Calcium-dependent binding of Myc to calmodulin
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Philipp Raffeiner1,3, Andrea Schraffl1, Thomas Schwarz2, Ruth Röck1, Karin Ledolter2, Markus Hartl1, Robert Konrat2, Eduard Stefan1, Klaus Bister1
1Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, A-6020 Innsbruck, Austria
2Max F. Perutz Laboratories, Department of Structural and Computational Biology, University of Vienna, A-1030 Vienna, Austria
3Present address: Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
Eduard Stefan, email: email@example.com
Klaus Bister, email: firstname.lastname@example.org
Keywords: oncogene, transcription factor, signal transduction, protein-protein interaction, second messenger signaling
Received: August 08, 2016 Accepted: November 21, 2016 Published: December 01, 2016
The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.
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