INVESTIGADORES
MORENO Silvia Margarita
artículos
Título:
Evaluation of in vivo activation of protein
Autor/es:
PORTELA, P.; ZAREMBERG, V.; MORENO, S.
Revista:
MICROBIOLOGY
Editorial:
SGM (UK)
Referencias:
Año: 2001 vol. 147 p. 1149 - 1159
ISSN:
0026-2617
Resumen:
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
4090-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 N/M
cAMP using the specific substrate kemptide in crude extracts (ii) and
permeabilized cells (iii). Upon overexpression of the R subunits, PKAdependent
phenotypes were less severe when compared with their own
background. However, a gradient in the degree of severity of phenotypes bcy1-
14 Sbcy1-16 S 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 N/M 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.