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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

Glis3 associates with Itch, Smurf2, and NEDD4.A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-PY461 mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.
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pone.0131303.g002: Glis3 associates with Itch, Smurf2, and NEDD4.A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-PY461 mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.

Mentions: Since several HECT E3 ubiquitin ligases were identified several times as possible Glis3 interacting proteins, we focused our study on the further characterization of these interactions. Co-immunoprecipitation was performed using HEK293T cells co-expressing FLAG-tagged Glis3 (p-CMV-3xFLAG-Glis3) and Myc-tagged Itch, Smurf2, or NEDD4, the putative WW-domain interacting proteins identified with the highest level of confidence in the Y2H analysis. Since co-expression of Itch with Glis3 resulted in substantially decreased levels of Glis3 protein, a catalytically inactive Itch mutant (C832G) was used to study the interaction [26]. Western blot analysis showed that Itch and Smurf2 co-immunoprecipitated with Glis3 (Fig 2A and 2B) although the interaction appeared much weaker with Smurf2 than with Itch. NEDD4 failed to detectably interact with Glis3 by co-IP (Fig 2C). WW-domains are known to interact with proteins through the recognition of proline-rich motifs, including PPLP or PPxY motifs or proline residues preceded by phosphorylated serine or threonine (pSP or pTP motifs) [27–30]. Examination of the N-terminal sequence of Glis3 revealed a single PPxY motif located between aa 458–461 (PY461) as well as 18 putative p(S/T)P motifs. The Glis3 PY461 motif was conserved across all species examined ranging from fishes to humans (S1A Fig). Mutation of the PY461 motif dramatically reduced Glis3 interaction with Itch and Smurf2 (Fig 2A and 2B).


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)

Glis3 associates with Itch, Smurf2, and NEDD4.A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-PY461 mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.
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Related In: Results  -  Collection

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pone.0131303.g002: Glis3 associates with Itch, Smurf2, and NEDD4.A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective PY461 mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-PY461 mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.
Mentions: Since several HECT E3 ubiquitin ligases were identified several times as possible Glis3 interacting proteins, we focused our study on the further characterization of these interactions. Co-immunoprecipitation was performed using HEK293T cells co-expressing FLAG-tagged Glis3 (p-CMV-3xFLAG-Glis3) and Myc-tagged Itch, Smurf2, or NEDD4, the putative WW-domain interacting proteins identified with the highest level of confidence in the Y2H analysis. Since co-expression of Itch with Glis3 resulted in substantially decreased levels of Glis3 protein, a catalytically inactive Itch mutant (C832G) was used to study the interaction [26]. Western blot analysis showed that Itch and Smurf2 co-immunoprecipitated with Glis3 (Fig 2A and 2B) although the interaction appeared much weaker with Smurf2 than with Itch. NEDD4 failed to detectably interact with Glis3 by co-IP (Fig 2C). WW-domains are known to interact with proteins through the recognition of proline-rich motifs, including PPLP or PPxY motifs or proline residues preceded by phosphorylated serine or threonine (pSP or pTP motifs) [27–30]. Examination of the N-terminal sequence of Glis3 revealed a single PPxY motif located between aa 458–461 (PY461) as well as 18 putative p(S/T)P motifs. The Glis3 PY461 motif was conserved across all species examined ranging from fishes to humans (S1A Fig). Mutation of the PY461 motif dramatically reduced Glis3 interaction with Itch and Smurf2 (Fig 2A and 2B).

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