Evaluation of in vivo activation of protein kinase A under non-dissociable conditions through the overexpression of wild-type and mutant regulatory subunits in Saccharomyces cerevisiae.
BCY1-encoded protein kinase A (PKA) wild-type and mutant regulatory (R) subunits from Saccharomyces cerevisiae were inducibly overexpressed in their corresponding background strains containing the same mutation in the bcy1 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: bcy1-16, with a complete deletion of cAMP-binding domain B; and bcy1-14, 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 bcy1-14>bcy1-16> 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, bcy1-16 or bcy1-14 overexpression); (ii) PKA activity assayed in the absence of cAMP was lower in the case of extracts from strains overexpressing wild-type or bcy1-16 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 in vivo activation of PKA, permeabilized cells from control strains and strains transformed with either wild-type, bcy1-16 or bcy1-14 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.
Portela
Paula
P
Departamento de Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina1.
Zaremberg
Vanina
V
Departamento de Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina1.
Moreno
Silvia
S
Departamento de Quı́mica Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, Piso 4, 1428 Buenos Aires, Argentina1.
D015153:Blotting, Western; D020134:Catalytic Domain; D002463:Cell Membrane Permeability; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D013379:Substrate Specificity; D015854:Up-Regulation
D020035:Holoenzymes; D009842:Oligopeptides; C037642:kemptide; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases