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Published on: August 2, 2018
Quantitative phosphoproteomics of proteasome inhibition in multiple myeloma cells
Feng Ge1, Chuan-Le Xiao, Li-Jun Bi
1Institute of Life and Health Engineering and National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, China.
Background:
The proteasome inhibitor bortezomib represents an important advance in the treatment of multiple myeloma (MM). Bortezomib inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells; however, the mechanism of cytotoxicity is not well understood.
Methodology/Principal Findings:
We investigated the differential phosphoproteome upon proteasome inhibition by using stable isotope labeling by amino acids in cell culture (SILAC) in combination with phosphoprotein enrichment and LC-MS/MS analysis. In total 233 phosphoproteins were identified and 72 phosphoproteins showed a 1.5-fold or greater change upon bortezomib treatment. The phosphoproteins with expression alterations encompass all major protein classes, including a large number of nucleic acid binding proteins. Site-specific phosphopeptide quantitation revealed that Ser38 phosphorylation on stathmin increased upon bortezomib treatment, suggesting new mechanisms associated to bortezomib-induced apoptosis in MM cells. Further studies demonstrated that stathmin phosphorylation profile was modified in response to bortezomib treatment and the regulation of stathmin by phosphorylation at specific Ser/Thr residues participated in the cellular response induced by bortezomib.
Conclusions/Significance:
Our systematic profiling of phosphorylation changes in response to bortezomib treatment not only advanced the global mechanistic understanding of the action of bortezomib on myeloma cells but also identified previously uncharacterized signaling proteins in myeloma cells.
Insights
Bortezomib, a proteasome inhibitor, induces cell death in multiple myeloma (MM) through poorly understood mechanisms. This study reveals bortezomib alters stathmin phosphorylation, suggesting new pathways for MM cell apoptosis.
Area of Science:
- Proteomics
- Cancer Biology
- Molecular Mechanisms
Background:
- Bortezomib is a proteasome inhibitor used to treat multiple myeloma (MM).
- Its precise mechanism of tumor cell death is not fully understood.
- Understanding bortezomib's action is crucial for optimizing MM therapy.
Purpose of the Study:
- To investigate the global changes in protein phosphorylation (phosphoproteome) induced by bortezomib in MM cells.
- To identify novel signaling pathways involved in bortezomib-induced apoptosis.
- To elucidate the role of stathmin phosphorylation in the cellular response to bortezomib.
Main Methods:
- Utilized stable isotope labeling by amino acids in cell culture (SILAC) for quantitative phosphoproteomics.
- Employed phosphoprotein enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis.
- Performed site-specific phosphopeptide quantitation and functional studies.
Main Results:
- Identified 233 phosphoproteins, with 72 showing significant changes (>1.5-fold) upon bortezomib treatment.
- Observed alterations in phosphoproteins across major classes, including nucleic acid binding proteins.
- Found increased Ser38 phosphorylation on stathmin, implicating it in bortezomib-induced apoptosis.
Conclusions:
- Systematic phosphoproteomic profiling advanced the mechanistic understanding of bortezomib's action in MM.
- Identified stathmin phosphorylation as a key regulator in the cellular response to bortezomib.
- Discovered previously uncharacterized signaling proteins involved in MM cell death pathways.
