The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain

Baumann, Claudia; Lindholm, Cecilia; Rimoldi, Donata; Lévy, Frédéric

doi:10.1111/j.1742-4658.2010.07698.x

Cited by 19 publications

(32 citation statements)

References 36 publications

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“…Itch knockout mice, similar to A20 and TAX1BP1 knockout mice, display severe immunological disorders associated in every organ in the body [3, 20]. In humans, Itch is strongly expressed in the gastrointestinal tract, pancreas, neuronal cells and lymphoid tissues [20].…”

Section: Discussionmentioning

confidence: 99%

Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain

Pranski

Sanford

Dalal

et al. 2012

Neuroscience Letters

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Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.

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Section: Discussionmentioning

confidence: 99%

Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain

Pranski

Sanford

Dalal

et al. 2012

Neuroscience Letters

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“…Furthermore, a 3-amino acid mutant (TAT-SH3-2M) unable to bind pSMAD3 was similarly unable to affect pSMAD3 actions (Figure 4, Figure 5, and Supplemental Figure 5). As the SNX9 SH3 domain is known to have a number of binding partners including dynamin 2, CDC42-associated kinase, WASp, and ITCH (19,20,22,23), we further addressed this issue by demonstrating a lack of SNX9 association with pSMAD1/5/8 (Supplemental Figure 1) and no inhibitory effect or effects of TAT-SH3 peptides on BMP4-, PDGF-, or EGF-responsive reporters (Figure 2A and Figure 4E) as well as SMAD2 or SMAD3 phosphorylation (Supplemental Figure 6) or serum-dependent growth ( Figure 6). …”

Section: Discussionmentioning

confidence: 99%

“…Once phosphorylated by TβRI, they most often form a heterotrimer consisting of 2 molecules of either SMAD2 or SMAD3 complexed with SMAD4 prior to undergoing nuclear translocation, where they function as comodulators of gene expression. Recently, we have extended this model and provide evidence that sorting nexin 9 (SNX9), a member of the PX/BAR subfamily of intracellular trafficking proteins (19)(20)(21)(22)(23), has an obligate role in mediating phosphorylated SMAD3 (pSMAD3) nuclear import following ligand treatment (24).…”

Section: Introductionmentioning

confidence: 99%

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

Kang¹,

Jung²,

Yin³

et al. 2017

Journal of Clinical Investigation

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ResultsA TAT-SNX9 peptide specifically blocks pSMAD3 nuclear import and profibrotic TGF-β signaling. We previously determined that SNX9 has an obligate role in mediating profibrotic TGF-β signaling dependent upon SMAD3 (24). In order to investigate colony formation, and cell migration; and (b) fibrotic changes associated with the bleomycin (BLM) and adenoviral models of lung fibrosis. Moreover, a 3-amino acid point mutant of the TAT-SNX9 peptide unable to bind pSMAD3 was ineffective in analogous in vitro and in vivo assays. Lysates from AKR-2B cells untreated (-) or stimulated (+) for 45 minutes with 5 ng/ml TGF-β were incubated with GST beads or the indicated fusion proteins immobilized on GST beads. Bound proteins were eluted and assessed by Western blot analysis for pSMAD3 or pSMAD2. Cell lysate reflects signal obtained from 10 μg total protein; the slower migrating band in the pSMAD3 lane is a nonspecific protein (right, representative of 3 separate experiments). (B) AKR-2B cells were transduced for 90 minutes with the indicated concentration of TAT peptide. Following washing and 1 hour TGF-β (5 ng/ml) treatment, nuclear fractions were isolated and assessed by Western blot analysis for pSMAD2, pSMAD3, or histone deacetylase 1 (HDAC) (left, representative of 3 separate experiments). Quantitation of nuclear pSMADs was performed with ImageJ software (NIH) and represents the mean ± SEM of 3 experiments (right). (C) Left panels: AKR-2B cells were incubated with vehicle (top panels) or transduced (bottom panels) as in B with TAT-SH3 or TAT-LC (1.8 μM). Following treatment with or without TGF-β (5 ng/ml) for 1 hour, immunofluorescence for SMAD3 or the HA-tagged TAT peptide was performed as described in Methods and nuclei were stained with DAPI. Upper and lower panels show 2 distinct microscopic fields for each condition. Original magnification in C: ×100. Right: quantitation of nuclear SMAD3 from 30 cells in each of 3 experiments. *P < 0.05; **P < 0.005; ***P < 0.0005, 1-way ANOVA followed by Dunnett's multiple comparisons test. The Journal of Clinical Investigation R E S E A R C H A R T I C L E2 5 4 3 jci.orgVolume 127 Number 7 July 2017 sion of this fragment could act in trans as a dominant inhibitor of pSMAD3 nuclear uptake. To address this issue, constructs were prepared expressing either the SNX9 SH3 or LC domains fused to the cell-penetrating TAT peptide from HIV (39). Subsequent to TAT peptide transduction, cultures were treated with TGF-β and nuclear accumulation of pSMAD2 or pSMAD3 determined. As shown in Figure 1B and Supplemental Figure 2, while the TAT-SH3 peptide inhibited nuclear import of pSMAD3 in a dose-dependent manner and increased cytoplasmic pSMAD3, it had no effect on pSMAD2. Furthermore, consistent with the inability of the LC domain to bind receptorregulated SMADs (R-SMADs) ( Figure 1A), it was similarly ineffective in modulating nuclear translocation ( Figure 1B). These biochemical findings were independently confirmed using immunofluorescence ( Figure 1C and Supplemental Figure 3). While t...

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“…Components of the endocytic machinery, including adapter or accessory molecules, are also the targets of Itch . Sorting nexin 9, which is involved in the clathrin‐mediated endocytic pathway, binds to Pro‐rich domain of Itch through its Src‐homology 3 (SH3) domain, resulting in its ubiquitylation and degradation by Itch .…”

Section: Subcellular Localizationmentioning

confidence: 99%

The E3 ligase Itch in immune regulation and beyond

Aki

Zhang

Liu

2015

Immunological Reviews

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Itch or itchy E3 ubiquitin ligase was initially discovered by genetic studies on the mouse coat color changes, and its deletion results in an itchy phenotype with constant skin scratching and multi-organ inflammation. It is a member of the homologous to E6-associated protein C-terminus (HECT)-type family of E3 ligases, with the protein-interacting WW-domains for the recruitment of substrate and the HECT domain for the transfer of ubiquitin to the substrate. Since its discovery, numerous studies have demonstrated that Itch is involved in the control of many aspects of immune responses including T-cell activation and tolerance and T-helper cell differentiation. Itch is also implicated in other biological contexts such as tumorigenesis, development, and stress responses. Many signaling pathways are regulated by Itch-promoted ubiquitylation of diverse target proteins. Itch is also involved in human diseases. Here, we discuss the major progress in understanding the biological significance of Itch-promoted protein ubiquitylation in the immune and other systems and in Itch-mediated regulation of signal transduction.

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The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain

Cited by 19 publications

References 36 publications

Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain

Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

The E3 ligase Itch in immune regulation and beyond

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