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Phosphorylation of v-mos Ser 47 by the mitotic form of p34cdc2
W L Bai1, B Singh, W L Karshin
1Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Abstract:
P85gag-mos is hyperphosphorylated during mitosis in normal rat kidney (NRK) cells transformed by Moloney murine sarcoma virus ts110. We now report that P85gag-mos is phosphorylated in vitro by the mitotic form of the cdc2 kinase (p34cdc2, known as M-phase kinase) derived from virus-transformed cells. The major site of P85gag-mos phosphorylation by the M-phase kinase in vitro lies within the amino-terminal portion of the viral mos protein sequence spanning residues 45-53, as determined by tryptic peptide mapping. A synthetic peptide corresponding to amino acids 37-55 of v-mos was specifically phosphorylated by the M-phase kinase, whereas v-mos peptides either lacking Ser 47 or substituted with Ala at residue 47 were not phosphorylated. Protein sequencing analyses established that the M-phase kinase specifically phosphorylates Ser 47. Tryptic phosphopeptide mapping of the in vivo-phosphorylated gag-mos protein from mitotic cells indicated that the 45-53 v-mos region was also phosphorylated within mitotic cells. These findings demonstrate that the M-phase kinase phosphorylates the viral mos protein at Ser 47. These results were unexpected in view of earlier reports regarding cdc2 kinase activation/stabilization by the c-mos kinase in maturing oocytes.
Insights
Mitotic cdc2 kinase (M-phase kinase) phosphorylates the viral mos protein at Serine 47. This phosphorylation occurs in Moloney murine sarcoma virus-transformed cells during mitosis, identifying a key regulatory event.
Area of Science:
- Cell Biology
- Molecular Biology
- Virology
Background:
- P85gag-mos protein undergoes hyperphosphorylation during mitosis in virus-transformed cells.
- The role of specific kinases in mos protein regulation during cell division is under investigation.
Purpose of the Study:
- To investigate the specific kinase responsible for P85gag-mos hyperphosphorylation during mitosis.
- To identify the precise phosphorylation site on the viral mos protein.
Main Methods:
- In vitro kinase assays using purified M-phase kinase (p34cdc2) from transformed cells.
- Tryptic peptide mapping to identify phosphorylation sites.
- Site-directed mutagenesis and synthetic peptide analysis to confirm phosphorylation site specificity.
- Protein sequencing to determine the exact residue phosphorylated.
Main Results:
- The mitotic form of cdc2 kinase (M-phase kinase) was identified as the enzyme phosphorylating P85gag-mos in vitro.
- The major phosphorylation site was mapped to the amino-terminal region (residues 45-53) of the viral mos protein.
- Serine 47 (Ser 47) was confirmed as the specific phosphorylation site.
- In vivo phosphorylation patterns in mitotic cells corroborated the in vitro findings.
Conclusions:
- M-phase kinase directly phosphorylates the viral mos protein at Ser 47 during mitosis.
- This phosphorylation event is crucial for understanding mos protein regulation in the context of viral transformation and cell cycle progression.
- The findings offer new insights into kinase-substrate interactions, potentially diverging from known roles in oocyte maturation.