Large-scale identification of c-MYC-associated proteins using a combined TAP/MudPIT approach.
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.
Koch
Heike B
HB
Molecular Oncology, Independent Max-Planck-Research Group, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
Zhang
Ru
R
Verdoodt
Berlinda
B
Bailey
Aaron
A
Zhang
Chang-Dong
CD
Yates
John R
JR
Menssen
Antje
A
Hermeking
Heiko
H
D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D025941:Protein Interaction Mapping; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D014407:Tumor Cells, Cultured
D020543:Proteome; D016271:Proto-Oncogene Proteins c-myc