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.