Selective subcellular redistributions of protein kinase C isoforms by chemical hypoxia

Abstract

The mechanisms of neuronal degeneration following hypoxia/ischemia remain undefined, but the processes include increases in neurotransmitter release, elevation of cytosolic-free calcium concentration, and changes in signal transduction pathways. Activation of the multigene family of protein kinase C (PKC) has been associated with the release of neurotransmitter and the survival of neurons. Therefore, to understand which PKC isozymes are involved in hypoxia/ischemia-induced neuronal degeneration, we examined PKC isozymes after chemical hypoxia (i.e., KCN exposure) in PC12 cells. Cell toxicity, as measured by lactate dehydrogenase (LDH) release, was increased significantly by KCN in glucose-free DMEM and was exaggerated by acute 12-O-tetradecanoyl phorbol-13-acetate (TPA) pretreatment. Under parallel conditions, KCN elevated cytosolic-free calcium ([Ca2+]i) in glucose-free but not in glucose containing DMEM, and TPA pretreatment did not exaggerate KCN's effect on [Ca2+]i. Thus, increases in [Ca2+]i are not sufficient for the synergistic toxic effect of KCN and TPA. In the glucose-free DMEM, selective PKC isozyme inhibitor Go 6976 at 10 nM completely inhibited KCN-induced LDH release and at higher concentrations (1 microM) inhibited the basal levels of LDH release. The protein levels of PKCs in the nuclear, membrane, and cytosolic fractions were measured by Western blot analysis using antibodies against specific isoforms. Two Ca2+-dependent (-alpha, -gamma) and four Ca2+-independent (-delta, -epsilon, -zeta, and -lambda) isozymes were identified and two isozymes (-beta and -theta) were not detected in the subcellular fractions of PC12 cells. Treatment of the cells with TPA significantly activated translocation of conventional PKC-gamma from the cytosol to the membrane and nuclear fractions and other PKC isozymes (-alpha, -delta, and -epsilon) from the cytosol to the membrane, but not atypical PKC-zeta and -lambda. Although only the levels in the nuclear PKC-gamma but not other PKC isozymes were increased significantly following KCN, the levels of cPKC-alpha and -gamma in the membrane mainly- and those and PKC-epsilon in the nucleus-were increased when KCN was combined with TPA. In addition, this condition (TPA + KCN) did not affect the TPA insensitive atypical isozymes, PKC-zeta or -lambda. Taking the results together, differential activation/translocation of PKC isozymes by KCN and TPA is important in the regulation of chemical hypoxia-induced cell injury in PC12 cells.

HMGB1 Is Phosphorylated by Classical Protein Kinase C and Is Secreted by a Calcium-Dependent Mechanism

“…A confocal microscopy study of cPKC in the nucleus after LPS stimuli and an in vitro kinase assay and immunoprecipitation analysis using purified nuclear cPKC from LPS-treated RAW264.7 cells showed that cPKC can migrate into the nucleus and bind to HMGB1 after LPS stimuli to phosphorylate HMGB1 protein with an unknown mechanism. PKC isoenzymes are located in the cytoplasm as an inactive state in resting cells and are redistributed after stimuli (20,21), and the redistribution of PKC␣ and PKC␥ to the nucleus after PMA stimuli or hypoxic stress had been suggested in previous reports (47,48).…”

εPKC Is Required for the Induction of Tolerance by Ischemic and NMDA-Mediated Preconditioning in the Organotypic Hippocampal Slice

“…Furthermore, the extracellular and intracellular calcium chelation was also efficacious in blocking the induction of tolerance by NMDA preconditioning. We conjecture that a critical level of calcium is necessary to stimulate calcium-dependent enzymes, including PKC (Huang et al, 1999;Katsura et al, 1999) and nitric oxide synthase (Gonzalez-Zulueta et al, 2000;Nandagopal et al, 2001), that will lead to a signal transduction pathway that could ultimately play a role in neuroprotection.…”

Ca2+/Calmodulin Kinase-dependent Activation of Hypoxia Inducible Factor 1 Transcriptional Activity in Cells Subjected to Intermittent Hypoxia

“…Preparation of Membrane Fractions-Membrane fractions were prepared as described previously (27). Cells were washed twice with phosphate-buffered saline containing 2 mM EDTA and incubated on ice with extraction buffer (20 mM Tris, 2 mM EDTA, 5 mM EGTA, 10 mM mercaptoethanol, 0.1 mM PMSF, 5 g/ml leupeptin, and 5 g/ml aprotinin) for 10 min.…”