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Phosphorylation of Rap1GAP during the cell cycle
I Janoueix-Lerosey1, M Fontenay, G Tobelem
1INSERM U-248, Faculté de Médecine Lariboisière-Saint-Louis, Paris, France.
Biochemical and Biophysical Research Communications
|July 29, 1994
Summary
Rap1GAP phosphorylation in Hela cells is regulated by p34cdc2 kinase during mitosis. This mitotic phosphorylation of Rap1 GTPase Activating Protein (Rap1GAP) by cdc2 kinase does not alter Rap1 activity but may affect protein interactions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rap1GAP (Rap1 GTPase Activating Protein) is a key regulator of the small GTP binding protein Rap1.
- Previous in vitro studies indicated Rap1GAP can be phosphorylated by p34cdc2 kinase, an enzyme crucial for cell cycle regulation.
Purpose of the Study:
- To investigate the in vivo phosphorylation of Rap1GAP during the cell cycle.
- To determine if p34cdc2 kinase phosphorylates Rap1GAP during mitosis in Hela cells.
Main Methods:
- Analysis of Rap1GAP electrophoretic mobility in interphasic and mitotic Hela cells.
- Co-immunoprecipitation of p34cdc2 with cyclin B1 from Hela cell lysates.
- In vitro kinase assays using purified p34cdc2 and wild-type or mutant Rap1GAP.
- Depletion of p34cdc2 from mitotic extracts to assess its role in Rap1GAP phosphorylation.
Main Results:
- Rap1GAP is phosphorylated in vivo in both interphasic and mitotic Hela cells, with reduced mobility in mitotic cells indicating hyperphosphorylation.
- Mitotic p34cdc2/cyclin B1 complexes efficiently phosphorylate wild-type Rap1GAP at serine 484, but not a mutant version.
- Depletion of p34cdc2 abolished Rap1GAP phosphorylation in mitotic cell lysates.
Conclusions:
- Rap1GAP is a direct in vivo substrate of p34cdc2 kinase during mitosis.
- Mitotic phosphorylation of Rap1GAP by cdc2 kinase does not affect its GTPase activating function for Rap1.
- This phosphorylation may regulate Rap1GAP interactions with other proteins involved in Rap1-mediated cellular functions.