Phosphorylation of the MADS-Box Transcription Factor MEF2C Enhances Its DNA Binding Activity

Jeffery D Molkentin,Li Li,Eric N Olson

doi:10.1074/jbc.271.29.17199

Abstract

Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors activate muscle gene expression by binding an A/T-rich DNA sequence in the control regions of muscle-specific genes. There are four MEF2 factors in vertebrates, MEF2A-D, which share homology in an amino-terminal MADS domain and an adjacent region known as the MEF2 domain, that together mediate DNA binding and dimerization. We show that serine 59 located between the MADS and MEF2 domains of MEF2C is phosphorylated in vivo and can be phosphorylated in vitro by casein kinase-II (CKII). Phosphorylation of this site enhanced the DNA binding and transcriptional activity of MEF2C by increasing its DNA binding activity 5-fold. In vivo 32P labeling experiments showed that serine 59 is the only phosphorylation site in the MADS and MEF2 domains. Mutagenesis of this serine to an aspartic acid resulted in an increase in DNA binding and transcriptional activity of MEF2C comparable to that observed when this site was phosphorylated, suggesting that phosphorylation augments DNA binding activity by introducing negative charge. This phosphorylation site, which corresponds to a CKII recognition site, is conserved in all known MEF2 factors in organisms ranging from flies to humans, consistent with its importance for the functions of MEF2C.

Highlights

Members of the myocyte enhancer factor-2 (MEF2)1 family of MADS (MCM1, Agamous, Deficiens, Serum response factor) box transcription factors have been implicated in the control of skeletal and cardiac muscle gene expression
While studying the DNA binding properties of MEF2C, we observed that the DNA-binding domain expressed in bacteria bound DNA less efficiently than the same region of MEF2C translated in a rabbit reticulocyte lysate
The labeled protein migrated as a single band of ϳ15 kDa. These results demonstrated that the region of sequence corresponding to the MADS and MEF2 domains of MEF2C is phosphorylated in vivo

Summary

Introduction

Members of the myocyte enhancer factor-2 (MEF2) family of MADS (MCM1, Agamous, Deficiens, Serum response factor) box transcription factors have been implicated in the control of skeletal and cardiac muscle gene expression (reviewed in Ref. 33). An adjacent 28-amino acid motif, known as the MEF2 domain, is unique to the MEF2 factors and influences dimerization, DNA binding, and interaction with members of the MyoD family of basic helix-loop-helix transcription factors (8, 9). Because of the extensive amino acid homology between the MADS domains of SRF and MEF2 factors, it is likely that the DNA-binding regions of these factors adopt similar conformations. While studying the DNA binding properties of MEF2, we observed that the bacterially-expressed protein bound DNA weakly, but that incubation with rabbit reticulocyte lysate significantly enhanced DNA binding activity. This suggested that MEF2 was subjected to a post-translational modification event. This site is conserved in all known members of the MEF2 family, consistent with its importance for MEF2 function

Methods

Results

Conclusion