Cyclic Amp-Dependent Protein Kinase in Rat Adrenal Mitochondrial Fraction

Abstract

Although it has been proposed that cyclic AMP is an intracellular mediator in the stimulatory effect of ACTH on steroidogenesis, and that the action of cyclic AMP is exerted through the activation of protein kinase, it still remains to be resolved whether cyclic AMP-dependent protein kinases present in adrenocortical cells bear any direct or indirect relationship to steroidogenesis. My previous observation that cyclic AMP acts directly on rat adrenal mitochondrial fraction to stimulate its steroidogenesis suggests that cyclic AMP-dependent protein kinase might be present in mitochondria per se. The present studies were undertaken to examine whether cyclic AMP-dependent protein kinase activity and the existence of its substrate could be demonstrated in rat adrenal mitochondrial fraction. Adrenal subcellular fractions were obtained from male Sprague-Dawley rats pretreated with dexamethasone. Protein kinase activity was determined in a 0.2 ml reaction mixture containing 4.1 mM phosphate buffer (pH 6.5), 10 mM magnesium acetate, 0.3 mM EGTA, 10 mM sodium fluoride, histone (0.2 mg/tube), ATP-γ-32P (2.5 μM, 0.2~3 μCi/tube) and subcellular fraction, with or without cyclic AMP. Incubation was carried out at 30°C for 5 min., and trichloroacetic acid-precipitable radioactive phosphate was assayed. Cyclic AMP-dependent protein kinase activity was detected in the mitochondrial fraction as well as in soluble and microsomal fractions. The possibility of contamination of the mitochondrial fraction by other fractions was checked by comparing enzyme activities of protein kinase and various marker enzymes, 21α-hydroxylase, glucose-6-phosphate dehydrogenase, β-glucuronidase and 11β-hydroxylase, in each fraction. Contamination of microsomal and soluble fractions was found negligible, although the lysosomal enzyme activity was to some extent detectable in the mitochondrial fraction. In order to examine the ability of mitochondrial proteins to serve as substrates for mitochondrial protein kinase, mitochondrial fraction was incubated with ATP-γ-32P and Mg++ without histone. 32P-phosphate incorporation into the mitochondrial protein was clearly demonstrated. When phosphorylated mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, several radioactive peaks with a predominant peak were observed. Cyclic AMP stimulated incorporation of phosphate into several protein bands on the gel without significant effect on the major peak. These findings suggest that cyclic AMP-dependent protein kinase is present in adrenal mitochondria, and that a small but significant amount of mitochondrial protein undergoes phosphorylation by endogenous protein kinase in response to cyclic AMP. Therefore, cyclic AMP, by acting directly on adrenal mitochondria, might regulate its metabolic processes including steroidogenesis, and thus might act as the intracellular messenger of ACTH action.