Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.
The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.
Fukuchi
Kunihiko
K
Department of Biochemistry, Showa University, School of Medicine, Tokyo 142-8555, Japan. kfukuchi@med.showa-u.ac.jp
Hagiwara
Tamio
T
Nakamura
Kentarou
K
Ichimura
Sachiko
S
Tatsumi
Kouichi
K
Gomi
Kunihide
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D003110:Colonic Neoplasms; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D025801:Ubiquitin