Regulation of Cardiac Specific nkx2.5 Gene Activity by Small Ubiquitin-like Modifier

Robert J. Schwartz,Jun Wang,Dinakar Iyer,Hua Zhang,Xin-Hua Feng

doi:10.1074/jbc.m709748200

Abstract

The cardiac specific homeobox gene nkx2.5, a member of the nk-2 class family, plays a central role in cardiogenesis and is a target of the small ubiquitin-like modifier (SUMO). Nkx2.5 was modified by SUMO on its 51st amino acid, a lysine residue conserved across species but absent in other nk-2 members. Conversion of this lysine to an arginine (K51R) substantially reduced Nkx2.5 DNA binding and also its transcriptional activity. Unexpectedly, mutant K51R was targeted by ubiquitin. E3 ligase PIAS proteins PIAS1, PIASx, and PIASy, but not PIAS3, enhanced SUMO-1 attachment to Nkx2.5 on the primary SUMO acceptor site. SUMO-2 linkage to Nkx2.5 was catalyzed only by PIASx and not by other PIAS proteins. SUMO conjugation stabilized the formation of Nkx2.5-containing complexes that led to robust transcriptional activation. Thus, SUMO modification serves as a positive regulator for Nkx2.5 transcriptional activity.

Highlights

Cardiac specific homeobox gene nkx2.5 (1, 2), a member of the nk-2 class of homeodomain (HD)2 factors, is required for early heart development and morphogenesis (3, 4)
small ubiquitin-like modifier (SUMO)-1 or Nkx2.5 alone had no significant effect on the tested promoter
HeLa cells transfected with Nkx2.5 in the presence or absence of SUMO-1 wild type (WT) or SUMO-1-⌬GG, a defective SUMO mutant

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructions—The cardiac ␣-actin promoterdriven luciferase reporter construct (Ca-actin-Luc) and Nkx2.5 expression vector were described previously (9). Ni2ϩ-NTA pulldowns for ubiquitination assays were performed by either transfecting His6-tagged Nkx2.5 WT or K51R in the presence or absence of ubiquitin expression vector or overlapping the conversion site and a second pair covering each transfecting Nkx2.5 WT or K51R with or without His6end of cDNA. To determine if exogenous Nkx2.5 was modified by deletion mutants were generated by PCR using corresponding endogenous SUMOs, Ni2ϩ-NTA was performed on COS-7 oligonucleotides covering the desired segments of cDNA, transfected with His6-tagged Nkx2.5, and protein blots were which were thereafter subcloned into pcDNA4B-V5/His vector tested with anti-Nkx2.5 antibody. Reporter constructs (200 WT or K51R mutant were used in binding assays performed at ng) and expression vectors were transfected into CV1 cells in room temperature, as described in detail (17). In supershift activity assays were executed with MonolightTM3010

RESULTS

To examine which PIAS enhanced

DISCUSSION