Polymeric structure of the cyclic AMP-dependent protein kinase from the dimorphic fungus Mucor rouxii and purification of its catalytic subunit

Abstract

The polymeric structure of the cyclic AMP-dependent protein kinase (E.C.2.7.1.37) from the dimorphic fungus Mucor rouxii was analyzed through studies of gel filtration and sucrose gradient centrifugation of the holoenzyme and its subunits and by photoaffinity labeling of the regulatory subunit. It was demonstrated that it is a tetramer composed by two regulatory subunits (R) of mol. wt. 75 000 and two catalytic subunits (C) of mol. wt. 41 000 forming a holoenzyme R2C2 of mol. wt. 242 000. Frictional coefficients of 1.55 and 1.62 for the holoenzyme and for the regulatory dimer, respectively, indicate a significant degree of dimensional asymmetry in both molecules. A procedure for the purification of the catalytic subunit of the kinase is presented. The holoenzyme could be bound to a cyclic AMP-agarose column and the catalytic subunit could be eluted by 0.5 M NaCl, well resolved from the bulk of protein. This particular behaviour of the holoenzyme in cyclic AMP-agarose chromatography allowed the inclusion of this step in the purification of the catalytic subunit and corroborated that the holoenzyme was not dissociated by cyclic AMP alone. The isolated catalytic subunit displays Michaelis-Menten behavior towards kemptide, protamine and histone and is inhibited by sulfhydryl reagents, indicating that the molecule has at least one cysteine residue essential for enzyme activity. The catalytic activity of the isolated C subunit is inactivated by the mammalian protein kinase inhibitor, and is inhibited by the regulatory subunit from homologous and heterologous sources. In general, the properties of the catalytic subunit suggest a structural similarity between Mucor and mammalian C subunits.

Heat shock effects on second messenger systems of Neurospora crassa

“…In order to test whether the observed transient increases in the cAMP content were also reflected in the activation of the cAMP-dependent PKA, we analyzed the nuclear translocation of the catalytic subunit of the PKA (Pastori et al 1985) in isolated nuclei. The nuclear fraction was first inspected by fluorescence microscopy after staining with 4-diamidino-2-phenylindole-dihydrochloride (DAPI) and showed stained nuclei of the same size as observed in fixed hyphae.…”

Gene expression inMucordimorphism

“…Measured activity of AC remains static during the phase transition, whereas activity of PDE displays an inverse relationship to internal cAMP concentration (12). The PDE is reversibly activated by a CAMP-dependent protein kinase (PK) and deactivated by a phosphoprotein phosphatase (PP) (22). Two PP activities and two heat-stable inhibitors of these enzymes have been characterized (23).…”

“…Other enzymes found to display changes in activity or in isoenzyme ratios include a-glucosidase, 0-glucosidase, alcohol dehydrogenase, pyruvate kinase, pyruvate decarboxylase, phosphofructokinase, trehalase, peptidases, and RNA polymerases, as well as several enzymes involved in cell wall synthesis and degradation (see reference 2 for citations of original reports). In some cases, the mechanism by which an enzyme activity is controlled during development is well understood, as for example PDE from M. rouxii, which is regulated by PK and two PP activities (22)(23)(24). In most cases, it is unknown how many genes specify a measured activity, what relative contributions to the total measured activity different copies of the gene may make, and what kind of mechanism regulates their expression.…”

Structural and Functional Analysis of the cAMP Binding Domain from the Regulatory Subunit of Mucor rouxii Protein Kinase A

“…cAMP-dependent protein kinases (PKA) 4 are tetrameric holoenzymes composed of a dimer of regulatory (R) subunits interacting and inhibiting with high affinity two catalytic (C) subunits. Cooperative binding of cAMP to the R subunit decreases the affinity between both subunits by several orders, promoting the activation of the catalytic moiety.…”

“…The protein kinase A from the fungus Mucor rouxii is present as a unique isoform and has been characterized and proved to be a tetrameric enzyme like those of higher eukaryotes (3,4), with two cAMP binding sites per R subunit monomer exhibiting site-analog selectivity (5). The M. rouxii R subunit seems particularly large, since the sedimentation coefficient of its dimer is 6.2 S (the holoenzyme has a sedimentation coefficient of 8.8 S) and the MW estimated through SDS-PAGE analysis of the photoaffinity-labeled subunit (4) and from Western blots (6) is in the range of 70 -75 kDa.…”