title: 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) 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.
authors.affiliation: Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, California, USA.
authors.forename: Sun Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Toth
authors.forename: Lai-Yee
authors.initials: LY
authors.lastname: Wong
authors.forename: Kevin
authors.initials: K
authors.lastname: Brulois
authors.forename: Jim
authors.initials: J
authors.lastname: Nguyen
authors.forename: June-Yong
authors.initials: JY
authors.lastname: Lee
authors.forename: Ebrahim
authors.initials: E
authors.lastname: Zandi
authors.forename: Jae U
authors.initials: JU
authors.lastname: Jung
mesh_terms: D019943:Amino Acid Substitution; D019288:Herpesvirus 8, Human / Q000472:pathogenicity; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
chemical_list: C491731:IKBKG protein, human; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D014443:Tyrosine; D012694:Serine; D051550:I-kappa B Kinase