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. 1979 Oct;76(10):5187–5191. doi: 10.1073/pnas.76.10.5187

Adrenocorticotropin (ACTH) induces phosphorylation of a cytoplasmic protein in intact isolated adrenocortical cells.

E J Podesta, A Milani, H Steffen, R Neher
PMCID: PMC413105  PMID: 228281

Abstract

In 32P incorporation experiments with intact adrenocortical cells, adrenocorticotropin (ACTH) or adenosine 3',5'-cyclic monophosphate (cAMP) induced a rapid and transient increase of approximately 300-500% in the phosphorylation of a 32P-containing cytoplasmic protein of about 150,000 daltons (APS150). Half-maximal stimulation of APS150 phosphorylation was observed with about 3 pM ACTH. Receptor-bound cAMP, corticosterone production, and the appearance of phosphorylated APS150 increased in parallel with respect to both time and ACTH concentration. All three responses were dependent on extracellular calcium. Inhibition of protein synthesis with cycloheximide suggested a half-life of APS150 of about 10 min. The time course of 32P incorporation into ACTH-induced APS150 in the absence and presence of nonradioactive phosphate shows that the phosphorylation of APS150 is under simultaneous control of cAMP-dependent protein kinase and of phosphoatase activity. Thus a rapid ACTH-dependent and cAMP-dependent protein phosphorylation in intact adrenocortical cells within steroidogenic ACTH concentrations has now been demonstrated.

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Selected References

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  1. Avruch J., Leone G. R., Martin D. B. Effects of epinephrine and insulin on phosphopeptide metabolism in adipocytes. J Biol Chem. 1976 Mar 10;251(5):1511–1515. [PubMed] [Google Scholar]
  2. Avruch J., Witters L. A., Alexander M. C., Bush M. A. Effects of glucagon and insulin on cytoplasmic protein phosphorylation in hepatocytes. J Biol Chem. 1978 Jul 10;253(13):4754–4761. [PubMed] [Google Scholar]
  3. Beckett G. J., Boyd G. S. Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation. Eur J Biochem. 1977 Jan;72(2):223–233. doi: 10.1111/j.1432-1033.1977.tb11243.x. [DOI] [PubMed] [Google Scholar]
  4. Bhargava G., Schwartz E., Koritz S. B. The stimulation by the adrenocorticotropic hormone of the phosphorylation of an adrenal mitochondrial preparation. Proc Soc Exp Biol Med. 1978 Jun;158(2):183–186. doi: 10.3181/00379727-158-40167. [DOI] [PubMed] [Google Scholar]
  5. Caron M. G., Goldstein S., Savard K., Marsh J. M. Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum. J Biol Chem. 1975 Jul 10;250(13):5137–5143. [PubMed] [Google Scholar]
  6. Cochet C., Job D., Chambaz M. Characterization of an endogenous substrate of protein kinases in the bovine adrenal cortex. FEBS Lett. 1977 Nov 1;83(1):59–62. doi: 10.1016/0014-5793(77)80641-8. [DOI] [PubMed] [Google Scholar]
  7. Dufau M. L., Tsuruhara T., Horner K. A., Podesta E., Catt K. J. Intermediate role of adenosine 3':5'-cyclic monophosphate and protein kinase during gonadotropin-induced steroidogenesis in testicular interstitial cells. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3419–3423. doi: 10.1073/pnas.74.8.3419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. FERGUSON J. J., Jr PROTEIN SYNTHESIS AND ADRENOCORTICOTROPIN RESPONSIVENESS. J Biol Chem. 1963 Aug;238:2754–2759. [PubMed] [Google Scholar]
  9. Garren L. D., Ney R. L., Davis W. W. Studies on the role of protein synthesis in the regulation of corticosterone production by adrenocorticotropic hormone in vivo. Proc Natl Acad Sci U S A. 1965 Jun;53(6):1443–1450. doi: 10.1073/pnas.53.6.1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hofmann K., Kim J. J., Finn F. M. The role of protein kinases in ACTH-stimulated steroidogenesis. Biochem Biophys Res Commun. 1978 Oct 30;84(4):1136–1143. doi: 10.1016/0006-291x(78)91702-3. [DOI] [PubMed] [Google Scholar]
  11. Ichii S. Adenosine 3',5'-monophosphate, adenosine 3',5'-monophosphate-binding protein and protein kinase in rat adrenal glands; effect of adrenocorticotrophin. Endocrinol Jpn. 1972 Jun;19(3):229–235. doi: 10.1507/endocrj1954.19.229. [DOI] [PubMed] [Google Scholar]
  12. Ichii S., Murakami N., Ikeda A. Phosphorylation in vitro of adrenal subcellular proteins by adenosine-3',5'-monophosphate-dependent protein kinase: effect of ACTH in vivo. Acta Endocrinol (Copenh) 1974 Feb;75(2):325–332. doi: 10.1530/acta.0.0750325. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Langan T. A. Protein kinases and protein kinase substrates. Adv Cyclic Nucleotide Res. 1973;3:99–153. [PubMed] [Google Scholar]
  15. Lowry P. J., McMartin C. Measurement of the dynamics of stimulation and inhibition of steroidogenesis in isolated rat adrenal cells by using column perfusion. Biochem J. 1974 Aug;142(2):287–294. doi: 10.1042/bj1420287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Miyamoto E., Kuo J. F., Greengard P. Cyclic nucleotide-dependent protein kinases. 3. Purification and properties of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain. J Biol Chem. 1969 Dec 10;244(23):6395–6402. [PubMed] [Google Scholar]
  17. Naghshineh S., Treadwell C. R., Gallo L. L., Vahouny G. V. Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex. J Lipid Res. 1978 Jul;19(5):561–569. [PubMed] [Google Scholar]
  18. Neher R., Milani A. Steroidogenesis in isolated adrenal cells: excitation by calcium. Mol Cell Endocrinol. 1978 Jan;9(3):243–253. doi: 10.1016/0303-7207(78)90067-9. [DOI] [PubMed] [Google Scholar]
  19. Podesta E. J., Dufau M. L., Catt K. J. Adenosine 3',5'-monophosphate-dependent protein kinase of Leydig cells: in vitro activation and relationship to gonadotropin action upon cyclic AMP and steroidogenesis. FEBS Lett. 1976 Nov;70(1):212–216. doi: 10.1016/0014-5793(76)80760-0. [DOI] [PubMed] [Google Scholar]
  20. Podesta E. J., Dufau M. L., Catt K. J. Characterization of two forms of cyclic 3', 5'-adenosine monophosphate-dependent protein kinase in rat testicular interstitial cells. Mol Cell Endocrinol. 1976 Jun-Jul;5(1-2):109–122. doi: 10.1016/0303-7207(76)90074-5. [DOI] [PubMed] [Google Scholar]
  21. Podesta E. J., Dufau M. L., Solano A. R., Catt K. J. Hormonal activation of protein kinase in isolated Leydig cells. Electrophoretic analysis of cyclic AMP receptors. J Biol Chem. 1978 Dec 25;253(24):8994–9001. [PubMed] [Google Scholar]
  22. Podesta E. J., Milani A., Steffen H., Neher R. Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine e 3':5'-cyclic monophosphate. Biochem J. 1979 May 15;180(2):355–363. doi: 10.1042/bj1800355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rae P. A., Gutmann N. S., Tsao J., Schimmer B. P. Mutations in cyclic AMP-dependent protein kinase and corticotropin (ACTH)-sensitive adenylate cyclase affect adrenal steroidogenesis. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1896–1900. doi: 10.1073/pnas.76.4.1896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Roos B. A. ACTH and cAMP stimulation of adrenal ribosomal protein phosphorylation. Endocrinology. 1973 Dec;93(6):1287–1293. doi: 10.1210/endo-93-6-1287. [DOI] [PubMed] [Google Scholar]
  25. Rubin C. S., Rosen O. M. Protein phosphorylation. Annu Rev Biochem. 1975;44:831–887. doi: 10.1146/annurev.bi.44.070175.004151. [DOI] [PubMed] [Google Scholar]
  26. Saez J. M., Evain D., Gallet D. Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans. J Cyclic Nucleotide Res. 1978 Aug;4(4):311–321. [PubMed] [Google Scholar]
  27. Schulster D., Jenner C. A counter-streaming centrifugation technique for the superfusion of adrenocortical cell suspensions stimulated by ACTH. J Steroid Biochem. 1975 Mar-Apr;6(3-4):389–394. doi: 10.1016/0022-4731(75)90162-4. [DOI] [PubMed] [Google Scholar]
  28. Simpson E. R., McCarthy J. L., Peterson J. A. Evidence that the cycloheximide-sensitive site of adrenocorticotropic hormone action is in the mitochondrion. Changes in pregnenolone formation, cholesterol content, and the electron paramagnetic resonance spectra of cytochrome P-450. J Biol Chem. 1978 May 10;253(9):3135–3139. [PubMed] [Google Scholar]
  29. Swallow R. L., Sayers G. A technic for the preparation of isolated rat adrenal cells. Proc Soc Exp Biol Med. 1969 May;131(1):1–4. doi: 10.3181/00379727-131-33789. [DOI] [PubMed] [Google Scholar]
  30. Walton G. M., Gill G. N., Abrass I. B., Garren L. D. Phosphorylation of ribosome-associated protein by an adenosine 3':5'-cyclic monophosphate-dependent protein kinase: location of the microsomal receptor and protein kinase. Proc Natl Acad Sci U S A. 1971 May;68(5):880–884. doi: 10.1073/pnas.68.5.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Walton G. M., Gill G. N. Adenosine 3',5'-monophosphate and protein kinase dependent phosphorylation of ribosomal protein. Biochemistry. 1973 Jul 3;12(14):2604–2611. doi: 10.1021/bi00738a009. [DOI] [PubMed] [Google Scholar]

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