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.
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