Calcium/calmodulin inhibition of transcriptional activity of E-proteins by prevention of their binding to DNA.
2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 39, 41004-41011 p.Article in journal (Refereed) Published
The Ca2+ sensor protein calmodulin can interact with the DNA binding basic helix-loop-helix (bHLH) domain of E12, E47, and SEF2-1 (E2-2), which belong to the E-protein subclass of bHLH transcription factors. This interaction inhibits the DNA binding of these bHLH proteins in vitro, and an ionophore that increases intracellular Ca2+ can inhibit transcriptional activation by the E-proteins. Here we have attempted to determine if these phenomena reflect a direct calmodulin-dependent inhibition of DNA binding by E-proteins in vivo. We show that calmodulin overexpression inhibits the transcriptional activity of E12, E47, and SEF2-1. We have compared calmodulin effects on DNA binding in vitro and on activation of transcription in vivo using a series of E12 mutants harboring defined alterations within the basic sequence of the bHLH domain that reduce their ability to bind calmodulin to varying degrees. We find a striking direct correlation between the ability of calmodulin to inhibit their DNA binding in vitro and the ability of overexpressed calmodulin or cellular Ca2+ mobilization to inhibit their transcriptional activity in vivo. Furthermore, E12 and overexpressed calmodulin were co-localized in the nucleus, and calmodulin pull-down experiments with cell extracts showed a Ca2+-dependent interaction between calmodulin and E12 but not with a calmodulin inhibition-deficient E12 mutant. Chromatin immunoprecipitation showed that calmodulin overexpression leads to decreased binding of E12 and E47, but not a calmodulin inhibition-deficient E12 mutant, to the DNA recognition sequence in vivo. The data suggest that Ca2+ signaling can inhibit the transcriptional activities of E-proteins through direct binding of Ca2+/calmodulin to the basic sequence of E-proteins, resulting in inhibition of their DNA binding. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.
Place, publisher, year, edition, pages
2004. Vol. 279, no 39, 41004-41011 p.
Amino Acid Sequence, Animals, Blotting; Western, Calcium/*metabolism, Calmodulin/chemistry/*metabolism, Cell Nucleus/metabolism, Cells; Cultured, Chromatin/metabolism, Cytoplasm/metabolism, DNA/*chemistry/metabolism, DNA; Complementary/metabolism, DNA-Binding Proteins/*metabolism, Humans, Mice, Microscopy; Fluorescence, Molecular Sequence Data, Mutagenesis; Site-Directed, Mutation, Nerve Tissue Proteins, Plasmids/metabolism, Precipitin Tests, Protein Binding, Protein Structure; Tertiary, Sepharose/chemistry, Sequence Homology; Amino Acid, Signal Transduction, TCF Transcription Factors, Thapsigargin/pharmacology, Trans-Activation (Genetics), Transcription Factors/*metabolism, Transcription; Genetic, Transfection
IdentifiersURN: urn:nbn:se:umu:diva-16488DOI: 10.1074/jbc.M408120200PubMedID: 15280352OAI: oai:DiVA.org:umu-16488DiVA: diva2:156161