Large-Scale Identification of c-MYC-Associated Proteins Using a Combined TAP/MudPIT Approach

• Published in: Cell cycle (Georgetown, Tex.) Tex.), 2007-01, Vol.6 (2), p.205-217
• Main Authors: Koch, Heike, Zhang, Ru, Verdoodt, Berlinda, Bailey, Aaron, Zhang, Chang-Dong, Yates, John R., Menssen, Antje, Hermeking, Heiko
• Format: Article
• Language: English
• Subjects: Binding; Biology; Bioscience; Calcium; Cancer; Cell; Cell Line; Cycle; HeLa Cells; Humans; Landes; Organogenesis; Protein Interaction Mapping; Proteins; Proteome - genetics; Proteome - isolation & purification; Proteome - metabolism; Proteomics - methods; Proto-Oncogene Proteins c-myc - genetics; Proto-Oncogene Proteins c-myc - isolation & purification; Proto-Oncogene Proteins c-myc - metabolism; Sequence Analysis, Protein - methods; Tumor Cells, Cultured
• ISSN: 1538-4101; 1551-4005
• DOI: 10.4161/cc.6.2.3742

The c-MYC oncogene encodes a transcription factor, which is sufficient and necessary for the induction of cellular proliferation. However, the c-MYC protein is a relatively weak transactivator suggesting that it may have other functions. To identify protein interactors which may reveal new functions or represent regulators of c-MYC we systematically identified proteins associated with c-MYC in vivo using a proteomic approach. We combined tandem affinity purification (TAP) with the mass spectral multidimensional protein identification technology (MudPIT). Thereby, 221 c-MYC-associated proteins were identified. Among them were 17 previously known c-MYC-interactors. Selected new c-MYC-associated proteins (DBC-1, FBX29, KU70, MCM7, Mi2-b/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1a, U5-116kD, ZNF281) were confirmed independently. For association with MCM7, SV40 Large T Antigen and DBC-1 the functionally important MYC-box II region was required, whereas FBX29 and Mi2-b interacted via MYC-box II and the BR-HLH-LZ motif. In addition, regulators of c-MYC activity were identified: ectopic expression of FBX29, an E3 ubiquitin ligase, decreased c-MYC protein levels and inhibited c-MYC transactivation, whereas knock-down of FBX29 elevated the concentration of c-MYC. Furthermore, sucrose gradient analysis demonstrated that c-MYC is present in numerous complexes with varying size and composition, which may accommodate the large number of new c-MYC-associated proteins identified here and mediate the diverse functions of c-MYC. Our results suggest that c-MYC, besides acting as a mitogenic transcription factor, regulates cellular proliferation by direct association with protein complexes involved in multiple synthetic processes required for cell division, as for example DNA-replication/repair and RNA-processing. Furthermore, this first comprehensive description of the c-MYC-associated sub-proteome will facilitate further studies aimed to elucidate the biology of c-MYC.