cDNA cloning, biochemical and phylogenetic characterization of beta- and beta'-subunits of Candida albicans protein kinase CK2.
We have previously reported that Candida albicans protein kinase CK2 is composed of two distinct catalytic (alpha- and alpha'-) and two distinct regulatory (beta- and beta'-) subunits. We report here the isolation of two cDNAs clones, CaCKB1 and CaCKB2, encoding C. albicans beta- and beta'-subunits, respectively. The predicted beta- and beta'-proteins have calculated molecular masses of 34 kDa and 31 kDa and show all major features conserved in beta-subunits of other organisms, including the N-terminal autophosphorylation site, the internal acidic region and a potential metal-binding motif. The deduced amino acid sequence of C. albicans beta-subunit displays 48% identity with that of Saccharomyces cerevisiae and has an unusually long C-terminal acidic region containing a putative autophosphorylation site. C. albicans beta' shows 54% sequence identity with its S. cerevisiae homologue. Semi-quantitative RT-PCR analyses indicate that the mRNAs corresponding to both subunits are present in similar amounts in the yeast and hyphal forms of the fungus. To evaluate the biochemical properties of C. albicans beta- and beta'-subunits, both proteins were expressed in Escherichia coli and purified. Experiments performed in vitro indicate that both recombinant subunits reconstitute a fully functional holoenzyme when incubated with stoichiometric amounts of human recombinant alpha-subunit, as judged by their ability to abolish basal phosphorylation of calmodulin by human recombinant alpha-subunit and the reversion of the inhibitory effect by polylysine. In addition, both regulatory subunits can be phosphorylated by human recombinant alpha subunit. Phylogenetic analysis of beta- and beta'-proteins of C. albicans and other organisms shows that the CKB gene duplication occurred before the split of the ascomycete and basidiomycete lineages.
Zelada
Alicia
A
Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, Avda. San Martín 4453, 1417 Buenos Aires, Argentina. zelada@agro.uba.ar
De Souza
Flávio S J
FS
Walz
Katherina
K
Giasson
Luc
L
Passeron
Susana
S
D000595:Amino Acid Sequence; D001665:Binding Sites; D002176:Candida albicans / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D047390:Casein Kinase II; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D019281:Dimerization; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D017931:DNA Primers; D021122:Protein Subunits; D011994:Recombinant Proteins; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases