NanoLC-MS/MS analysis provides new insights into the phosphorylation pattern of Cdc25B in vivo: full overlap with sites of phosphorylation by Chk1 and Cdk1/cycB kinases in vitro.
NanoLC-MS/MS analysis was used to characterize the phosphorylation pattern in vivo of CDC25B3 (phosphatase splice variant 1) expressed in a human cell line and to compare it to the phosphorylation of CDC25B3 by Cdk1/cyclin B and Chk1 in vitro. Cellular CDC25B3 was purified from U2OS cells conditionally overexpressing the phosphatase. Eighteen sites were detectably phosphorylated in vivo. Nearly all existing (S/T)P sites were phosphorylated in vivo and in vitro. Eight non(S/T)P sites were phosphorylated in vivo. All these sites could be phosphorylated by kinase Chk1, which phosphorylated a total of 11 sites in vitro, with consensus sequence (R/K) X(2-3) (S/P)-non P. Nearly half of the sites identified in this study were not previously described and were not homologous to sites reported to be phosphorylated in other CDC25 species. We also show that in vivo a significant part of CDC25B molecules can be hyperphosphorylated, with up to 13 phosphates per phosphatase molecule.
Bouché
Jean-Pierre
JP
LBCMCP-CNRS-IFR109, Institut d'Exploration Fonctionnelle des Génomes, University of Toulouse, France. bouche@cict.fr
Froment
Carine
C
Dozier
Christine
C
Esmenjaud-Mailhat
Charlotte
C
Lemaire
Matthieu
M
Monsarrat
Bernard
B
Burlet-Schiltz
Odile
O
Ducommun
Bernard
B
D000595:Amino Acid Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D036103:Nanotechnology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D020687:cdc25 Phosphatases / Q000378:metabolism*
D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; C517008:CDC25B protein, human; D020687:cdc25 Phosphatases