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Identification of ICER’s ubiquitination sites to distinguish its Novel Ubiquitin Ligase (LB186)
Author(s) -
Woldeamanuel Fanaye,
Molina Carlos
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb186
Subject(s) - ubiquitin , ubiquitin ligase , subcellular localization , lysine , repressor , microbiology and biotechnology , mutant , cytosol , biochemistry , biology , arginine , chemistry , amino acid , transcription factor , gene , cytoplasm , enzyme
Inducible cAMP Early Repressor (ICER) is a dominant negative transcriptional repressor that expresses tumor suppressor activity. In cancer cells, ICER is abnormally expressed and becomes modified by the posttranslational modification ubiquitination. Consequentially, ICER is degraded or mislocalized from the nucleus to the cytosol where it is non‐ functional. When exogenous ICER is introduced into cancer cells, it inhibits the transformed phenotype of cancer cells. The goal of this project is to identify the ubiquitinated residues of ICER to ultimately rescue this protein from degradation and cytosolic localization. Since lysine residues are sites for ubiquitination, mutant forms of each residue were constructed using site‐directed mutagenesis. All lysine residues were substituted to arginine to create an ICER molecule without lysines. Another 10 independent mutants were generated where all but one specific lysine was substituted by arginine. All clones were subcloned in a mammalian expression vector. By using these altered forms of ICER, we have found that ICER is ubiquitinated in several lysines. This data suggested alternative modes of regulating ICER degradation and subcellular localization. The long‐term goal of this project is to identify ways of inhibiting ICER degradation in cancer cells as a new mode for cancer treatment.