Abolishment of the Tyr-15 inhibitory phosphorylation site on cdc2 reduces the radiation-induced G(2) delay, revealing a potential checkpoint in early mitosis.
cdc2 is inactivated before mitosis by phosphorylation at its inhibitory sites, Thr-14 and Tyr-15. Irradiation prevents HeLa cells from completing the G(2)-M transition, and they arrest in G(2). Whereas phosphorylation at both of these sites occurs during the G(2) arrest, the individual role of each site in the G(2) delay has not previously been investigated. We have shown that the radiation-induced G(2) delay is preserved in wild-type or cdc2-AY-transfected cells (which retain Tyr-15); this delay is abolished in cdc2-TF- or cdc2-AF-transfected cells (which lack Tyr-15). Thus Tyr-15, but not Thr-14, appears to be essential for development of a G(2) delay after radiation. Abolishment of the G(2) delay by mutation at Tyr-15 resulted in the accumulation of cells with condensed chromatin and disrupted lamin B, suggesting that these cells may be blocked at a second G(2)-M checkpoint in early mitosis (i.e., prophase). These data suggest (a) that the two inhibitory phosphorylation sites have distinct functions and that Tyr-15 phosphorylation, in particular, has a key role in the radiation-induced G(2) delay, and (b) that a second G(2)-M checkpoint exists in early mitosis and that activation of this checkpoint by radiation prevents cells that enter mitosis from progressing further.
Fletcher
Lynda
L
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Cheng
Yi
Y
Muschel
Ruth J
RJ
D001665:Binding Sites; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D019926:Cyclin B / Q000096:biosynthesis / Q000235:genetics; D056744:Cyclin B1; D016195:G2 Phase / Q000502:physiology / Q000528:radiation effects*; D006367:HeLa Cells; D006801:Humans; D034921:Lamin Type B; D034882:Lamins; D008938:Mitosis / Q000502:physiology / Q000528:radiation effects; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
C534699:CCNB1 protein, human; D002843:Chromatin; D019926:Cyclin B; D056744:Cyclin B1; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D014443:Tyrosine; D016203:CDC2 Protein Kinase