The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif.
The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.
Fukuchi
Kunihiko
K
Department of Clinical Pathology, Showa University, School of Medicine, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8666, Japan. kfukuchi@med.showa-u.ac.jp
Nakamura
Kentarou
K
Ichimura
Sachiko
S
Tatsumi
Kouichi
K
Gomi
Kunihide
K
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D045744:Cell Line, Tumor; D019925:Cyclin A / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005720:Gamma Rays; D006801:Humans; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding / Q000528:radiation effects; D017384:Sequence Deletion
C493956:CDKN1A protein, human; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D000409:Alanine