1887

Abstract

-encoded protein kinase A (PKA) wild-type and mutant regulatory (R) subunits from were inducibly overexpressed in their corresponding background strains containing the same mutation in the gene. The aim of this approach was to shift the catalytic activity of PKA within the cell to the undissociated holoenzyme form(s) in order to evaluate whether the wild-type or the mutant forms of the holoenzyme could display catalytic activity. Two mutants of R subunits were used: , with a complete deletion of cAMP-binding domain B; and , with a small deletion in the carboxy terminus of cAMP-binding domain A. Their overexpression caused an increase in the level of R subunits in the range 40–90-fold, as detected by cAMP-binding activity, Coomasie-stained SDS-PAGE and Western blot analysis. The change in PKA activity attained by overexpression of R was assessed in three ways: (i) through the analysis of PKA-dependent phenotypes, and (ii, iii) by measurement of PKA activity −/+ cAMP using the specific substrate kemptide in crude extracts (ii) and permeabilized cells (iii). Upon overexpression of the R subunits, PKA-dependent phenotypes were less severe when compared with their own background. However, a gradient in the degree of severity of phenotypes >> wild-type was observed in the background strains and was maintained in the strains overexpressing the R subunits. cAMP levels measured in background and in R-overexpressing strains showed an increase of around two orders accompanying the overexpression of the R subunits. Three main conclusions could be drawn from the PKA activity measurements −/+ cAMP in crude extracts: (i) catalytic activity was not increased in compensation for the increase in R subunits in any of the three cases (wild-type, or overexpression); (ii) PKA activity assayed in the absence of cAMP was lower in the case of extracts from strains overexpressing wild-type or R subunits when compared with the corresponding extracts without overexpression; and (iii) in these two cases, the great excess of R subunits in the crude extracts displayed additional inhibitory capacity towards exogenously added catalytic (C) subunits. To provide an estimate of the activation of PKA, permeabilized cells from control strains and strains transformed with either wild-type, or R subunits were used to measure PKA activity in the presence of variable concentrations of cAMP. There were two main observations from the results: (i) the activity of PKA detected in the absence of exogenous cAMP was decreased in the strains overexpressing the R subunits when compared to their corresponding backgrounds, and (ii) the sensitivity to activation by cAMP was decreased or almost nil. The biochemical and genetic results obtained are consistent with the hypothesis that within the cell it is possible to have catalytically active, cAMP-bound, undissociated PKA holoenzyme.

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2001-05-01
2019-12-12
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References

  1. Cannon, J. F., Gibbs, J. B. & Tatchell, K. ( 1986; ). Suppressors of the ras2 mutation of Saccharomyces cerevisiae. Genetics 113, 247-264.
    [Google Scholar]
  2. Cannon, J. F., Gitan, R. & Tatchell, K. ( 1990; ). Yeast cAMP-dependent protein kinase regulatory subunit mutations display a variety of phenotypes. J Biol Chem 265, 11897-11904.
    [Google Scholar]
  3. Chester, V. ( 1968; ). Heritable glycogen storage deficiency in yeast and its induction by ultraviolet light. J Gen Microbiol 51, 49-56.[CrossRef]
    [Google Scholar]
  4. Døskeland, S. O., Maronde, E. & Gjersten, B. T. ( 1993; ). The genetic subtypes of cAMP-dependent protein kinase – functionally different or redundant? Biochim Biophys Acta 1178, 249-258.[CrossRef]
    [Google Scholar]
  5. Fantes, P. A. ( 1981; ). Isolation of cell size mutants of a fission yeast by a new selective method: characterization of mutants and implications for division control mechanisms. J Bacteriol 146, 746-754.
    [Google Scholar]
  6. Francis, S. H. & Corbin, J. D. ( 1994; ). Structure and function of cyclic nucleotide dependent protein kinases. Annu Rev Physiol 56, 237-272.[CrossRef]
    [Google Scholar]
  7. Griffioen, G., Anghileri, P., Imre, E., Baroni, M. D. & Ruis, H. ( 2000; ). Nutritional control of nucleocytoplasmic localization of cAMP-dependent protein kinase catalytic and regulatory subunits in Saccharomyces cerevisiae. J Biol Chem 275, 1449-1456.[CrossRef]
    [Google Scholar]
  8. Houge, G., Vintermyr, O. K. & Døskeland, S. O. ( 1990; ). The expression of cAMP-dependent protein kinase subunits in primary rat hepatocyte cultures. Cyclic AMP down-regulates its own effector system by decreasing the amount of catalytic subunit and increasing the mRNAs for the inhibitory (R) subunits of cAMP-dependent protein kinase. Mol Endocrinol 4, 481-488.[CrossRef]
    [Google Scholar]
  9. Huang, L. J. & Taylor, S. S. ( 1998; ). Dissecting cAMP binding domain A in the RIα subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit. J Biol Chem 273, 26739-26746.[CrossRef]
    [Google Scholar]
  10. Ito, H., Fukuda, Y., Murata, K. & Kimura, A. ( 1983; ). Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153, 163-168.
    [Google Scholar]
  11. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. ( 1987; ). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol 154, 367-382.
    [Google Scholar]
  12. Londesborough, J. & Lukkari, T. M. ( 1980; ). The pH and temperature dependence of the activity of the high K m cyclic nucleotide phosphodiesterase of baker’s yeast. J Biol Chem 255, 9262-9267.
    [Google Scholar]
  13. Ma, P., Wera, S., Van Dijck, P. & Thevelein, J. M. ( 1999; ). The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10, 91-104.[CrossRef]
    [Google Scholar]
  14. Maggese, M. C., Galvagno, M. A., Cantore, M. L. & Passeron, S. ( 1982; ). In situ measurement of cAMP related enzymes in the dimorphic fungus Mucor rouxii. Cell Biol Int Rep 6, 1101-1108.[CrossRef]
    [Google Scholar]
  15. Mazón, M. J., Beherens, M. M., Morgado, E. & Portillo, F. ( 1993; ). Low activity of the yeast cAMP-dependent protein kinase catalytic subunit TPK3 is due to the poor expression of the TPK3 gene. Eur J Biochem 213, 501-506.[CrossRef]
    [Google Scholar]
  16. Moreno, S., Pastori, R. & Passeron, S. ( 1983; ). Protein kinase of Mucor rouxii. Unshielding of new cyclic AMP-binding sites upon dissociation of the ternary complex holoenzyme-cyclic AMP. Mol Cell Biochem 52, 13-16.
    [Google Scholar]
  17. Nikawa, J., Cameron, S., Toda, T., Ferguson, K. & Wigler, M. ( 1987; ). Rigorous feed back control of cAMP levels in Saccharomyces cerevisiae. Genes Dev 1, 931-937.[CrossRef]
    [Google Scholar]
  18. Ogur, M., St John, R. & Nagai, S. ( 1957; ). Tetrazolium overlay technique for population studies of respiration deficiency in yeast. Science 125, 982-991.
    [Google Scholar]
  19. Robertson, L. S., Causton, H. C., Young, R. A. & Fink, G. R. ( 2000; ). The yeast A kinases differentially regulate iron uptake and respiratory function. Proc Natl Acad Sci USA 97, 5984-5988.[CrossRef]
    [Google Scholar]
  20. Roskoski, R. ( 1983; ). Assay of protein kinase. Methods Enzymol 99, 3-6.
    [Google Scholar]
  21. Saraswat, L. D., Filutowicz, M. & Taylor, S. S. ( 1986; ). Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli. J Biol Chem 261, 11091-11096.
    [Google Scholar]
  22. Schwechheimer, K. & Hofmann, F. ( 1977; ). Properties of regulatory subunit of cyclic AMP-dependent protein kinase (peak I) from rabbit skeletal muscle prepared by urea treatment of the holoenzyme. J Biol Chem 252, 7690-7696.
    [Google Scholar]
  23. Sherman, F., Fink, G. & Hicks, J. B. (1981). Methods in Yeast Genetics: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Task n, K., Sk lhegg, B. S., Task n, K. A. & 13 other authors ( 1997; ). Structure, function, and regulation of human cAMP-dependent protein kinases. Adv Second Messengers Phosphoprotein Res 31, 191–204.
    [Google Scholar]
  25. Tatchell, K. ( 1986; ). genes and growth control in Saccharomyces cerevisiae. J Bacteriol 166, 364-367.
    [Google Scholar]
  26. Thevelein, J. M. ( 1992; ). The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Leeuwenhoek 62, 109-130.[CrossRef]
    [Google Scholar]
  27. Thevelein, J. M. ( 1994; ). Signal transduction in yeast. Yeast 10, 1753-1790.[CrossRef]
    [Google Scholar]
  28. Thevelein, J. M. & de Winde, J. H. ( 1999; ). Novel sensing mechanisms and targets for the cAMP-protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol 33, 904-918.[CrossRef]
    [Google Scholar]
  29. Thevelein, J. M., Beullens, M., Honshoven, F., Hoebeeck, G., Detremerie, K., Den Hollander, J. A. & Jans, A. W. A. ( 1987; ). Regulation of the cAMP levels in the yeast Saccharomyces cerevisiae: intracellular pH and the effect of membrane depolarizing compounds. J Gen Microbiol 133, 2191-2196.
    [Google Scholar]
  30. Toda, T., Cameron, S., Sass, P., Zoller, M. & Wigler, M. ( 1987; ). Three different genes in Saccharomyces cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell 50, 277-287.[CrossRef]
    [Google Scholar]
  31. Zaremberg, V. & Moreno, S. ( 1996; ). Analysis of the mechanism of activation of cAMP-dependent protein kinase through the study of mutants of yeast regulatory subunit. Eur J Biochem 237, 136-142.[CrossRef]
    [Google Scholar]
  32. Zhao, J., Hoye, E., Boylan, S., Walsh, D. A. & Trewhella, J. ( 1998; ). Quaternary structures of a catalytic subunit-regulatory subunit dimeric complex and the holoenzyme of the cAMP-dependent protein kinase by neutron contrast variation. J Biol Chem 273, 30448-30459.[CrossRef]
    [Google Scholar]
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