 4), we directly examined directly the role of mTORC2 in mesangial cell hypertrophy. Specific inhibition of rictor by shRNAs to block mTORC2 activity significantly abrogated the protein synthesis in mesangial cells induced by high glucose (Fig. 5A). Similarly, shRNAs against rictor markedly inhibited high glucose-induced mesangial cell hypertrophy (Fig. 5B). These results suggest an involvement of mTORC2 in mesangial cell hypertrophy. Interestingly, inhibition of mTORC2 under basal condition increased both protein synthesis and hypertrophy of mesangial cells (Fig. 5, A and B).

However, we have shown above that mTORC2 is upstream of mTORC1, which in turn is regulated by Akt kinase (Fig. 4). Therefore, to directly test the involvement of Akt and mTORC1 downstream of mTORC2, we overexpressed constitutively active mutant of Akt (Myr Akt) along with shRNA against rictor. As expected, shRictor significantly inhibited the high glucose-induced protein synthesis and mesangial cell hypertrophy (Fig. 5, C and D). Expression of constitutively active Akt kinase significantly reversed the inhibition of protein synthesis and hypertrophy by shRictor in the presence of high glucose (Fig. 5, C and D). Similarly, expression of constitutively active mTORC1 (CA mTOR) prevented the shRictor-induced suppression of high glucose-induced protein synthesis and hypertrophy (Fig. 5, C and D). These results conclusively indicate that both Akt and mTORC1 act downstream of mTORC2 to regulate mesangial cell hypertrophy in response to high glucose.

mTORC2-regulated PKCβII controls Akt Ser-473 phosphorylation for the induction of mesangial cell hypertrophy.

It is known that mTORC2 phosphorylates Akt Ser-473 for its full activation (64). We showed above that mTORC2 phosphorylates PKCβII at its hydrophobic motif site Ser-660 (Fig. 4A). Also, our results above conclusively demonstrate that active PKCβII is necessary for Akt Ser-473 phosphorylation in response to high glucose in mesangial cells (Fig. 3, F, G, and H). Therefore, we investigated the connection between mTORC2 and PKCβII for Akt Ser-473 phosphorylation. As expected, when mTORC2 was blocked by downregulation of rictor, phosphorylation of Akt at Ser-473 by high glucose was inhibited (Fig. 6A). Interestingly, coexpression of constitutively active PKCβII reversed the shRictor-induced inhibition of high glucose-stimulated phosphorylation of Akt (Fig. 6A, compare lane 8 with lane 4). Importantly, expression of constitutively active PKCβII alone under low glucose condition was sufficient to induce Akt Ser-473 phosphorylation (Fig. 6A, compare lane 5 with lane 1). Moreover, under basal mTORC2-inhibited condition in the presence of shRictor, PKCβII increased phosphorylation of Akt at Ser-473 (Fig. 6A, compare lane 7 with lane 1). These results suggest that downstream of mTORC2, PKCβII is the kinase for phosphorylation of Akt hydrophobic motif site Ser-473 in the presence of high glucose.

We have shown that mTORC2 and PKCβII independently regulate high glucose-induced mesangial cell hypertrophy (Figs. 1, F–K, and 5). To examine the direct contribution of PKCβII downstream of mTORC2, we coexpressed CA PKCβII along with shRNAs against rictor. As expected downregulation of rictor to inhibit mTORC2 activity blocked high glucose-induced protein synthesis and hypertrophy in mesangial cells (Fig. 6, B and C). Coexpression of CA PKCβII with shRNAs against Rictor reversed the shRictor-mediated inhibition of high glucose-induced protein synthesis and hypertrophy (Fig. 6, B and C; compare bar 8 with bar 4). Furthermore, CA PKCβII alone or along with shRNAs against Rictor significantly induced the protein synthesis and hypertrophy of mesangial cells (Fig. 6, B and C; compare bars 5 and 7 with bar 1). Together our results indicate that not only mTORC2 but PKCβII downstream of mTORC2 contributes to the high glucose-induced mesangial cell hypertrophy.

Phosphorylation of PKCβII Ser-660 in kidneys of OVE26 type 1 diabetic mice.

Activation of PKCβII in diabetic rat kidney has been reported (28). Our results above show that hydrophobic motif site phosphorylation of PKCβII is necessary for mesangial cell hypertrophy (Fig. 1, J and K). To examine the relevance of our observations in vivo, we used OVE26 mouse model, which displays type 1 diabetes within 3 days after birth (15). The transgenic OVE26 mouse exhibits pathologic features of diabetic nephropathy, including renal hypertrophy, augmented mesangial volume, and matrix expansion (84). We examined phosphorylation of PKCβII at Ser-660 in the renal cortexes of diabetic OVE26 mice. The results show a significant increase in phosphorylation of PKCβII at Ser-660 (Fig. 7, A and B). These results indicate that activation of mTORC2 may phosphorylate PKCβII. We directly examined the activation of mTORC2 in the renal cortical lysates of the diabetic mice. Rictor antibody was used to immunoprecipitate mTORC2 followed by an immunecomplex kinase assay using recombinant PKCβII protein as substrate in vitro in the presence of ATP. The reaction product was analyzed by immunoblotting with PKCβII phsopho-Ser-660 antibody. The results show significantly increased phosphorylation of PKCβII at Ser-660 in the diabetic mice compared with the mTORC2 immunoprecipitates from control mice (Fig. 7, C and D). These results indicate that mTORC2 is activated in the diabetic kidney.

We have shown above that PKCβII promoted phosphorylation of Akt in mesangial cells (Fig. 3, F–H); we tested this phenomenon. Increase in PKCβII phosphorylation was associated with enhanced phosphorylation of Akt at both hydrophobic motif and catalytic loop sites in diabetic renal cortexes (Fig. 7, E and F). Since activation of Akt results in phosphorylation of its substrate PRAS40, we determined its phosphorylation in the diabetic renal cortex. As evident in Fig. 7, G and H, PRAS40 phosphorylation was significantly increased in the diabetic animals. PRAS40 phosphorylation and hence inactivation results in activation of mTORC1, which was associated with mesangial cell hypertrophy (14). Therefore, we determined its activation by measuring phosphorylation of two of its substrates S6 kinase and 4EBP-1 in vivo. Phosphorylation of both S6 kinase and 4EBP-1 was significantly enhanced in the renal cortex of diabetic animals compared with those in controls (Fig. 7, I–L). These results indicate a possible role of PKCβII hydrophobic motif site phosphorylation in pathology of diabetic nephropathy.