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Large-scale identification of c-MYC-associated proteins using a combined TAP/MudPIT approach

Academic Article
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Overview

authors

  • Koch, H. B.
  • Zhang, R.
  • Verdoodt, B.
  • Bailey, A.
  • Zhang, C. D.
  • Yates III, John
  • Menssen, A.
  • Hermeking, H.

publication date

  • January 2007

journal

  • Cell Cycle  Journal

abstract

  • 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-beta/CHD4, RNA Pol II, RFC2, RFC3, SV40 Large T Antigen, TCP1alpha, 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-beta 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.

subject areas

  • Cell Line
  • HeLa Cells
  • Humans
  • Protein Interaction Mapping
  • Proteome
  • Proteomics
  • Proto-Oncogene Proteins c-myc
  • Sequence Analysis, Protein
  • Tumor Cells, Cultured
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Research

keywords

  • DNA replication
  • MudPIT analysis
  • c-MYC
  • proteomics
  • proto-oncogene
  • tandem affinity purification
  • ubiquitination
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Identity

International Standard Serial Number (ISSN)

  • 1538-4101

Digital Object Identifier (DOI)

  • 10.4161/cc.6.2.3742

PubMed ID

  • 17314511
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Additional Document Info

start page

  • 205

end page

  • 217

volume

  • 6

issue

  • 2

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