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Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like.

ABSTRACT: The organizer anchors the primary embryonic axis, and balance between dorsal (organizer) and ventral domains is fundamental to body patterning. LNX (ligand of Numb protein-X) is a RING finger and four PDZ domain-containing E3 ubiquitin ligase. LNX serves as a binding platform and may have a role in cell fate determination, but its in vivo functions are unknown. Here we show that Lnx-l (Lnx-like) functions as a critical regulator of dorso-ventral axis formation in zebrafish. Depletion of Lnx-l using specific antisense morpholinos (MOs) caused strong embryonic dorsalization. We identified Bozozok (Boz, also known as Dharma or Nieuwkoid) as a binding partner and substrate of Lnx-l. Boz is a homeodomain-containing transcriptional repressor induced by canonical Wnt signalling that is critical for dorsal organizer formation. Lnx-l induced K48-linked polyubiquitylation of Boz, leading to its proteasomal degradation in human 293T cells and in zebrafish embryos. Dorsalization induced by Boz overexpression was suppressed by raising the level of Lnx-l, but Lnx-l failed to counteract dorsalization caused by mutant Boz lacking a critical motif for Lnx-l binding. Furthermore, dorsalization induced by depletion of Lnx-l was alleviated by attenuation of Boz expression. We conclude that Lnx-l modulates Boz activity to prevent the invasion of ventral regions of the embryo by organizer tissue. These studies introduce a ubiquitin ligase, Lnx-l, as a balancing modulator of axial patterning in the zebrafish embryo.

SUBMITTER: Ro H

PROVIDER: S-EPMC2759713 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like.

Ro Hyunju H Dawid Igor B IB

Nature cell biology 20090809 9

The organizer anchors the primary embryonic axis, and balance between dorsal (organizer) and ventral domains is fundamental to body patterning. LNX (ligand of Numb protein-X) is a RING finger and four PDZ domain-containing E3 ubiquitin ligase. LNX serves as a binding platform and may have a role in cell fate determination, but its in vivo functions are unknown. Here we show that Lnx-l (Lnx-like) functions as a critical regulator of dorso-ventral axis formation in zebrafish. Depletion of Lnx-l us ...[more]

PMID: 19668196

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Project description: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.

Dataset Information

Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like.

Publications

Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like.

Similar Datasets

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