The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-xL. Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-xL the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-xL as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Δψm) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Δψm and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-xL may represent a potential of the modulation of mitochondrial function during apoptosis.
- cytochrome c
- membrane potential
Abbreviations used: ANT, adenine nucleotide translocase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; JNK, c-Jun N-terminal kinase; arJNK1, active dual-phosphorylated recombinant JNK1; rJNK1, inactive non-phosphorylated recombinant JNK1; aipJNK1, active immunoprecipitated JNK; ipJNK, inactive immunoprecipitated JNK; CsA, cyclosporin A; ERK, extracellular signal-regulated kinase; imPT, mitochondrial inner-membrane permeability transition; Δψm, mitochondrial inner-membrane potential; omPT, mitochondrial outer-membrane permeability transition; Smac, second mitochondria-derived activator of caspases; TMRM, tetramethylrhodamine methyl ester; VDAC, voltage-dependent anion channel; 2D IEF, two-dimensional isoelectric focusing; GST, glutathione S-transferase; ATF-2, activating transcription factor 2; DTT, dithiothreitol; MIB, mitochondrial isolation buffer; MKB, mitochondria kinase buffer; RCR, respiratory control ratio; PCNA, proliferating cell nuclear antigen; pAb, polyclonal antibody; Hsp, heat-shock protein.
- The Biochemical Society, London ©2003