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Silvia Moreno

Publications and source records attributed to Silvia Moreno.

24 records · Page 2Linked to original sources
PubMed2003

Activation state of protein kinase A as measured in permeabilised Saccharomyces cerevisiae cells correlates with PKA-controlled phenotypes in vivo.

Protein kinase A (PKA) activity was measured in situ in permeabilised Saccharomyces cerevisiae cells in the absence and the presence of cAMP. Four strains genetically predicted to have differential PKA-dependent phenotypes were used: a wild-type strain and a strain containing a bcy1-14 mutation (with almost constitutively active PKA), and the same strains with overexpression of the wild-type or mutant BCY1 gene, respectively. Cells were grown on galactose or glucose. The measured phenotypic characteristics were: trehalose and glycogen levels and the activity of a reporter gene under control of the NTH1 promoter. The 'endogenous' PKA activity (measured in situ in the absence of cAMP) showed the best correlation with the PKA-dependent phenotypes determined in vivo. We propose that this parameter offers a good estimate for the degree of activation of PKA in vivo.

Cyclic AMP↗
PubMed2002

In vivo and in vitro phosphorylation of two isoforms of yeast pyruvate kinase by protein kinase A.

Saccharomyces cerevisiae pyruvate kinase 1 (Pyk1) was demonstrated to be associated to an immunoprecipitate of yeast protein kinase A holoenzyme (HA-Tpk1.Bcy1) and to be phosphorylated in a cAMP-dependent process. Both glutathione S-transferase (GST)-Pyk1 and GST-Pyk2 were phosphorylated in vitro by the bovine heart protein kinase A (PKA) catalytic subunit and by immobilized yeast HA-Tpk1. The specificity constant for the phosphorylation of GST-Pyk1 and GST-Pyk2 by bovine catalytic subunit was in the range of the value for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide). Both fusion proteins were phosphorylated in vivo, in intact cells overexpressing the protein, or in vitro using crude extracts, as source of protein kinase A, when a wild type strain was used but were not phosphorylated when using a strain with only one TPK gene with an attenuated mutation (tpk1(w1)). The effect of phosphorylation on Pyk activity was assayed in partially purified preparations from three strains, containing different endogenous protein kinase A activity levels. Pyk1 activity was measured at different phosphoenolpyruvate concentrations in the absence or in the presence of the activator fructose 1,6-bisphosphate at 1.5 mm. Preliminary kinetic results derived from the comparison of Pyk1 obtained from extracts with the highest versus those from the lowest protein kinase A activity indicate that the enzyme is more active upon phosphorylation conditions; in the absence of the activator it shows a shift in the titration curve for phosphoenolpyruvate to the left and an increase in the Hill coefficient, whereas in the presence of fructose 1,6-bisphosphate it shows an n(H) value of 1.4, as compared with an n(H) of 2 for the Pyk1 obtained from extracts with almost null protein kinase A activity.

Animals↗
PubMed2002

The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter.

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

3T3 Cells↗
PubMed2001

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.

Blotting, Western↗
PubMed2000

Threshold level of protein kinase A activity and polarized growth in Mucor rouxii.

A model system to study the involvement of cAMP-mediated regulation of a cellular process such as hyphal morphogenesis was investigated. Impairment of polarized growth was observed when Mucor rouxii sporangiospores were grown in the presence of N(6)-cAMP analogues and of SQ 65,442, a cAMP phosphodiesterase inhibitor. Together with an effect on isodiametric growth, there was increased pigmentation, increased cell fragility and loss of cell adhesiveness. The total effect on morphology was attained even after adding the compounds shortly before germ-tube emergence; when added after this time growth continued in a non-polarized form and rounding of the germ tip was observed. The morphological effect was observed under all the nutritional and environmental conditions studied (aerobic conditions and defined medium with maltose or glucose, Casamino acids medium with glucose, or rich medium; anaerobic conditions with rich medium; and following a shift from anaerobiosis to aerobiosis). The time of germ-tube emergence, and the size of the cell at this time, was dependent on the growth medium. Protein kinase A (PKA) specific activity was followed during the germination process under three growth conditions. It was found that the total activity of PKA correlated with differentiation and not with growth, and that the total specific activity at the time of germination was the same, independent of the culture medium. The time of germ-tube emergence correlated with the time of attainment of a threshold level of PKA total specific activity. The concentration of dibutyryl-cAMP needed to promote isodiametric growth correlated with the total units of activity of PKA to be activated per cell. It was concluded that PKA is involved in the morphogenetic process of the fungus grown under all the nutritional and ambient conditions tested.

Cell Division↗