A Rare Human Sequence Variant Reveals Myocardin Autoinhibition

Joshua F Ransom,Isabelle N King,Vidu Garg,Deepak Srivastava

doi:10.1074/jbc.m805909200

Abstract

Myocardin (MYOCD) is a transcriptional co-activator that promotes cardiac or smooth muscle gene programs through its interaction with myocyte-enhancing factor (MEF2) or serum-response factor (SRF). Isoforms of MYOCD with a truncated amino terminus show increased activity when compared with those with the full-length amino terminus, but how this is achieved remains unknown. We identified a rare human sequence variation in MYOCD in a patient with congenital heart disease that resulted in a missense mutation at codon 259 (K259R). This variation created a hypomorphic cardiac isoform with impaired SRF binding and transactivation capacity but did not impair the smooth muscle isoform of MYOCD, which lacks the amino terminus. Consistent with differential effects of the amino terminus on the K259R mutation, we found that the cardiac-specific amino terminus acted in an autoinhibitory fashion to bind MYOCD via specific negatively charged residues and thereby repressed SRF-dependent MYOCD activity. This effect was exaggerated in the MYOCD-K259R mutant. The amino terminus was sufficient to impair MYOCD-dependent fibroblast conversion into smooth muscle cells as well as cardiomyocyte hypertrophy. These findings identify a novel mechanism that regulates levels of MYOCD-dependent activation of the SRF genetic program differentially in cardiac and smooth muscle.

Highlights

Myocardin (MYOCD)2 was the first recognized member of a family of transcriptional co-activators that binds to serum-response factor (SRF) to activate cardiac or smooth muscle gene programs [1]
This residue is conserved in MRTF-A, but MRTF-B contains an arginine at the corresponding codon
We found that mutation of the conserved arginines (R71A, R115A) of the two RPEL motifs had no effect on the ability of the MYOCD homology domain (MHD) to inhibit MYOCD activity, suggesting that the RPEL motifs are not necessary for autoinhibition (Fig. 3B)

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—FLAG-MYOCD truncations, atrial natriuretic factor-luciferase (ANF-Luc), and smooth muscle 22␣-luciferase (SM22␣-Luc) have been described [1]. Western analysis was performed for all transfected proteins to ensure equal expression. Transfections were performed with FuGENE 6 (Roche Applied Science) according to the manufacturer’s instructions. Immunocytochemistry and quantitative realtime (RT) PCR (TaqMan, Applied Biosystems) were performed. The cardiomyocytes were enzymatically separated with 1 ␮g/ml pancreatin, plated on 0.1% gelatin-coated dishes, and infected 1 day later with lentivirus (pLenti Gateway System, Invitrogen) overexpressing protein under control of the EF1 promoter. Quantitative RT-PCR—Whole RNA was purified with TRI Reagent (Applied Biosystems), treated with DNA-free DNase (Ambion), and synthesized into cDNA with poly(dT) primers (Superscriptase III kit, Invitrogen) according to the manufacturer’s instructions. RT-PCR was performed with Applied Biosystems 2ϫ master mix and TaqMan primers (see supplemental Table S1) as described [16]. Fluorescein isothiocyanate- or TRITCconjugated goat anti-mouse secondary antibodies were used for immunocytochemistry, and alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit antibodies were used for Western analysis (Jackson Immunologicals)

RESULTS

Cardiac but Not Smooth Muscle

DISCUSSION