Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Plant Journal Wiley

Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)

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Publisher
Wiley
Copyright
Copyright © 2002 Wiley Subscription Services, Inc., A Wiley Company
ISSN
0960-7412
eISSN
1365-313X
DOI
10.1046/j.1365-313X.2002.01298.x
Publisher site
See Article on Publisher Site

Abstract

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.

Journal

The Plant JournalWiley

Published: May 1, 2002

Keywords: ; ; ; ; ;

References

  • The cell biology of the COP/DET/FUS proteins. Regulating proteolysis in photomorphogenesis and beyond?
    Hardtke, C.S.; Deng, X.W.
  • Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1
    Holm, M.; Hardtke, C.S.; Gaudet, R.; Deng, X.W.
  • Nuclear and cytosolic events of light‐induced, phytochrome‐regulated signaling in higher plants
    Nagy, F.; Schäfer, E.
  • The N‐terminal fragment of Arabidopsis photomorphogenic repressor COP1 maintains partial function and acts in a concentration‐dependent manner
    Stoop‐Myer, C.; Torii, K.U.; McNellis, T.W.; Coleman, J.E.; Deng, X.W.
  • Functional dissection of Arabidopsis COP1 reveals specific roles of its three structural modules in light control of seedling development
    Torii, K.U.; McNellis, T.W.; Deng, X.W.
  • Role of a COP1 interactive protein in mediating light‐regulated gene expression in Arabidopsis
    Yamamoto, Y.Y.; Matsui, M.; Ang, L.H.; Deng, X.W.

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