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

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-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.

c-Myc protein is degraded in response to UV irradiation

“…It has been shown recently that the F-box E3 ubiquitin ligase FBW8 (also known as FBXW8 or FBX29) interacts with c-MYC via both the MBII motif and the CT region and regulates c-MYC protein levels and transactivation. 44 Deletion of both these domains of c-MYC did not impair its degradation induced by UV (Fig. 3B), ruling out a role for FBW8, and by the same way of SKP2, in this phenomenon.…”

A Conserved Proliferating Cell Nuclear Antigen-interacting Protein Sequence in Chk1 Is Required for Checkpoint Function

“…The protein mix was digested with trypsin, and the resulting peptides were separated and identified using mass spectrometry-based Mud-PIT (47,48). This resulted in the identification of known Chk1 interactors (e.g.…”

c-MYC-Induced Genomic Instability

“…The idea that MYC interferes with gene products that mediate DNA break repair was proposed by Karlsson et al after their observation of severe magnitude increases in chromosome breaks in normal human foreskin fibroblasts through DSB assays (Karlsson et al 2003). It was later determined by tandem affinity purification with mass spectral multidimensional protein identification technology proteomics that MYC regulates nonhomologous end-joining (NHEJ) factors (Koch et al 2007), and a recent investigation concluded that MYC directly suppresses the NHEJ pathway causing inhibition of DSB repair and V(D)J recombination through inhibitory MYC box II binding with Ku70 in vitro and in vivo (Li et al 2012). It has also been reported that MYC inhibits homologous recombination through regulation of RAD51 in various cancer cell lines (Luoto et al 2010).…”