Novel Phosphorylations of IKKγ/NEMO

ABSTRACT Central to NF-κB signaling pathways is IKKγ/NEMO, a regulatory subunit of the cytoplasmic IκB kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi’s sarcoma-associated herpesvirus (KSHV) FA...

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Autores principales:	Sun Hwa Lee, Zsolt Toth, Lai-Yee Wong, Kevin Brulois, Jim Nguyen, June-Yong Lee, Ebrahim Zandi, Jae U. Jung
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2012
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/662479506ebb433b92ebc775a6f8161a
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spelling	oai:doaj.org-article:662479506ebb433b92ebc775a6f8161a2021-11-15T15:39:11ZNovel Phosphorylations of IKKγ/NEMO10.1128/mBio.00411-122150-7511https://doaj.org/article/662479506ebb433b92ebc775a6f8161a2012-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00411-12https://doaj.org/toc/2150-7511ABSTRACT Central to NF-κB signaling pathways is IKKγ/NEMO, a regulatory subunit of the cytoplasmic IκB kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi’s sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (vFLIP) activates the NF-κB signaling pathway by directly interacting with IKKγ/NEMO. However, the exact functions of IKKγ/NEMO phosphorylation and its KvFLIP interaction in NF-κB activation remain elusive. Here, we report two novel phosphorylation sites of IKKγ/NEMO and their negative effect on the IKKγ/NEMO-mediated NF-κB signaling pathway. First, the Src family protein tyrosine kinases (SF-PTKs), including Src, Fyn, Lyn, and Fgr, interact with and phosphorylate tyrosine residue 374 (Y374) of IKKγ/NEMO. Mutation of the Y374 residue to phenylalanine (Y374F) specifically abolished SF-PTK-mediated tyrosine phosphorylation, leading to increased tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Moreover, our mass spectrometry analysis found that the serine 377 residue (S377) of IKKγ/NEMO underwent robust phosphorylation upon KvFLIP expression. Replacement of the IKKγ/NEMO S377 residue by alanine (S377A) or glutamic acid (S377E) resulted in a significant increase or decrease of NF-κB activity and TNF-α-mediated IL-6 cytokine production, respectively. Our study thus demonstrates that the Y374 or S377 residue located at the C-terminal proline-rich domain of human IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This study suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway. IMPORTANCE Since unchecked regulation of NF-κB has been linked to uncontrolled proliferation and cell death, the downregulation of the NF-κB signaling pathway is as important as its activation. Specifically, the phosphorylation-mediated modification of IKKγ/NEMO is a critical regulatory mechanism of NF-κB activity. Here, we report two novel phosphorylations of IKKγ/NEMO and their negative effects on the NF-κB signaling pathway. First, the Src family protein tyrosine kinase interacts with and phosphorylates tyrosine residue 374 of IKKγ/NEMO, suppressing tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Additionally, Kaposi’s sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (KvFLIP) expression induces a robust phosphorylation of the serine 377 residue of IKKγ/NEMO, resulting in a significant decrease of NF-κB activity. Our study thus demonstrates that the Y374 or S377 residue of IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This also suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway.Sun Hwa LeeZsolt TothLai-Yee WongKevin BruloisJim NguyenJune-Yong LeeEbrahim ZandiJae U. JungAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 3, Iss 6 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Sun Hwa Lee Zsolt Toth Lai-Yee Wong Kevin Brulois Jim Nguyen June-Yong Lee Ebrahim Zandi Jae U. Jung Novel Phosphorylations of IKKγ/NEMO
description	ABSTRACT Central to NF-κB signaling pathways is IKKγ/NEMO, a regulatory subunit of the cytoplasmic IκB kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi’s sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (vFLIP) activates the NF-κB signaling pathway by directly interacting with IKKγ/NEMO. However, the exact functions of IKKγ/NEMO phosphorylation and its KvFLIP interaction in NF-κB activation remain elusive. Here, we report two novel phosphorylation sites of IKKγ/NEMO and their negative effect on the IKKγ/NEMO-mediated NF-κB signaling pathway. First, the Src family protein tyrosine kinases (SF-PTKs), including Src, Fyn, Lyn, and Fgr, interact with and phosphorylate tyrosine residue 374 (Y374) of IKKγ/NEMO. Mutation of the Y374 residue to phenylalanine (Y374F) specifically abolished SF-PTK-mediated tyrosine phosphorylation, leading to increased tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Moreover, our mass spectrometry analysis found that the serine 377 residue (S377) of IKKγ/NEMO underwent robust phosphorylation upon KvFLIP expression. Replacement of the IKKγ/NEMO S377 residue by alanine (S377A) or glutamic acid (S377E) resulted in a significant increase or decrease of NF-κB activity and TNF-α-mediated IL-6 cytokine production, respectively. Our study thus demonstrates that the Y374 or S377 residue located at the C-terminal proline-rich domain of human IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This study suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway. IMPORTANCE Since unchecked regulation of NF-κB has been linked to uncontrolled proliferation and cell death, the downregulation of the NF-κB signaling pathway is as important as its activation. Specifically, the phosphorylation-mediated modification of IKKγ/NEMO is a critical regulatory mechanism of NF-κB activity. Here, we report two novel phosphorylations of IKKγ/NEMO and their negative effects on the NF-κB signaling pathway. First, the Src family protein tyrosine kinase interacts with and phosphorylates tyrosine residue 374 of IKKγ/NEMO, suppressing tumor necrosis factor alpha (TNF-α)-induced NF-κB activity. Additionally, Kaposi’s sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1β (IL-1β) converting enzyme (FLICE) inhibitory protein (KvFLIP) expression induces a robust phosphorylation of the serine 377 residue of IKKγ/NEMO, resulting in a significant decrease of NF-κB activity. Our study thus demonstrates that the Y374 or S377 residue of IKKγ/NEMO undergoes phosphorylation upon TNF-α treatment or KvFLIP expression, respectively, resulting in the suppression of IKKγ/NEMO activity to induce NF-κB activation. This also suggests the potential phosphorylation-mediated feedback negative regulation of IKKγ/NEMO activity in the NF-κB signaling pathway.
format	article
author	Sun Hwa Lee Zsolt Toth Lai-Yee Wong Kevin Brulois Jim Nguyen June-Yong Lee Ebrahim Zandi Jae U. Jung
author_facet	Sun Hwa Lee Zsolt Toth Lai-Yee Wong Kevin Brulois Jim Nguyen June-Yong Lee Ebrahim Zandi Jae U. Jung
author_sort	Sun Hwa Lee
title	Novel Phosphorylations of IKKγ/NEMO
title_short	Novel Phosphorylations of IKKγ/NEMO
title_full	Novel Phosphorylations of IKKγ/NEMO
title_fullStr	Novel Phosphorylations of IKKγ/NEMO
title_full_unstemmed	Novel Phosphorylations of IKKγ/NEMO
title_sort	novel phosphorylations of ikkγ/nemo
publisher	American Society for Microbiology
publishDate	2012
url	https://doaj.org/article/662479506ebb433b92ebc775a6f8161a
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Novel Phosphorylations of IKKγ/NEMO

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