Drug modification of protein thiophosphorylation in permeabilized chromaffin cells

Treatment of permeabilized chromaffin cells with low concentrations of the ATP analog adenosine-5?-O-(3-thiotriphosphate)[(35)S] results in (35)S incorporation into a small number of cellular proteins. Of these proteins, a 47 kilodalton protein is most heavily thiophosphorylated. Permeabilized cells were treated with various drugs known to influence cell functions, more specifically chromaffin granule function, to determine the kinase responsible for thiophosphorylation of the 47 kilodalton protein and if its thiophosphorylation is associated with a specific cell function. Several drugs which influence the activity of cell kinases were examined for their effect on secretion and thiophosphorylation of the 47 kilodalton protein. There was no qualitative effect of cAMP, cGMP or trifluoperazine on thiophosphorylation of the protein. Both cyclic nucleotides slightly enhanced secretion, while trifluoperazine enhanced only unstimulated catecholamine release. Phorbol 12-myristate 13-acetate had no effect on secretion or (35)S incorporation into cell proteins. Only the free calcium concentration of the medium influenced thiophosphorylation of the 47 kilodalton protein, with increased calcium producing increased thiophosphorylation. Drugs affecting chromaffin vesicle functions were used to assess the relationship between specific functions and thiophosphorylation of the protein. Inhibition of nucleotide translocation with atractyloside or 4,4?diisothiocyanatostilbene-2,2?disulfonic acid or inhibition of the proton translocating ATPase by N-ethylmaleimide inhibited thiophosphorylation of the 47 kilodalton protein, with little effect on secretion. Treatment with rotenone markedly enhanced secretion and thiophosphorylation of the protein. Calcium ionophores had no effect on thiophosphorylation of the protein. Dichloroacetic acid, which inhibits phosphorylation of mitochondrial pyruvate dehydrogenase, had no effect on secretion and a variable effect on thiophosphorylation of the 47 kilodalton protein. The data suggest that thiophosphorylation of the protein may be associated with nucleotide translocation across the vesicle membrane.