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 wit...
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| Publicado: |
2001
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13500872_v147_n5_p1149_Portela http://hdl.handle.net/20.500.12110/paper_13500872_v147_n5_p1149_Portela |
| Aporte de: |
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paper:paper_13500872_v147_n5_p1149_Portela |
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dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
BCY1 CAMP Holoenzyme In situ activity cyclic AMP cyclic AMP binding protein cyclic AMP dependent protein kinase gene product holoenzyme kemptide protein bcy1 protein subunit regulator protein unclassified drug animal cell article binding affinity biochemistry carboxy terminal sequence catalysis cell membrane permeability controlled study enzyme activation enzyme active site enzyme activity gene deletion gene mutation gene overexpression genetic transformation hypothesis in vivo study inhibition kinetics mutant nonhuman phenotype polyacrylamide gel electrophoresis priority journal protein domain Saccharomyces cerevisiae Western blotting Animalia Saccharomyces cerevisiae |
| spellingShingle |
BCY1 CAMP Holoenzyme In situ activity cyclic AMP cyclic AMP binding protein cyclic AMP dependent protein kinase gene product holoenzyme kemptide protein bcy1 protein subunit regulator protein unclassified drug animal cell article binding affinity biochemistry carboxy terminal sequence catalysis cell membrane permeability controlled study enzyme activation enzyme active site enzyme activity gene deletion gene mutation gene overexpression genetic transformation hypothesis in vivo study inhibition kinetics mutant nonhuman phenotype polyacrylamide gel electrophoresis priority journal protein domain Saccharomyces cerevisiae Western blotting Animalia Saccharomyces cerevisiae Portela, Paula Zaremberg, Vanina Moreno de Colonna, Silvia 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 |
| topic_facet |
BCY1 CAMP Holoenzyme In situ activity cyclic AMP cyclic AMP binding protein cyclic AMP dependent protein kinase gene product holoenzyme kemptide protein bcy1 protein subunit regulator protein unclassified drug animal cell article binding affinity biochemistry carboxy terminal sequence catalysis cell membrane permeability controlled study enzyme activation enzyme active site enzyme activity gene deletion gene mutation gene overexpression genetic transformation hypothesis in vivo study inhibition kinetics mutant nonhuman phenotype polyacrylamide gel electrophoresis priority journal protein domain Saccharomyces cerevisiae Western blotting Animalia Saccharomyces cerevisiae |
| description |
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. |
| author |
Portela, Paula Zaremberg, Vanina Moreno de Colonna, Silvia |
| author_facet |
Portela, Paula Zaremberg, Vanina Moreno de Colonna, Silvia |
| author_sort |
Portela, Paula |
| title |
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 |
| title_short |
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 |
| title_full |
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 |
| title_fullStr |
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 |
| title_full_unstemmed |
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 |
| title_sort |
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 |
| publishDate |
2001 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13500872_v147_n5_p1149_Portela http://hdl.handle.net/20.500.12110/paper_13500872_v147_n5_p1149_Portela |
| work_keys_str_mv |
AT portelapaula evaluationofinvivoactivationofproteinkinaseaundernondissociableconditionsthroughtheoverexpressionofwildtypeandmutantregulatorysubunitsinsaccharomycescerevisiae AT zarembergvanina evaluationofinvivoactivationofproteinkinaseaundernondissociableconditionsthroughtheoverexpressionofwildtypeandmutantregulatorysubunitsinsaccharomycescerevisiae AT morenodecolonnasilvia evaluationofinvivoactivationofproteinkinaseaundernondissociableconditionsthroughtheoverexpressionofwildtypeandmutantregulatorysubunitsinsaccharomycescerevisiae |
| _version_ |
1768546081810939904 |
| spelling |
paper:paper_13500872_v147_n5_p1149_Portela2023-06-08T16:10:45Z 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 Portela, Paula Zaremberg, Vanina Moreno de Colonna, Silvia BCY1 CAMP Holoenzyme In situ activity cyclic AMP cyclic AMP binding protein cyclic AMP dependent protein kinase gene product holoenzyme kemptide protein bcy1 protein subunit regulator protein unclassified drug animal cell article binding affinity biochemistry carboxy terminal sequence catalysis cell membrane permeability controlled study enzyme activation enzyme active site enzyme activity gene deletion gene mutation gene overexpression genetic transformation hypothesis in vivo study inhibition kinetics mutant nonhuman phenotype polyacrylamide gel electrophoresis priority journal protein domain Saccharomyces cerevisiae Western blotting Animalia 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. Fil:Portela, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Zaremberg, V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Moreno, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2001 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_13500872_v147_n5_p1149_Portela http://hdl.handle.net/20.500.12110/paper_13500872_v147_n5_p1149_Portela |