Protein kinase C-α inhibits the repair of oxidative phosphorylation afterS-(1,2-dichlorovinyl)-l-cysteine injury in renal cells

Abstract

Previously, we showed that physiological functions of renal proximal tubular cells (RPTC) do not recover following S-(1,2-dichlorovinyl)-l-cysteine (DCVC)-induced injury. This study investigated the role of protein kinase C-alpha (PKC-alpha) in the lack of repair of mitochondrial function in DCVC-injured RPTC. After DCVC exposure, basal oxygen consumption (Qo(2)), uncoupled Qo(2), oligomycin-sensitive Qo(2), F(1)F(0)-ATPase activity, and ATP production decreased, respectively, to 59, 27, 27, 57, and 68% of controls. None of these functions recovered. Mitochondrial transmembrane potential decreased 53% after DCVC injury but recovered on day 4. PKC-alpha was activated 4.3- and 2.5-fold on days 2 and 4, respectively, of the recovery period. Inhibition of PKC-alpha activation (10 nM Go6976) did not block DCVC-induced decreases in mitochondrial functions but promoted the recovery of uncoupled Qo(2), oligomycin-sensitive Qo(2), F(1)F(0)-ATPase activity, and ATP production. Protein levels of the catalytic beta-subunit of F(1)F(0)-ATPase were not changed by DCVC or during the recovery period. Amino acid sequence analysis revealed that alpha-, beta-, and epsilon-subunits of F(1)F(0)-ATPase have PKC consensus motifs. Recombinant PKC-alpha phosphorylated the beta-subunit and decreased F(1)F(0)-ATPase activity in vitro. Serine but not threonine phosphorylation of the beta-subunit was increased during late recovery following DCVC injury, and inhibition of PKC-alpha activation decreased this phosphorylation. We conclude that during RPTC recovery following DCVC injury, 1). PKC-alpha activation decreases F(0)F(1)-ATPase activity, oxidative phosphorylation, and ATP production; 2). PKC-alpha phosphorylates the beta-subunit of F(1)F(0)-ATPase on serine residue; and 3). PKC-alpha does not mediate depolarization of RPTC mitochondria. This is the first report showing that PKC-alpha phosphorylates the catalytic subunit of F(1)F(0)-ATPase and that PKC-alpha plays an important role in regulating repair of mitochondrial function.

Human ATP synthase beta is phosphorylated at multiple sites and shows abnormal phosphorylation at specific sites in insulin-resistant muscle

“…Here, we demonstrate that human ATPsyn-β is phosphorylated at multiple specific sites in vivo. These results extend our previous work [21] and that of several other studies that have used various phospholabelling techniques or phosphopeptide identification by MS, which have provided indirect evidence for serine/ threonine/tyrosine phosphorylation of ATPsyn-β in yeast, plants and mammalian tissues and cell lines [27][28][29][30][31][32][33][34][35].…”

“…Consumption of soy proteins, which have been linked to a decreased risk of type 2 diabetes and certain types of cancer, caused decreased phosphorylation of ATPsyn-β and increased ATP synthase activity in the liver of rats [31]. In rat renal cells, PRKCAmediated serine phosphorylation of ATPsyn-β was associated with a decreased ATP synthase activity in vitro [30]. Moreover, in plant mitochondria, the binding of recombinant 14-3-3 protein to phosphorylated serine/threonineresidues on ATPsyn-β inhibited ATP synthase activity [36].…”

Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-l-cysteine in primary cultures of human proximal tubular cells

“…Figure 6). As found previously in hPT cells [32][33][34], rPT cells [9,[12][13][14][15][17][18][19][20]26,31], and rabbit proximal tubules [21][22][23][24][25], hPT cells in the current study exhibited decreases in respiration. Modest effects were observed at 10 μM DCVC and none were detected at 1 or 5 μM DCVC.…”

“…Changes in mitochondrial Ca 2+ ion homeostasis also appear to be a key step in DCVC-induced renal toxicity [13,[18][19][20]. The importance of mitochondria in DCVC-induced renal toxicity is further highlighted by findings that preservation of mitochondrial function is associated with recovery and repair of epithelial cells from rabbit proximal tubules after sublethal injury from DCVC [21][22][23][24][25]. Other studies in primary cultures of rPT cells, which enable assessment of longer term exposures at lower concentrations as compared to studies that can be conducted in suspensions of freshly isolated cells, showed that DCVC primarily caused necrosis at relatively high concentrations but that concentrations as low as 10 μM increased expression of vimentin [26].…”

Nephrotoxic effect of subchronic exposure to <i>S</i>-(1,2-dichlorovinyl)-<i>L</i>-cysteine in mice

“…Studies using freshly isolated rat proximal tubular cells have indicated that DCVC is a potent cytotoxin, leading to primary mitochondrial dysfunction (Lash et al, 1986a) and inhibition of mitochondrial Ca 2+ sequestration (Vamvakas et al, 1992). The importance of mitochondria in DCVC-induced nephrotoxicity is further highlighted by the studies of rabbit proximal tubules which have demonstrated that the preservation of mitochondrial function is associated with recovery and repair in damaged epithelial cells (Liu et al, 2004;Nowak et al, 1999;Nowak, 2003;Shaik et al, 2007Shaik et al, , 2008.…”