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Large-Scale Identification of c-MYC-Associated Proteins Using a Combined TAP/MudPIT Approach
Heike Koch, Ru Zhang, Berlinda Verdoodt, Aaron Bailey, Chang-Dong Zhang, John R. Yates, Antje Menssen and Heiko Hermeking
volume 6 | issue 2
15 January 2007Pages: 205 - 217
This is an open-access article
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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.
Authors
Heike Koch
MPI of Biochemistry; Molecular Oncology; Bavaria, Germany
Ru Zhang
MPI of Biochemistry; Molecular Oncology; Bavaria, Germany
Berlinda Verdoodt
Max-Planck-Institute of Biochemistry, Martinsried, Germany
Aaron Bailey
The Scripps Research Institute; Cell Biology; La Jolla, California USA
Chang-Dong Zhang
MPI of Biochemistry; Molecular Oncology; Bavaria, Germany
John R. Yates
The Scripps Research Institute; Cell Biology; La Jolla, California USA
Antje Menssen
Max-Planck-Institute of Biochemistry, Martinsried, Germany
Heiko Hermeking
Max-Planck-Institute of Biochemistry, Martinsried, Germany
This is an open-access article
Download PDFIf the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.