Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit.
The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.
Zelada
Alicia
A
Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, Argentina.
Castilla
Rocijo
R
Passeron
Susana
S
Giasson
Luc
L
Cantore
Marija L
ML
D001665:Binding Sites; D002176:Candida albicans / Q000201:enzymology*; D002384:Catalysis; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation
D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D005656:Fungal Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases