Physiological Ca 2+ level and Ca 2+-induced Permeability Transition Pore control protein phosphorylation in rat brain mitochondria

Abstract

Phosphorylation of several low molecular mass proteins (3.5, 17, 23 and 29 kDa) was observed in rat brain mitochondria (RBM) at ATP concentration close to that in the mitochondrial matrix. Furthermore, regulatory effects of Ca 2+ on phosphorylation of these proteins were investigated. Protein phosphorylation was found to be modulated by Ca 2+ in the physiological concentration range (10 −8 to 10 −6 M free Ca 2+). Incorporation of 32 P from [ γ- 32 P ]ATP into the 17 kDa protein was dramatically increased within the 10 −7 to 10 −6 M free Ca 2+ range, whereas an opposite effect was observed for the 3.5 kDa polypeptide. Strong de-phosphorylation of the 3.5 kDa polypeptide and enhanced 32 P -incorporation into the 17 and 23 kDa proteins were found with supra-threshold Ca 2+ loads and these effects were eliminated or reduced in the presence of cyclosporin A, an inhibitor of Permeability Transition Pore (PTP) opening. In the presence of calmidazolium (Cmz), a calmodulin antagonist, enhanced levels of phosphorylation of the 17 and 3.5 kDa polypeptides were observed and the 17 kDa protein phosphorylation was suppressed by H-8, a protein kinase A inhibitor. It is concluded that Ca 2+ in physiological concentrations, as a second messenger, can control phosphorylation of the low molecular mass phospoproteins in RBM, in addition to well known regulation of some Krebs cycle dehydrogenases by Ca 2+. The protein phosphorylation was strongly dependent on the Ca 2+-induced PTP opening.