The N-terminal 15-48 region of cyclin kinase inhibitor p21 is a determinant of basal expression.
The cyclin kinase inhibitor, p21, inhibits or arrests cell cycle progression in response to DNA damage and regulates the progression of apoptosis, either negatively or positively depending on the situation. The stability of p21 is regulated by its phosphorylation or through binding with partner molecules, and, when cells grow without DNA damage, the level of p21 is regulated by proteasome degradation. In this study, we analyzed the mechanism by which the basal expression level of p21 is stabilized. The transient expression of various p21 deletion mutants revealed a specific mutant with a deletion of 15-48 aa (Delta15-48C) that was extremely unstable. This mutant was stabilized by the proteasome inhibitor, lactacystin. Since the cysteine in the region of the alanine mutant did not destabilize p21, possible disulfide bonds formed by cysteines in the region are not responsible for the stabilization. The Delta15-48C was unstable in the cells stably expressing the 1-60 aa region, indicating that the 1-60 aa region did not function in trans. Fusion of the 1-60 aa fragment to the N-terminal of Delta15-48C stabilized the product, indicating that the 1-60 aa region in the molecule is effective for the stabilization. We constructed cells that stably expressed Delta15-48C. The Delta15-48C was unstable, but was stabilized by lactacystin. Irradiation (5 Gy) enhanced the expression of Delta15-48C without elevation of mRNA levels and increased the binding with cyclin A or CDK2. Taken together, the 15-48 aa region of p21 is essential for basal expression by preventing degradation by the proteasome, which is distinct from the mechanism induced by DNA damage.
Nomura
Norihiro
N
Department of Clinical Pathology, Showa University, School of Medicine, Shinagawa, Tokyo, Japan.
Yamaguchi
Fumihiro
F
Fukuchi
Kunihiko
K
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D005720:Gamma Rays; D005786:Gene Expression Regulation / Q000528:radiation effects; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism