Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8).
Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2-dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N-terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5's susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8's ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull-down native CIP8 from the extract of dark-grown seedlings, but not from the extract of light-grown seedlings in a column-binding assay, implying a requirement for light-regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co-operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8-CIP8 module might interact with COP1 in vivo, thereby participating in proteasome-mediated degradation of HY5.
Hardtke
Christian S
CS
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8104, USA.
Okamoto
Haruko
H
Stoop-Myer
Chatanika
C
Deng
Xing Wang
XW
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000378:metabolism*; D050976:Basic-Leucine Zipper Transcription Factors; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II; D003624:Darkness; D016350:Leucine Zippers / Q000502:physiology; D008025:Ligases / Q000378:metabolism*; D008027:Light; D009024:Morphogenesis / Q000528:radiation effects; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
D029681:Arabidopsis Proteins; D050976:Basic-Leucine Zipper Transcription Factors; C121940:CIP8 protein, Arabidopsis; D002352:Carrier Proteins; C109254:HY5 protein, Arabidopsis; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D044763:Ubiquitin-Conjugating Enzymes; C409686:ubiquitin-conjugating enzyme UBC8; C471983:AT2G32950 protein, Arabidopsis; D044767:Ubiquitin-Protein Ligases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D008025:Ligases