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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

Mass spectrometry analysis identified several putative Glis3-interacting proteins and Glis3 post-translational modifications.A. Schematic representation of dual tagged Glis3 N-terminus stably expressed in HEK293F cells used for the tandem affinity pulldown and GeLC-MS analysis. Posttranslationally modified sites identified by MS analysis are indicated. N = N-terminal conserved region; ZFD = zinc finger domain; TAD = transactivation domain. B. Representative Coomassie blue stained gel showing proteins immunoprecipitated by a tandem FLAG/HA pulldown. Protein complexes from HEK293F cells stably expressing pIRES2-EGFP-FLAG-HA empty vector or pIRES2-EGFP-FLAG-Glis3-ΔC480-HA were immobilized using anti-M2 FLAG and anti-HA antibody conjugated to agarose or magnetic beads, respectively. Eluted proteins were separated by SDS-PAGE and the gel was stained with Coomassie blue and analysed by GeLC-MS. Arrows indicate bands corresponding to approximate molecular weight of Glis3 interacting proteins identified by MS. * indicates over-expressed FLAG-Glis3-ΔC480-HA. C. Table showing posttranslational modifications of Glis3-ΔC480 identified by MS analysis. Modified amino acid positions are underlined. Specificity could not be determined between phosphorylation at S305, 307, or 310. Me = methylation; PO4 = phosphorylation.
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pone.0131303.g001: Mass spectrometry analysis identified several putative Glis3-interacting proteins and Glis3 post-translational modifications.A. Schematic representation of dual tagged Glis3 N-terminus stably expressed in HEK293F cells used for the tandem affinity pulldown and GeLC-MS analysis. Posttranslationally modified sites identified by MS analysis are indicated. N = N-terminal conserved region; ZFD = zinc finger domain; TAD = transactivation domain. B. Representative Coomassie blue stained gel showing proteins immunoprecipitated by a tandem FLAG/HA pulldown. Protein complexes from HEK293F cells stably expressing pIRES2-EGFP-FLAG-HA empty vector or pIRES2-EGFP-FLAG-Glis3-ΔC480-HA were immobilized using anti-M2 FLAG and anti-HA antibody conjugated to agarose or magnetic beads, respectively. Eluted proteins were separated by SDS-PAGE and the gel was stained with Coomassie blue and analysed by GeLC-MS. Arrows indicate bands corresponding to approximate molecular weight of Glis3 interacting proteins identified by MS. * indicates over-expressed FLAG-Glis3-ΔC480-HA. C. Table showing posttranslational modifications of Glis3-ΔC480 identified by MS analysis. Modified amino acid positions are underlined. Specificity could not be determined between phosphorylation at S305, 307, or 310. Me = methylation; PO4 = phosphorylation.

Mentions: To determine the potential importance of the 500 aa N-terminal region of Glis3 in regulating the function of the protein, tandem affinity purification (TAP) followed by gel-enhanced liquid chromatography mass spectrometry was used to identify partners that may interact with the region. The N-terminal region up to amino acid 480, including an N-terminal 3xFLAG and a C-terminal HA epitope (Fig 1A), was stably expressed in HEK293-F cells. Subsequently, TAP was performed on cell lysates and eluted proteins were separated by SDS-PAGE and analyzed by Coomassie blue staining. In addition to Glis3 (indicated by an * in Fig 1B), GeLC-MS identified a number of proteins that potentially interact with the Glis3 N-terminus, including SUFU, a previously verified interacting partner [19]. Other putative partners of interest that were identified with high confidence in more than one experiment included protein arginine methyltransferase 5 (Prmt5) and Itchy E3 ubiquitin protein ligase (Itch). S2 Table contains a full list of co-immunoprecipitated proteins. Further, GeLC-MS identified several putative post-translational modifications (PTM), two phosphorylation and two methylation sites, in the Glis3 N-terminus (Fig 1C).


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)

Mass spectrometry analysis identified several putative Glis3-interacting proteins and Glis3 post-translational modifications.A. Schematic representation of dual tagged Glis3 N-terminus stably expressed in HEK293F cells used for the tandem affinity pulldown and GeLC-MS analysis. Posttranslationally modified sites identified by MS analysis are indicated. N = N-terminal conserved region; ZFD = zinc finger domain; TAD = transactivation domain. B. Representative Coomassie blue stained gel showing proteins immunoprecipitated by a tandem FLAG/HA pulldown. Protein complexes from HEK293F cells stably expressing pIRES2-EGFP-FLAG-HA empty vector or pIRES2-EGFP-FLAG-Glis3-ΔC480-HA were immobilized using anti-M2 FLAG and anti-HA antibody conjugated to agarose or magnetic beads, respectively. Eluted proteins were separated by SDS-PAGE and the gel was stained with Coomassie blue and analysed by GeLC-MS. Arrows indicate bands corresponding to approximate molecular weight of Glis3 interacting proteins identified by MS. * indicates over-expressed FLAG-Glis3-ΔC480-HA. C. Table showing posttranslational modifications of Glis3-ΔC480 identified by MS analysis. Modified amino acid positions are underlined. Specificity could not be determined between phosphorylation at S305, 307, or 310. Me = methylation; PO4 = phosphorylation.
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pone.0131303.g001: Mass spectrometry analysis identified several putative Glis3-interacting proteins and Glis3 post-translational modifications.A. Schematic representation of dual tagged Glis3 N-terminus stably expressed in HEK293F cells used for the tandem affinity pulldown and GeLC-MS analysis. Posttranslationally modified sites identified by MS analysis are indicated. N = N-terminal conserved region; ZFD = zinc finger domain; TAD = transactivation domain. B. Representative Coomassie blue stained gel showing proteins immunoprecipitated by a tandem FLAG/HA pulldown. Protein complexes from HEK293F cells stably expressing pIRES2-EGFP-FLAG-HA empty vector or pIRES2-EGFP-FLAG-Glis3-ΔC480-HA were immobilized using anti-M2 FLAG and anti-HA antibody conjugated to agarose or magnetic beads, respectively. Eluted proteins were separated by SDS-PAGE and the gel was stained with Coomassie blue and analysed by GeLC-MS. Arrows indicate bands corresponding to approximate molecular weight of Glis3 interacting proteins identified by MS. * indicates over-expressed FLAG-Glis3-ΔC480-HA. C. Table showing posttranslational modifications of Glis3-ΔC480 identified by MS analysis. Modified amino acid positions are underlined. Specificity could not be determined between phosphorylation at S305, 307, or 310. Me = methylation; PO4 = phosphorylation.
Mentions: To determine the potential importance of the 500 aa N-terminal region of Glis3 in regulating the function of the protein, tandem affinity purification (TAP) followed by gel-enhanced liquid chromatography mass spectrometry was used to identify partners that may interact with the region. The N-terminal region up to amino acid 480, including an N-terminal 3xFLAG and a C-terminal HA epitope (Fig 1A), was stably expressed in HEK293-F cells. Subsequently, TAP was performed on cell lysates and eluted proteins were separated by SDS-PAGE and analyzed by Coomassie blue staining. In addition to Glis3 (indicated by an * in Fig 1B), GeLC-MS identified a number of proteins that potentially interact with the Glis3 N-terminus, including SUFU, a previously verified interacting partner [19]. Other putative partners of interest that were identified with high confidence in more than one experiment included protein arginine methyltransferase 5 (Prmt5) and Itchy E3 ubiquitin protein ligase (Itch). S2 Table contains a full list of co-immunoprecipitated proteins. Further, GeLC-MS identified several putative post-translational modifications (PTM), two phosphorylation and two methylation sites, in the Glis3 N-terminus (Fig 1C).

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