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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

The PY461 motif and the ZFD are required for Itch-mediated degradation of Glis3.A. HEK293T cells were transfected with the indicated FLAG-Glis3 construct and Myc-Itch or empty vector. After 48 h, the cells were harvested and proteins examined by Western blot analysis using an anti-M2 FLAG-HRP or anti-GAPDH antibody. Bands were quantified as described in Materials and Methods. The average intensity of the bands of the Itch plus samples (n = 3) were normalized against that of GAPDH and plotted relative to the average intensity of the bands of the Itch minus samples. Representative images are shown below the histogram. B. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc-Itch or empty vector. After 48 h, cells were harvested and proteins examined by Western blot analysis with anti-M2 FLAG-HRP, anti-Myc, and goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG-Glis3 or the indicated ZF mutant along with p-mIP-696-Luc luciferase reporter. After 48 h cells were harvested and assayed for luciferase and ß-galactosidase activity and the normalized relative luciferase values (nRLU) were plotted. Each bar represents the mean +/- SEM. * indicates statistically different value from corresponding Myc empty vector control p < 0.02. # indicates statistically different value from corresponding WT Glis3 control p < 0.02. D. HEK293T cells were transfected with FLAG-Glis3 or the indicated zinc finger mutant along with CMV-HA-Ubiquitin and Myc Itch or empty vector. Cells were treated with 10 μM MG132 for 7 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins examined by Western blot analysis using anti-M2 FLAG-HRP, anti-HA, and anti-Myc, and goat anti-mouse-HRP antibodies. E. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc Itch or empty vector. Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-M2 FLAG antibody followed by staining with anti-mouse Alexa 488. Protein localization was examined by fluorescence microscopy.
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pone.0131303.g007: The PY461 motif and the ZFD are required for Itch-mediated degradation of Glis3.A. HEK293T cells were transfected with the indicated FLAG-Glis3 construct and Myc-Itch or empty vector. After 48 h, the cells were harvested and proteins examined by Western blot analysis using an anti-M2 FLAG-HRP or anti-GAPDH antibody. Bands were quantified as described in Materials and Methods. The average intensity of the bands of the Itch plus samples (n = 3) were normalized against that of GAPDH and plotted relative to the average intensity of the bands of the Itch minus samples. Representative images are shown below the histogram. B. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc-Itch or empty vector. After 48 h, cells were harvested and proteins examined by Western blot analysis with anti-M2 FLAG-HRP, anti-Myc, and goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG-Glis3 or the indicated ZF mutant along with p-mIP-696-Luc luciferase reporter. After 48 h cells were harvested and assayed for luciferase and ß-galactosidase activity and the normalized relative luciferase values (nRLU) were plotted. Each bar represents the mean +/- SEM. * indicates statistically different value from corresponding Myc empty vector control p < 0.02. # indicates statistically different value from corresponding WT Glis3 control p < 0.02. D. HEK293T cells were transfected with FLAG-Glis3 or the indicated zinc finger mutant along with CMV-HA-Ubiquitin and Myc Itch or empty vector. Cells were treated with 10 μM MG132 for 7 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins examined by Western blot analysis using anti-M2 FLAG-HRP, anti-HA, and anti-Myc, and goat anti-mouse-HRP antibodies. E. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc Itch or empty vector. Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-M2 FLAG antibody followed by staining with anti-mouse Alexa 488. Protein localization was examined by fluorescence microscopy.

Mentions: Since Itch interacts with and ubiquitinates Glis3, it was of interest to determine which regions were necessary for Itch-directed degradation of Glis3. Mutation of the PY461 motif stabilized Glis3 in the presence of Itch as did deletion of the N-terminus (ΔN496), which includes the PY461 motif (Fig 7A). In contrast, deletion of the C-terminus beyond the ZFD (ΔC653) had a negligible effect on Itch-mediated degradation of Glis3. These observations are consistent with our data showing that interaction of Itch with the Glis3 N-terminus, but not its C-terminus, which includes the transactivation domain of Glis3, is required for the Itch-mediated degradation of Glis3. Interestingly, even though the PY461 motif is central to Glis3 degradation by Itch, the Glis3-ΔC480 mutant, which lacks the ZFD, was stable in the presence of Itch despite its interaction with and ubiquitination by Itch. These findings indicated that in addition to the PY461 motif, the ZFD played a role in Itch-mediated destruction. To further analyze the requirement of the ZFD in the Itch dependent degradation of Glis3, the effect of disruption of the tetrahedral configuration of each zinc finger on Glis3 stability was examined. As seen in Fig 7B, mutation of either ZF3 or ZF4, but not ZF1, ZF2, or ZF5 inhibited the degradation of Glis3 by Itch. Since DNA binding and transactivation was ablated in all five zinc finger mutants ([33] and Fig 7C), loss of these two functions could not account for the requirement of ZF3 and ZF4 in Itch-mediated degradation. Additionally, ZF3 and ZF4 mutants were capable of interacting with Itch (data not shown) and were polyubiquitinated by Itch (Fig 7D) suggesting that the ZF mutations did not change the ubiquitination of Glis3 by Itch, but instead might affect the recognition of Itch-modified Glis3 by the proteasome. The Glis3 ZFD has been shown to be required for nuclear localization [33]; however, Glis3 nuclear localization was not affected by the ZF3 or ZF4 mutations (Fig 7E) suggesting that the observed stability of the mutants in the presence of Itch is not due to exclusion from the nucleus. Collectively, these data suggest that the ZFD of Glis3 is required for Itch-mediated degradation of the protein in addition to the N-terminal PY461 motif in a manner that is not dependent on Glis3 nuclear localization or DNA binding.


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)

The PY461 motif and the ZFD are required for Itch-mediated degradation of Glis3.A. HEK293T cells were transfected with the indicated FLAG-Glis3 construct and Myc-Itch or empty vector. After 48 h, the cells were harvested and proteins examined by Western blot analysis using an anti-M2 FLAG-HRP or anti-GAPDH antibody. Bands were quantified as described in Materials and Methods. The average intensity of the bands of the Itch plus samples (n = 3) were normalized against that of GAPDH and plotted relative to the average intensity of the bands of the Itch minus samples. Representative images are shown below the histogram. B. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc-Itch or empty vector. After 48 h, cells were harvested and proteins examined by Western blot analysis with anti-M2 FLAG-HRP, anti-Myc, and goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG-Glis3 or the indicated ZF mutant along with p-mIP-696-Luc luciferase reporter. After 48 h cells were harvested and assayed for luciferase and ß-galactosidase activity and the normalized relative luciferase values (nRLU) were plotted. Each bar represents the mean +/- SEM. * indicates statistically different value from corresponding Myc empty vector control p < 0.02. # indicates statistically different value from corresponding WT Glis3 control p < 0.02. D. HEK293T cells were transfected with FLAG-Glis3 or the indicated zinc finger mutant along with CMV-HA-Ubiquitin and Myc Itch or empty vector. Cells were treated with 10 μM MG132 for 7 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins examined by Western blot analysis using anti-M2 FLAG-HRP, anti-HA, and anti-Myc, and goat anti-mouse-HRP antibodies. E. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc Itch or empty vector. Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-M2 FLAG antibody followed by staining with anti-mouse Alexa 488. Protein localization was examined by fluorescence microscopy.
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Related In: Results  -  Collection

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pone.0131303.g007: The PY461 motif and the ZFD are required for Itch-mediated degradation of Glis3.A. HEK293T cells were transfected with the indicated FLAG-Glis3 construct and Myc-Itch or empty vector. After 48 h, the cells were harvested and proteins examined by Western blot analysis using an anti-M2 FLAG-HRP or anti-GAPDH antibody. Bands were quantified as described in Materials and Methods. The average intensity of the bands of the Itch plus samples (n = 3) were normalized against that of GAPDH and plotted relative to the average intensity of the bands of the Itch minus samples. Representative images are shown below the histogram. B. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc-Itch or empty vector. After 48 h, cells were harvested and proteins examined by Western blot analysis with anti-M2 FLAG-HRP, anti-Myc, and goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG-Glis3 or the indicated ZF mutant along with p-mIP-696-Luc luciferase reporter. After 48 h cells were harvested and assayed for luciferase and ß-galactosidase activity and the normalized relative luciferase values (nRLU) were plotted. Each bar represents the mean +/- SEM. * indicates statistically different value from corresponding Myc empty vector control p < 0.02. # indicates statistically different value from corresponding WT Glis3 control p < 0.02. D. HEK293T cells were transfected with FLAG-Glis3 or the indicated zinc finger mutant along with CMV-HA-Ubiquitin and Myc Itch or empty vector. Cells were treated with 10 μM MG132 for 7 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins examined by Western blot analysis using anti-M2 FLAG-HRP, anti-HA, and anti-Myc, and goat anti-mouse-HRP antibodies. E. HEK293T cells were transfected with FLAG Glis3 or the indicated zinc finger mutant and Myc Itch or empty vector. Cells were fixed in 4% paraformaldehyde, permeabilized, and stained with anti-M2 FLAG antibody followed by staining with anti-mouse Alexa 488. Protein localization was examined by fluorescence microscopy.
Mentions: Since Itch interacts with and ubiquitinates Glis3, it was of interest to determine which regions were necessary for Itch-directed degradation of Glis3. Mutation of the PY461 motif stabilized Glis3 in the presence of Itch as did deletion of the N-terminus (ΔN496), which includes the PY461 motif (Fig 7A). In contrast, deletion of the C-terminus beyond the ZFD (ΔC653) had a negligible effect on Itch-mediated degradation of Glis3. These observations are consistent with our data showing that interaction of Itch with the Glis3 N-terminus, but not its C-terminus, which includes the transactivation domain of Glis3, is required for the Itch-mediated degradation of Glis3. Interestingly, even though the PY461 motif is central to Glis3 degradation by Itch, the Glis3-ΔC480 mutant, which lacks the ZFD, was stable in the presence of Itch despite its interaction with and ubiquitination by Itch. These findings indicated that in addition to the PY461 motif, the ZFD played a role in Itch-mediated destruction. To further analyze the requirement of the ZFD in the Itch dependent degradation of Glis3, the effect of disruption of the tetrahedral configuration of each zinc finger on Glis3 stability was examined. As seen in Fig 7B, mutation of either ZF3 or ZF4, but not ZF1, ZF2, or ZF5 inhibited the degradation of Glis3 by Itch. Since DNA binding and transactivation was ablated in all five zinc finger mutants ([33] and Fig 7C), loss of these two functions could not account for the requirement of ZF3 and ZF4 in Itch-mediated degradation. Additionally, ZF3 and ZF4 mutants were capable of interacting with Itch (data not shown) and were polyubiquitinated by Itch (Fig 7D) suggesting that the ZF mutations did not change the ubiquitination of Glis3 by Itch, but instead might affect the recognition of Itch-modified Glis3 by the proteasome. The Glis3 ZFD has been shown to be required for nuclear localization [33]; however, Glis3 nuclear localization was not affected by the ZF3 or ZF4 mutations (Fig 7E) suggesting that the observed stability of the mutants in the presence of Itch is not due to exclusion from the nucleus. Collectively, these data suggest that the ZFD of Glis3 is required for Itch-mediated degradation of the protein in addition to the N-terminal PY461 motif in a manner that is not dependent on Glis3 nuclear localization or DNA binding.

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