title: QUICK identification and SPR validation of signal transducers and activators of transcription 3 (Stat3) interacting proteins.
abstract: Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved in the pathogenesis of various human diseases and is constitutively active in human multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry analysis allowed us to distinguish specific Stat3 interacting proteins from background proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting proteins - 14-3-3ζ, PRKCB and Hsp90 - were further confirmed by reciprocal co-immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results therefore not only uncover a number of Stat3 interacting proteins that possess a variety of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells.
authors.affiliation: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zheng
authors.forename: Qiu
authors.initials: Q
authors.lastname: Zhong
authors.forename: Qian
authors.initials: Q
authors.lastname: Xiong
authors.forename: Mingkun
authors.initials: M
authors.lastname: Yang
authors.forename: Jia
authors.initials: J
authors.lastname: Zhang
authors.forename: Chongyang
authors.initials: C
authors.lastname: Li
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Bi
authors.forename: Feng
authors.initials: F
authors.lastname: Ge
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D009101:Multiple Myeloma / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D009363:Neoplasm Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human