Limits...
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 directly interacts with Glis3 through its WW-domains.A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WWI containing only the four WW-domains. Co-immunoprecipitation was performed as described in Fig 2A–2C. B. FLAG-Glis3 or the PY461 mutant and Myc Empty or Myc-Itch were transcribed and translated in vitro and an in vitro pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the PY461 motif of Glis3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4493090&req=5

pone.0131303.g003: Itch directly interacts with Glis3 through its WW-domains.A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WWI containing only the four WW-domains. Co-immunoprecipitation was performed as described in Fig 2A–2C. B. FLAG-Glis3 or the PY461 mutant and Myc Empty or Myc-Itch were transcribed and translated in vitro and an in vitro pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the PY461 motif of Glis3.

Mentions: Members of the Nedd4/Rsp5p family contain in addition to the centrally located WW-domains, an N-terminal C2 domain and a C-terminal HECT domain [31]. To determine whether these domains influence the interaction between Itch and Glis3, IP was performed with Glis3 and Itch lacking its C2 or HECT domains. The results showed that deletion of either the C2 or the HECT domain (Fig 3A) had little effect on the interaction between Itch and Glis3 and therefore are not required for the interaction. In addition, the four WW domains from Itch were sufficient for interaction with Glis3 although the interaction was considerably weaker than observed with full-length Itch and might be due to differences in the WW domain conformation and therefore affinity for Glis3 between full-length Itch and the Itch WW domain only. The WW-domains of Smurf2 and NEDD4 were also sufficient for interaction with Glis3 in a PY461 motif dependent manner (S1C and S1D Fig). These data indicate that the association of Glis3 with these HECT ubiquitin ligases is mediated through a direct interaction of the WW domains with the PY461-motif of Glis3. In order to determine whether the interaction required secondary proteins associated with either Itch or Glis3, an in vitro pulldown was performed using in vitro transcribed and translated proteins (TnT-FLAG Glis3 and TnT-Myc Itch). As seen in Fig 3B, TnT-Myc-Itch immunoprecipitated with TnT-FLAG Glis3 and a weaker interaction was observed between TnT-Myc Itch and TnT-FLAG PY461 mutant suggesting that the interaction is direct and does not require intermediate proteins. Collectively, these results demonstrate that the HECT E3 ubiquitin ligases interact directly with the PY461 motif of Glis3 through their WW-domains and that mutation of two residues of the core PPxY motif greatly, but not completely, disrupt their interaction. A schematic summary of the interaction between Glis3 and Itch is shown in Fig 3C.


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 directly interacts with Glis3 through its WW-domains.A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WWI containing only the four WW-domains. Co-immunoprecipitation was performed as described in Fig 2A–2C. B. FLAG-Glis3 or the PY461 mutant and Myc Empty or Myc-Itch were transcribed and translated in vitro and an in vitro pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the PY461 motif of Glis3.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131303.g003: Itch directly interacts with Glis3 through its WW-domains.A. HEK293T cells were transfected with FLAG-Glis3 and Myc empty vector, Itch, Itch-Δ-C2, Itch-Δ-HECT, or Itch-WWI containing only the four WW-domains. Co-immunoprecipitation was performed as described in Fig 2A–2C. B. FLAG-Glis3 or the PY461 mutant and Myc Empty or Myc-Itch were transcribed and translated in vitro and an in vitro pulldown assay was performed using anti-Myc antibody as described in Methods and Materials. G. Schematic showing the interaction between Itch with the PY461 motif of Glis3.
Mentions: Members of the Nedd4/Rsp5p family contain in addition to the centrally located WW-domains, an N-terminal C2 domain and a C-terminal HECT domain [31]. To determine whether these domains influence the interaction between Itch and Glis3, IP was performed with Glis3 and Itch lacking its C2 or HECT domains. The results showed that deletion of either the C2 or the HECT domain (Fig 3A) had little effect on the interaction between Itch and Glis3 and therefore are not required for the interaction. In addition, the four WW domains from Itch were sufficient for interaction with Glis3 although the interaction was considerably weaker than observed with full-length Itch and might be due to differences in the WW domain conformation and therefore affinity for Glis3 between full-length Itch and the Itch WW domain only. The WW-domains of Smurf2 and NEDD4 were also sufficient for interaction with Glis3 in a PY461 motif dependent manner (S1C and S1D Fig). These data indicate that the association of Glis3 with these HECT ubiquitin ligases is mediated through a direct interaction of the WW domains with the PY461-motif of Glis3. In order to determine whether the interaction required secondary proteins associated with either Itch or Glis3, an in vitro pulldown was performed using in vitro transcribed and translated proteins (TnT-FLAG Glis3 and TnT-Myc Itch). As seen in Fig 3B, TnT-Myc-Itch immunoprecipitated with TnT-FLAG Glis3 and a weaker interaction was observed between TnT-Myc Itch and TnT-FLAG PY461 mutant suggesting that the interaction is direct and does not require intermediate proteins. Collectively, these results demonstrate that the HECT E3 ubiquitin ligases interact directly with the PY461 motif of Glis3 through their WW-domains and that mutation of two residues of the core PPxY motif greatly, but not completely, disrupt their interaction. A schematic summary of the interaction between Glis3 and Itch is shown in Fig 3C.

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