RING domain dimerization is essential for RNF4 function

Liew, C.W.; Sun, Hongbo; Hunter, Tony; Day, Catherine L.

doi:10.1042/bj20100957

Cited by 81 publications

(64 citation statements)

References 22 publications

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“…Immunoprecipitated Arkadia and RNF4 proteins from transfected 293T cells were assayed for their ubiquitin ligase activities against an HA-di-SUMO2-GST substrate (Fig. 1A) (31). Arkadia indeed promoted the ubiquitylation of di-SUMO2, in a manner similar to RNF4 although less strongly, and such modification was specifically dependent on both the SUMO-binding and the RING domains of Arkadia (Fig.…”

Section: Resultsmentioning

confidence: 96%

“…Flag-tagged proteins were each precipitated from 293T cell lysates with 20 l (bed volume) of anti-Flag antibody-agarose beads. The washed beads were used as the source of E3 in two parallel sets of ubiquitin ligase assays (14,31). The reaction mixture included either hemagglutinin (HA)-tagged ubiquitin for detection of nonspecific ubiquitin conjugation or a combination of untagged ubiquitin with HA-tagged di-SUMO2-GST fusion protein for the detection of SUMO-targeted ubiquitin ligase activity.…”

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confidence: 99%

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Multiple Arkadia/RNF111 Structures Coordinate Its Polycomb Body Association and Transcriptional Control

Sun

Liu

Hunter

2014

Molecular and Cellular Biology

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The RING domain protein Arkadia/RNF111 is a ubiquitin ligase in the transforming growth factor ␤ (TGF␤) pathway. We previously identified Arkadia as a small ubiquitin-like modifier (SUMO)-binding protein with clustered SUMO-interacting motifs (SIMs) that together form a SUMO-binding domain (SBD). However, precisely how SUMO interaction contributes to the function of Arkadia was not resolved. Through analytical molecular and cell biology, we found that the SIMs share redundant function with Arkadia's M domain, a region distinguishing Arkadia from its paralogs ARKL1/ARKL2 and the prototypical SUMO-targeted ubiquitin ligase (STUbL) RNF4. The SIMs and M domain together promote both Arkadia's colocalization with CBX4/Pc2, a component of Polycomb bodies, and the activation of a TGF␤ pathway transcription reporter. Transcriptome profiling through RNA sequencing showed that Arkadia can both promote and inhibit gene expression, indicating that Arkadia's activity in transcriptional control may depend on the epigenetic context, defined by Polycomb repressive complexes and DNA methylation. Posttranslational modification by the small ubiquitin-like modifier (SUMO) family proteins targets a large number of proteins in multiple cellular processes, such as transcription, DNA replication, and DNA damage repair, in eukaryotes (1-3). As in other forms of posttranslational modification, SUMO is recognized by a specific structure, the SUMO-interacting motif (SIM). Thus, sumoylation promotes a regulated protein complex assembly through a direct interaction between SUMO and SIM (4, 5). Sumoylation activity has been found in microscopically visible nuclear bodies (NBs), such as promyelocytic leukemia (PML) and Polycomb (Pc) bodies (6-9). These nuclear bodies contain sumoylation enzymes, sumoylated proteins, and SIM-containing proteins (10-12), suggesting that both SUMO conjugation and SUMO binding regulate the dynamic formation of nuclear bodies (4, 6).Recently, we applied a computational string search to SIM identification and found a group of proteins all containing clustered SIMs (13). This includes a RING domain protein called Arkadia, or RNF111, that has a cluster of at least three SIMs in a region between residues 253 and 415 that we now call a SUMObinding domain (SBD). This work echoed our previous finding of the SUMO-targeted ubiquitin ligases (STUbLs) RNF4 and its fission yeast homologs, Rfp1 and Rfp2, all containing multiple SIMs closely positioned as a cluster (14). Sequence gazing on Arkadia has also led to similar findings by other groups (15, 16). Among the three Arkadia SIMs, SIM3 is the most critical, and its particular sequence (VVDLT) resembles a VIDLT-type high-affinity SIM (17); SIM1 and SIM3 together contribute to the bulk of SUMO affinity of the entire SBD, and their simultaneous mutation results in complete loss of SUMO binding (13).Arkadia also has a pair of partial paralogs found in all vertebrates, namely, Arkadia-like 1 (ARKL1) and 2 (ARKL2, or RNF165) (18). They resemble the N-and C-terminal half of Arkadia,...

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Section: Resultsmentioning

confidence: 96%

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confidence: 99%

Multiple Arkadia/RNF111 Structures Coordinate Its Polycomb Body Association and Transcriptional Control

Sun

Liu

Hunter

2014

Molecular and Cellular Biology

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“…S4). Because RING-fingers are often implicated in mediating proteinprotein interactions, in particular dimerization (23), and isolated CesA RING-fingers have been shown to form homo-and heterodimers in vitro (24), it is likely that these domains stabilize membraneembedded CesA oligomers.…”

Section: Pttcesa8's N-terminal Cytosolic Domain Is Dispensable For Camentioning

confidence: 99%

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Purushotham

Cho

Díaz-Moreno

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.biopolymer | cellulose | glycosyltransferase | plant cell wall | membrane transport

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“…Many RING-type E3 ligases dimerize via their RING domains (24,45,47,48). We expressed MARCH1 with a RING-less variant and performed coimmunoprecipitation experiments (Fig.…”

Section: March1 Forms Homo-and Heterodimersmentioning

confidence: 99%

Autoregulation of MARCH1 Expression by Dimerization and Autoubiquitination

Bourgeois-Daigneault

Thibodeau

2012

The Journal of Immunology

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Some members of the membrane-associated RING-CH family of E3 ubiquitin ligases (MARCHs) are membrane-bound and target major players of the immune response. MARCH1 ubiquitinates and downregulates MHC class II expression in APCs. It is induced by IL-10 and despite a strong increase in mRNA expression in human primary monocytes, the protein remains hardly detectable. To gain insights into the posttranslational regulation of MARCH1, we investigated whether its expression is itself regulated by ubiquitination. Our results demonstrate that MARCH1 is ubiquitinated in transfected human cell lines. Polyubiquitin chain-specific Abs revealed the presence of K48-linked polyubiquitin chains. A mutant devoid of lysine residues in the N- and C-terminal regions was less ubiquitinated and had a prolonged half-life. Reduced ubiquitination was also observed for an inactive mutated form of the molecule (M1WI), suggesting that MARCH1 is capable of autoubiquitination. Immunoprecipitation and energy transfer experiments demonstrated that MARCH1 homodimerizes and also forms heterodimers with others family members. Coexpression of MARCH1 decreased the protein levels of the inactive M1WI, suggesting a transubiquitination process. Taken together, our results suggest that MARCH1 may regulate its own expression through dimerization and autoubiquitination.

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RING domain dimerization is essential for RNF4 function

Cited by 81 publications

References 22 publications

Multiple Arkadia/RNF111 Structures Coordinate Its Polycomb Body Association and Transcriptional Control

Multiple Arkadia/RNF111 Structures Coordinate Its Polycomb Body Association and Transcriptional Control

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Autoregulation of MARCH1 Expression by Dimerization and Autoubiquitination

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