THE PHORBOL ESTER 12-O-TETRADECANOYL-PHORBOL-13-ACETATE STIMULATES THE DEPHOSPHORYLATION OF MITOCHONDRIAL FERREDOXIN IN CULTURED CHICK KIDNEY-CELLS

The tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetat e (TPA), presumably through activation of protein kinase C, decreases the production of 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and incr eases that of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] by primary cul tures of chick kidney cells. We have previously shown that the regulat ion of the cellular output of 1,25(OH)2D3 and 24,25(OH)2D3 by PTH and 1,25(OH)2D3 can be quantitatively accounted for by altered hydroxylase activities within isolated mitochondria. In the present paper, we exa mined the effects of TPA and 1-oleoyl-2-acetyl-glycerol (OAG) on the s tate of mitochondrial protein phosphorylation and on 25-hydroxyvitamin D3 [25(OH)D3] metabolism. There was a good correlation between 25(OH) Ds- 1alpha- and 24-hydroxylase activities in mitochondria isolated fro m cells pretreated with either TPA or OAG and the pattern of 1- and 24 -hydroxylation of 25(OH)D3. The most notable change in protein phospho rylation in the molecular mass range of 10-60 kilodaltons (kDa) was a dramatic decrease in the phosphorylation of a 12.5-kDa mitochondrial m atrix protein after treatment of kidney cells with TPA or OAG. The ami no acid composition of the 12.5-kDa protein was similar to bovine and human ferredoxins and it comigrated with bovine and human ferredoxins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 12.5 -kDa phosphoprotein was immunoprecipitated specifically by an antipept ide polyclonal antibody for chick ferredoxin. The dephosphorylation of ferredoxin in response to TPA was both rapid and transient, with the phosphate content of the 12.5-kDa protein reduced by 70% after a 5-min exposure and returning to control levels by 20 min. A similar transie nce was observed with regard to the rapid effects of TPA on 1alpha-hyd roxylase activity, again showing maximal inhibition at 5 min. The resu lts of our studies are consistent with the idea that ferredoxin phosph orylation plays a role in the regulation of steroid hydroxylation.