RESUMO
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.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Quinases/metabolismo , Espectrometria de Massas em Tandem/métodos , Fosfatases cdc25/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Quinase 1 do Ponto de Checagem , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Nanotecnologia , FosforilaçãoRESUMO
CDC25C phosphatase is a key actor in cell cycle progression that controls the activation of CDK1-cyclin B at mitosis. Its activity is known to be highly regulated by a number of signalling pathway-activated kinases resulting in its phosphorylation on multiple residues. In this study, we have purified CDC25C from cells and have used a proteomic approach to identify new regulatory phosphorylations. Here, we report the identification by mass spectrometry of a peptide monophosphorylated on serine 263. We demonstrate by cell imaging that mutation of S263 to alanine leads to a nuclear accumulation of CDC25C that is further reinforced by leptomycin-B. We propose that phosphorylation at S263 is part of the regulatory mechanism that modulates nuclear import of CDC25C, thus preventing cytoplasm to nucleus shuttling.
