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1.
FEBS Lett ; 589(17): 2233-40, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26183205

ABSTRACT

Immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide exhibit high responsive rates for newly identified or relapsed multiple myeloma patients. However, their mechanisms of action are not completely understood. One mechanism involves the ubiquitination and degradation of two transcription factors, IKZF1 and IKZF3. Whether there are other degradation pathways for IKZF1 in myeloma cells remains unknown. Here, we found that although IKZF1 ubiquitination was reduced, its stability was also significantly reduced in MM1.S and OPM2 cells treated with kinase inhibitors, 5,6-dichlorobenzimidazole riboside (DRB) or roscovitine. Through pharmacological inhibition and biochemical approaches we demonstrated that instead of undergoing the ubiquitin-proteasome pathway, IKZF1 was degraded through apoptosis induced by kinase inhibition. This result may provide a new direction in developing therapeutic treatments for myeloma patients.


Subject(s)
Apoptosis/drug effects , Dichlororibofuranosylbenzimidazole/pharmacology , Ikaros Transcription Factor/metabolism , Purines/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , HEK293 Cells , Humans , Immunoblotting , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Proteolysis/drug effects , Roscovitine , Time Factors , Ubiquitination/drug effects
2.
Biochem Biophys Res Commun ; 461(4): 653-8, 2015 Jun 12.
Article in English | MEDLINE | ID: mdl-25918018

ABSTRACT

Neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8) is a ubiquitin-like modifier, which forms covalent conjugates on lysines of its substrates. This post-translational modification, neddylation, plays important roles in tumor cell proliferation and viability. Ubiquitin can form diverse polyubiquitin chains, on its seven lysines, which play important functions in various biological processes. However, the roles of lysines in NEDD8 have not been explored. Here, we generated nine NEDD8 point mutants, each with one lysine replaced by an arginine, to study the putative function of lysines in NEDD8. Our experiments discover that Lys27 in NEDD8 is a critical residue for protein neddylation. Replacement of this residue with arginine almost completely eliminates the conjugation of NEDD8 to its substrates. Furthermore, we find that the K27R mutant impairs NEDD8 conjugation to the E2 enzyme, which normally forms thioester bonds for further transferring NEDD8 to its ligases and substrates. Therefore, this mutation completely inhibits global protein neddylation, including neddylation of cullin family proteins, resulting in decreased activity of cullin-RING E3 ligases. This work sheds new light on the roles of NEDD8 lysines on neddylation cascades and provides a dominant negative mutant for the study of neddylation and its biological functions.


Subject(s)
Arginine/metabolism , Lysine/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Ubiquitins/metabolism , Arginine/chemistry , Enzyme Activation , HEK293 Cells , Humans , Lysine/chemistry , Mutagenesis, Site-Directed , NEDD8 Protein , Structure-Activity Relationship
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