Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Abstract

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

A whole-genome RNAi screen identifies an 8q22 gene cluster that inhibits death receptor-mediated apoptosis

“…For example, although we were unable to detect any EDD1-dependent posttranslational modification of FAS, it is possible that EDD1 and GRHL2 regulate the expression and/or stability of a key transcriptional component(s). This scenario is consistent with the nuclear localization of both genes, as well as the fact that both EDD1 and GRHL2 have previously been linked to the transcriptional regulation of other genes (22,30,32,33). Additional studies will be required to define the downstream targets that mediate the effect of EDD1 and GRHL2 on apoptosis signaling.…”

Functional Roles of the E3 Ubiquitin Ligase UBR5 in Cancer

“…This is in contrast to the previously well-defined role of GSK-3B, which is to phosphorylate b-catenin when Wnt signaling is absent, resulting in ubiquitination by the SCF ubiquitin ligase complex and subsequent b-catenin degradation (76). UBR5 also binds and stabilizes the transcriptional co-activator Myocardin, upregulating smooth muscle gene transcription independent of E3 ligase function (77).…”

EDD Inhibits ATM-mediated Phosphorylation of p53

“…EDD is also involved in transcriptional regulation and interacts with progesterone receptor as a coactivator (13). These E3 ligase-independent activities of EDD are further supported by a recent report that EDD enhances transactivation of smooth muscle-specific promoters by the myocardin family of proteins (14). Recent data from proteomics analysis also suggested that the EDD may be a substrate of ATM (15,16).…”

“…Other E3-independent functions of EDD are also identified. For example, EDD was reported to regulate progesterone receptor and myocardin through their physical interactions independently of its E3 ligase activity (13,14). In these cases, EDD functions as a transcription coactivator with progesterone receptor and myocardin.…”