Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-epsilon. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-epsilon mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, alpha-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, DeltaPsi(m), ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-epsilon did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-epsilon mediate oxidant-induced mitochondrial dysfunction through independent pathways.

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.