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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, Smurf2, and NEDD4 polyubiquitinate Glis3.A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective PY461 mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.
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pone.0131303.g004: Itch, Smurf2, and NEDD4 polyubiquitinate Glis3.A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective PY461 mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.

Mentions: To determine whether the E3 ubiquitin ligases were capable of promoting Glis3 ubiquitination as a result of their interaction, FLAG-Glis3 or the PY461 mutant was co-expressed in HEK293T cells with HA-tagged ubiquitin and Myc-tagged Itch, Smurf2, NEDD4, or empty vector and the effect on Glis3 ubiquitination examined. As seen in Fig 4A, only Itch substantially enhanced Glis3 ubiquitination. Furthermore, consistent with the data in Fig 1, mutation of the PY461 motif significantly reduced, but did not totally eliminate Itch-mediated ubiquitination of Glis3 (Fig 4A and S2A Fig). The latter is consistent with our observation that mutation of the PY461 motif did not totally abolish the interaction with Glis3 (Fig 2B) suggesting that Itch might weakly interact with Glis3 through an additional flanking residues. Likewise, Itch also enhanced the ubiquitination of Glis3-ΔC480, which was greatly reduced by mutation of the PY461 motif (Fig 4B and S2B Fig). Again, Smurf2 and NEDD4 had little effect on the ubiquitination of Glis3-ΔC480. To establish whether the effect of Itch on Glis3 ubiquitination was due to its ability to transfer ubiquitin, the catalytic cysteine in the HECT domain was mutated and its ability to promote Glis3 ubiquitination was assessed. Fig 4C shows that the catalytically inactive mutant was virtually incapable of promoting Glis3 ubiquitination. Finally, in order to determine what type of polyubiquitin chain is transferred to Glis3 by Itch, HA-ubiquitin containing a mutation that converts either Lys48 or Lys63 to Arg was used in a pulldown with Itch and Glis3. The results indicate that mutation of ubiquitin K48 had only a small effect on Glis3 polyubiquitination by Itch, while the K63R ubiquitin mutation significantly reduced Itch-mediated Glis3 ubiquitination, but did not totally eliminate it (S3 Fig). Collectively these data indicate that Itch induces K63-linked rather than K48-linked polyubiquitination of Glis3 and that this process is dependent upon the catalytic domain of Itch.


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, Smurf2, and NEDD4 polyubiquitinate Glis3.A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective PY461 mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493090&req=5

pone.0131303.g004: Itch, Smurf2, and NEDD4 polyubiquitinate Glis3.A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective PY461 mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.
Mentions: To determine whether the E3 ubiquitin ligases were capable of promoting Glis3 ubiquitination as a result of their interaction, FLAG-Glis3 or the PY461 mutant was co-expressed in HEK293T cells with HA-tagged ubiquitin and Myc-tagged Itch, Smurf2, NEDD4, or empty vector and the effect on Glis3 ubiquitination examined. As seen in Fig 4A, only Itch substantially enhanced Glis3 ubiquitination. Furthermore, consistent with the data in Fig 1, mutation of the PY461 motif significantly reduced, but did not totally eliminate Itch-mediated ubiquitination of Glis3 (Fig 4A and S2A Fig). The latter is consistent with our observation that mutation of the PY461 motif did not totally abolish the interaction with Glis3 (Fig 2B) suggesting that Itch might weakly interact with Glis3 through an additional flanking residues. Likewise, Itch also enhanced the ubiquitination of Glis3-ΔC480, which was greatly reduced by mutation of the PY461 motif (Fig 4B and S2B Fig). Again, Smurf2 and NEDD4 had little effect on the ubiquitination of Glis3-ΔC480. To establish whether the effect of Itch on Glis3 ubiquitination was due to its ability to transfer ubiquitin, the catalytic cysteine in the HECT domain was mutated and its ability to promote Glis3 ubiquitination was assessed. Fig 4C shows that the catalytically inactive mutant was virtually incapable of promoting Glis3 ubiquitination. Finally, in order to determine what type of polyubiquitin chain is transferred to Glis3 by Itch, HA-ubiquitin containing a mutation that converts either Lys48 or Lys63 to Arg was used in a pulldown with Itch and Glis3. The results indicate that mutation of ubiquitin K48 had only a small effect on Glis3 polyubiquitination by Itch, while the K63R ubiquitin mutation significantly reduced Itch-mediated Glis3 ubiquitination, but did not totally eliminate it (S3 Fig). Collectively these data indicate that Itch induces K63-linked rather than K48-linked polyubiquitination of Glis3 and that this process is dependent upon the catalytic domain of Itch.

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