The CK2 phosphorylation of catalytic domain of Cdc34 modulates its activity at the G1 to S transition in Saccharomyces cerevisiae.
The ubiquitin-conjugating enzyme Cdc34 was recently shown to be phosphorylated by CK2 on the C-terminal tail. Here we present novel findings indicating that in budding yeast CK2 phosphorylates Cdc34 within the N-terminal catalytic domain. Specifically, we show, by direct mass spectrometry analysis, that Cdc34 is phosphorylated in vitro and in vivo by CK2 on Ser130 and Ser167, and that the phosphoserines 130 and 167 are not present after CK2 inactivation in a cka1Deltacka2-8(ts) strain. CK2 phosphorylation of Ser130 and Ser167 strongly stimulates Cdc34 ubiquitin charging in vitro. The Cdc34(S130AS167A) mutant shows a basal ubiquitin charging activity which is indistinguishable from that of wild type but is not activated by CK2 phosphorylation and its expression fails to complement a cdc34-2(ts) yeast strain, supporting a model in which activation of Cdc34 involves CK2-mediated phosphorylation of its catalytic domain.
Coccetti
Paola
P
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy. paola.coccetti@unimib.it
Tripodi
Farida
F
Tedeschi
Gabriella
G
Nonnis
Simona
S
Marin
Oriano
O
Fantinato
Sonia
S
Cirulli
Claudia
C
Vanoni
Marco
M
Alberghina
Lilia
L
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D005434:Flow Cytometry; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016196:S Phase / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; C577449:CDC34 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome