Threonine phosphorylation is associated with mitosis in HeLa cells.
Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.
Zhao
J Y
JY
Department of Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston.
Kuang
J
J
Adlakha
R C
RC
Rao
P N
PN
D000906:Antibodies; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008938:Mitosis; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D000906:Antibodies; D013912:Threonine; D014443:Tyrosine; D012694:Serine