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Visualization of Myc/Max/Mad Family Dimers and the Competition for Dimerization in Living Cells

Asya V. Grinberg, Chang-Deng Hu, and Tom K. Kerppola^*

Howard Hughes Medical Institute and Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0650

Received 3 December 2003/ Returned for modification 11 January 2004/ Accepted 26 February 2004

Myc and Mad family proteins play opposing roles in the control of cell growth and proliferation. We have visualized the subcellular locations of complexes formed by Myc/Max/Mad family proteins using bimolecular fluorescence complementation (BiFC) analysis. Max was recruited to different subnuclear locations by interactions with Myc versus Mad family members. Complexes formed by Max with Mxi1, Mad3, or Mad4 were enriched in nuclear foci, whereas complexes formed with Myc were more uniformly distributed in the nucleoplasm. Mad4 was localized to the cytoplasm when it was expressed separately, and Mad4 was recruited to the nucleus through dimerization with Max. The cytoplasmic localization of Mad4 was determined by a CRM1-dependent nuclear export signal located near the amino terminus. We compared the relative efficiencies of complex formation among Myc, Max, and Mad family proteins in living cells using multicolor BiFC analysis. Max formed heterodimers with the basic helix-loop-helix leucine zipper (bHLHZIP) domain of Myc (bMyc) more efficiently than it formed homodimers. Replacement of two amino acid residues in the leucine zipper of Max reversed the relative efficiencies of homo- and heterodimerization in cells. Surprisingly, Mad3 formed complexes with Max less efficiently than bMyc, whereas Mad4 formed complexes with Max more efficiently than bMyc. The distinct subcellular locations and the differences between the efficiencies of dimerization with Max indicate that Mad3 and Mad4 are likely to modulate transcription activation by Myc at least in part through distinct mechanisms.

Supplemental material for this article may be found at http://mcb.asm.org/.

Present address: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy, West Lafayette, IN 47907-1333.

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