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

Microbiology
Paula PortelaS Moreno

Abstract

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. Howev...Continue Reading

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Citations

Aug 2, 2001·Yeast
Dec 22, 2015·Journal of Structural Biology·Nicolás González BardeciSilvia Moreno
Nov 22, 2001·Experimental Cell Research·M R SorolS Moreno
Jun 14, 2002·The Journal of Biological Chemistry·Paula PortelaSilvia Rossi

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