In vitro ubiquitination of cyclin D1 by ROC1-CUL1 and ROC1-CUL3.
Overexpression of cyclin D1 has been implicated in a variety of tumors, such as breast cancers, gastrointestinal cancers and lymphomas. Both gene amplification and protein degradation mediated by ubiquitin (Ub)-dependent proteolysis regulate the abundance of cyclin D1. Here we report that ROC1 interacted with all three D type cyclins in vivo but did not bind to other cyclins tested. The ROC1-CUL1 and ROC1-CUL3, but not ROC1-CUL2, -CUL3 and -CUL4, immunocomplexes promoted polyubiquitination of bacterially purified cyclin D1 in vitro. RING finger mutations of ROC1 eliminated the Ub ligase activity toward cyclin D1. In all cases the ubiquitination of cyclin D1 was accompanied by autoubiquitination of the cullins. The results suggest the involvement of ROC1-cullin ligases in cyclin D1 ubiquitination and a potential mechanism whereby the cullin subunit is ubiquitinated itself while ubiquitinating a substrate.
Maeda
I
I
Department of Surgery, St. Marianna University School of Medicine, 2-16-1 Sugao, Niyamae-ku, Kawasaki 216-8511, Japan.
Ohta
T
T
Koizumi
H
H
Fukuda
M
M
D064173:Anaphase-Promoting Complex-Cyclosome; D000936:Antigen-Antibody Complex / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins; D019938:Cyclin D1 / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D021122:Protein Subunits; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D016335:Zinc Fingers
D000936:Antigen-Antibody Complex; C503645:CUL3 protein, human; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D014452:Ubiquitins; D019938:Cyclin D1; C097310:UBE2D3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases