Phosphorylation of Rap1GAP during the cell cycle.
Rap1GAP (for Rap1 GTPase Activating Protein) is an 89 kD protein that highly stimulates the intrinsic GTPase activity of the small GTP binding protein Rap1. It has been shown that Rap1GAP is phosphorylated in vitro by purified p34cdc2 kinase, which regulates the G2/M transition of the cell cycle. In this work, we have studied the phosphorylation of Rap1GAP during the cell cycle and showed that Rap1GAP is phosphorylated in vivo in interphasic and mitotic Hela cells; the electrophoretic mobility of Rap1GAP from mitotic cells is reduced compared with that from interphasic cells, suggesting that the mitotic form of the protein is hyperphosphorylated. As the cdc2 kinase is specifically active during mitosis, we sought to investigate whether it actually phosphorylates Rap1GAP during this phase of the cell cycle. We show that p34cdc2 co-immunoprecipitated from mitotic Hela cell lysates with an anti human cyclin B1 antibody, but not from interphasic cell lysates, is able to phosphorylate efficiently wild-type Rap1GAP, but not a mutant in which the putative consensus site for phosphorylation by the cdc2 kinase (serine 484) has been altered. Moreover, depletion of p34cdc2 from mitotic extracts abolishes the phosphorylation of Rap1GAP by such lysates. These results therefore strongly suggest that Rap1GAP is indeed a substrate of the cdc2 kinase during mitosis. This phosphorylation does not affect the stimulation of the GTPase activity of Rap1 by Rap1GAP but may play a role in regulating the interaction of Rap1GAP with other proteins involved in the cellular functions regulated by Rap1 and Rap1GAP.
Janoueix-Lerosey
I
I
INSERM U-248, Faculté de Médecine Lariboisière-Saint-Louis, Paris, France.
Fontenay
M
M
Tobelem
G
G
Tavitian
A
A
Polakis
P
P
de Gunzburg
J
J
D000595:Amino Acid Sequence; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D007399:Interphase; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020668:rap GTP-Binding Proteins
D006657:Histones; D016203:CDC2 Protein Kinase; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins