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.

FIG 1

The Y374 or S377 phosphorylation of IKKγ/NEMO exerts negative effects on TNF-α-mediated NF-κB activation. (A) A schematic representation of the location of Y374 and S377 in the C-terminal proline-rich domain of human IKKγ/NEMO. Individual mutations were generated by site-directed mutagenesis. IKKγ/NEMO-knockout (IKKγ/NEMO^−/−) mouse embryo fibroblasts (MEFs) were transfected with puroIRES vector carrying the HA-tagged IKKγ/NEMO WT or mutant gene. IKKγ/NEMO^−/− MEFs stably expressing the IKKγ/NEMO WT or mutant were then established by puromycin resistance (2 µg/ml). Expression of the IKKγ/NEMO WT or mutant was confirmed by immunoblotting with anti-HA antibody. (B) IKKγ/NEMO^−/− MEFs stably expressing either HA-tagged IKKγ/NEMO WT or mutant were stimulated with mouse TNF-α (10 ng/ml) for indicated times. Whole-cell lysates (WCLs) were then analyzed by immunoblotting with anti-phospho-IκBα (p-IκBα) antibody. Immunoblotting with antiactin antibody served as a loading control. (C) IKKγ/NEMO^−/− MEFs stably expressing either HA-tagged IKKγ/NEMO WT or mutant were stimulated with mouse TNF-α (10 ng/ml) for the indicated times. Supernatants were analyzed by ELISA for IL-6 cytokine production. Experiments were repeated twice, and the average of triplicate samples from one experiment is shown with the error bars denoting the standard error of the mean.

FIG 2

Mutation of the Y374 or S377 residue of human IKKγ/NEMO shows no effect on IKK complex formation or self-oligomerization. (A) IKKγ/NEMO^−/− MEFs stably expressing either HA-tagged IKKγ/NEMO WT or mutant were subjected to immunoprecipitation (IP) with rabbit anti-HA antibody, followed by immunoblotting with antibodies against IKKα and IKKβ. (B) HEK 293T cells were transfected with HA-tagged IKKγ/NEMO WT or mutants together with FLAG-tagged WT IKKγ/NEMO. WCL was immunoprecipitated (IP) with anti-FLAG or anti-HA antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody. WCL was analyzed by immunoblotting with the indicated antibodies to confirm the expression of IKKγ/NEMO.

FIG 3

Mutation of the Y374 or S377 residue of human IKKγ/NEMO does not affect its ubiquitination. (A) HEK 293T cells were transfected with HA-tagged IKKγ/NEMO WT or mutants together with FLAG-tagged ubiquitin (Ub). WCL was used for the purification of the IKKγ/NEMO complex using agarose beads conjugated with mouse anti-HA antibody, followed by immunoblotting with rabbit anti-FLAG or rabbit anti-HA antibody. WCL was analyzed by immunoblotting with the indicated antibodies to confirm expression of IKKγ/NEMO. (B) IKKγ/NEMO^−/− MEFs stably expressing either HA-tagged IKKγ/NEMO WT or mutant were treated with TNF-α for 10 min, followed by immunoprecipitation with agarose beads conjugated with mouse anti-HA antibody and immunoblotting with rabbit anti-HA antibody (upper panel). A longer exposure of the blot marked with an asterisk is shown in the lower panel (both panels are marked with an asterisk). WCL was analyzed by immunoblotting with anti-HA antibody to confirm the IKKγ/NEMO expression.

FIG 4

Expression of SF-PTKs leads to the Y374 phosphorylation of human IKKγ/NEMO. (A) Either IKKγ/NEMO WT or the Y374F mutant was coexpressed with each member of the Src family protein tyrosine kinases (SF-PTKs) in HEK 293T cells. WCL was then analyzed by immunoblotting with phosphotyrosine (pY)-specific antibody. HA (IKKγ/NEMO), Myc (Fyn and Lck), and V5 (Fgr, Src, and Lyn) tagging antibodies were used to demonstrate expression levels. (B) HA-tagged IKKγ/NEMO was overexpressed in HEK 293T cells along with SF-PTKs as described above and purified with rabbit anti-HA antibody in RIPA buffer, followed by immunoblotting with horseradish peroxidase (HRP)-conjugated anti-pY antibody. IP, immunoprecipitation. (C) HA-tagged WT IKKγ/NEMO was coexpressed with Myc-Fyn, Myc-Lck, V5-Fgr, V5-Src, or V5-Lyn in HEK 293T cells. WCL was subjected to immunoprecipitation with either anti-Myc or anti-V5 antibody, followed by immunoblotting with horseradish peroxidase-conjugated anti-HA antibody.

FIG 5

The Y374 or S377 phosphorylation of IKKγ/NEMO differentially contributes to NF-κB activation and intracellular Ca²⁺ influx. (A) IKKγ/NEMO-deficient Jurkat T cells stably complemented with HA-tagged IKKγ/NEMO WT or mutant were stimulated with PMA (P) and ionomycin (I) for indicated times. WCL was analyzed by immunoblotting with anti-p-IκBα antibody. Immunoblotting with antiactin antibody served as a loading control. (B) IKKγ/NEMO-deficient Jurkat T cells stably complemented with HA-tagged IKKγ/NEMO WT or mutant were loaded with Ca²⁺ indicator for 1 h at 37°C. Intracellular Ca²⁺ influx upon PMA and ionomycin (P + I) treatment was monitored for 5 min by flow cytometry analysis. (C) IKKγ/NEMO-deficient Jurkat T cells stably complemented with HA-tagged IKKγ/NEMO WT or mutant were subjected to immunoprecipitation (IP) with rabbit anti-HA antibody, followed by immunoblotting with antibodies against IKKα, IKKβ, and HA.