Abstract
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.
Highlights
The myc oncogene was originally discovered as the oncogenic principle (v-myc) in the genome of avian acute leukemia virus MC29, derived from the chicken proto-oncogene c-myc by retroviral transduction [1,2,3]
We have previously described the identification of a Myc www.impactjournals.com/oncotarget target gene (BASP1, termed CAP-23/NAP-22) that is repressed in Myc-transformed cells and, when expressed ectopically, inhibits cell transformation by Myc [11]
For the initial analyses of possible protein-protein interactions (PPIs) between Myc and CaM, recombinant glutathione S-transferase(GST)-fusion proteins containing chicken Max, CaM, or BASP1 as bait segments were synthesized (Figure 1A). These GST-fusion proteins were used in pull-down assays performed with whole cell extracts from quail embryo fibroblasts (QEF) transformed by MC29
Summary
The myc oncogene was originally discovered as the oncogenic principle (v-myc) in the genome of avian acute leukemia virus MC29, derived from the chicken proto-oncogene c-myc by retroviral transduction [1,2,3]. It has been reported that CaM can bind to and modulate the activity of transcriptional regulators of the bHLH class like E12, E47, or SEF2-1 [17,18,19]. In view of this and emanating from the identification of the Myc target BASP1 encoding a CaM-binding protein, we searched for a possible connection of the bHLHLZ proteins Myc and Max with CaM, and analyzed the observed interactions in structural and functional detail
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