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HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity.

ZeRuth GT, Williams JG, Cole YC, Jetten AM - PLoS ONE (2015)

Bottom Line: However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation.Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity.This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

No MeSH data available.


Related in: MedlinePlus

Itch targets Glis3 for degradation through the 26s proteasome.A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.
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pone.0131303.g006: Itch targets Glis3 for degradation through the 26s proteasome.A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.

Mentions: Given that Itch interacted with and promoted Glis3 ubiquitination, we examined whether this targeted Glis3 for proteolytic degradation. We co-transfected HEK293T cells with FLAG-Glis3 and Myc-tagged-Itch, -Smurf2, -NEDD4, or empty vector and monitored Glis3 protein stability by Western blot analysis after timed cycloheximide treatment. Co-expression of Itch significantly reduced murine Glis3 protein stability relative to cells transfected with empty vector and internal controls (Fig 5A). Murine Itch was likewise capable of promoting the degradation of human GLIS3 as well as Danio rerio glis3 suggesting that the function is likely conserved throughout vertebrate evolution (Fig 5B). As expected, Smurf2 and NEDD4 did not have any significant effect on Glis3 protein stability (Fig 5C and 5D). Protein ubiquitination typically promotes degradation by targeting the ubiquitinated proteins to the 26S proteasome or less frequently, the lysosome [32]. To determine by which mechanism Itch promoted Glis3 degradation, the effect of lysosomal or proteasomal inhibitors on Glis3 protein degradation was examined. As seen in Fig 6A, the general proteasome inhibitor MG132 significantly reduced degradation of Glis3 by Itch, whereas the lysosomal inhibitors, NH4Cl and chloroquine, failed to protect Glis3 against Itch-directed degradation as did leupeptin, an inhibitor of trypsin-like and cysteine proteases (Fig 6A). Furthermore, while MG132 stabilized Glis3 protein expression in the presence of exogenous Itch, it had no observable effect on Glis3 co-expressed with the catalytically inactive Itch mutant (Fig 6B). Collectively, these results suggest that Itch targets Glis3 for degradation via the 26S proteasome.


HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity.

ZeRuth GT, Williams JG, Cole YC, Jetten AM - PLoS ONE (2015)

Itch targets Glis3 for degradation through the 26s proteasome.A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4493090&req=5

pone.0131303.g006: Itch targets Glis3 for degradation through the 26s proteasome.A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.
Mentions: Given that Itch interacted with and promoted Glis3 ubiquitination, we examined whether this targeted Glis3 for proteolytic degradation. We co-transfected HEK293T cells with FLAG-Glis3 and Myc-tagged-Itch, -Smurf2, -NEDD4, or empty vector and monitored Glis3 protein stability by Western blot analysis after timed cycloheximide treatment. Co-expression of Itch significantly reduced murine Glis3 protein stability relative to cells transfected with empty vector and internal controls (Fig 5A). Murine Itch was likewise capable of promoting the degradation of human GLIS3 as well as Danio rerio glis3 suggesting that the function is likely conserved throughout vertebrate evolution (Fig 5B). As expected, Smurf2 and NEDD4 did not have any significant effect on Glis3 protein stability (Fig 5C and 5D). Protein ubiquitination typically promotes degradation by targeting the ubiquitinated proteins to the 26S proteasome or less frequently, the lysosome [32]. To determine by which mechanism Itch promoted Glis3 degradation, the effect of lysosomal or proteasomal inhibitors on Glis3 protein degradation was examined. As seen in Fig 6A, the general proteasome inhibitor MG132 significantly reduced degradation of Glis3 by Itch, whereas the lysosomal inhibitors, NH4Cl and chloroquine, failed to protect Glis3 against Itch-directed degradation as did leupeptin, an inhibitor of trypsin-like and cysteine proteases (Fig 6A). Furthermore, while MG132 stabilized Glis3 protein expression in the presence of exogenous Itch, it had no observable effect on Glis3 co-expressed with the catalytically inactive Itch mutant (Fig 6B). Collectively, these results suggest that Itch targets Glis3 for degradation via the 26S proteasome.

Bottom Line: However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation.Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity.This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Section, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, United States of America.

ABSTRACT
The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.

No MeSH data available.


Related in: MedlinePlus