The association of cyclin A and cyclin kinase inhibitor p21 in response to γ-irradiation requires the CDK2 binding region, but not the Cy motif

• Published in: Biochimica et biophysica acta 2003-10, Vol.1642 (3), p.163-171
• Main Authors: Fukuchi, Kunihiko, Nakamura, Kentarou, Ichimura, Sachiko, Tatsumi, Kouichi, Gomi, Kunihide
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Alanine - metabolism; Amino Acid Motifs; Binding Sites; CDC2-CDC28 Kinases - metabolism; CDK2; Cell Line, Tumor; Cy motif; Cyclin A; Cyclin A - metabolism; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclins - chemistry; Cyclins - genetics; Cyclins - metabolism; DNA damage; Gamma Rays; Humans; p21; Phosphorylation; Precipitin Tests; Protein Binding - radiation effects; Sequence Deletion
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2003.08.001

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 γ-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 γ-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.