LARGE-SCALE IDENTIFICATION OF C-MYC-ASSOCIATED PROTEINS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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, Mif2-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 MYCbox 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-MYCassociated 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 subproteome will facilitate further studies aimed to elucidate the biology of c-MYC.