Novel Lys63-linked ubiquitination of IKKβ induces STAT3 signaling

Abstract

NFκB signaling plays a significant role in human disease, including breast and ovarian carcinoma, insulin resistance, embryonic lethality and liver degeneration, rheumatoid arthritis, aging and Multiple Myeloma (MM). Inhibitor of κB (IκB) kinase β (IKKβ) regulates canonical Nuclear Factor κB (NFκB) signaling in response to inflammation and cellular stresses. NFκB activation requires Lys63-linked (K63-linked) ubiquitination of upstream proteins such as NEMO or TAK1, forming molecular complexes with membrane-bound receptors. We demonstrate that IKKβ itself undergoes K63-linked ubiquitination. Mutations in IKKβ at Lys171, identified in Multiple Myeloma and other cancers, lead to a dramatic increase in kinase activation and K63-linked ubiquitination. These mutations also result in persistent activation of STAT3 signaling. Liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS) analysis identified Lys147, Lys418, Lys555 and Lys703 as predominant ubiquitination sites in IKKβ. Specific inhibition of the UBC13-UEV1A complex responsible for K63-linked ubiquitination establishes Lys147 as the predominant site of K63-ubiquitin conjugation and responsible for STAT3 activation. Thus, IKKβ activation leads to ubiquitination within the kinase domain and assemblage of a K63-ubiquitin conjugated signaling platform. These results are discussed with respect to the importance of upregulated NFκB signaling known to occur frequently in MM and other cancers.

Keywords: IKKβ; K-63 ubiquitination; NFκB; STAT3; cell proliferation; inflammatory signaling; mass spectrometry; multiple myeloma; oncogenesis; polyubiquitination.

Figure 1.

Phosphorylation of IKKβ Induced by Mutations at Lys171. (A) Schematic of IKKβ with the activation loop within the N-terminal kinase domain expanded to show amino acids critical for phosphorylation and signaling. The ubiqutin-like domain (ULD), the scaffold/dimerization domain (SDD) which contains the leucine zipper (LZ) and helix-looop-helix (HLH) regions, and NEMO binding domain (NBD) are indicated. (B) IKKβ mutant proteins were expressed in HEK293 cells and the IKK complex was immunoprecipitated with IKKγ antisera and assayed for in vitro phosphorylation. Samples were separated by SDS-PAGE and detected by autoradiography. IKKβ expression is shown by immunoblotting for IKKβ (middle panel). Assays were quantitated relative to ³²P incorporation of the IKKβ S177E/S181E mutant, +/− sem. (bottom) (C) HEK293T cells expressing IKKβ mutants were analyzed for activation loop serine phosphorylation using Phospho-IKKα/β antisera (top). The membrane was reprobed for IKKβ (middle panel). Serine 177/181 phosphorylation was quantitated relative to the K171T mutant, +/− sem. (bottom panel). (D) HEK293T cells expressing IKKβ WT or K171E were treated with 50 μg/ml cycloheximide (CHX) for 2, 5, 7, 12 and 24 h. Lysates were examined for IKKβ serine phosphorylation and total IKKβ as in (C) (top 2 panels). The membrane was reprobed for β-tubulin (third panel). Serine 177/181 phosphorylation was quantitated relative to K171E at time zero, +/− sem. (bottom).

Figure 2.

IKKβ Ubiquitination Regulated by Lys171. (A) HEK293T cells expressing IKKβ WT or Lys171 mutants, HA-Ub and HA-NEMO, were lysed and immunoprecipitated with IKKβ antisera. Samples were immunoblotted for K63-linkage specific polyubiquitin (top panel). The membrane was reprobed for IKKβ (bottom panel). (B) Cells as in Panel A were treated with 10 ng/ml of TNFα for 2, 5, 8, 12 and 24 h. Samples were immunoprecipitated and blotted as decribed in (A) (top 2 panels). Duplicate samples were immunoprecipitated with HA-probe antisera and immunoblotted for IKKβ (third panel). The membrane was reprobed for K63-linkage specific polyubiquitin (bottom). (C) Cells as in Panel A were treated with 50 μg/ml CHX for 2, 5, 7, 12 and 24 h. IKKβ immunoprecipitates were blotted as in (A). (D) Cells as in Panel A were treated with +/− 10ng/ml TNFα and +/− 10 μM MG132 for 12 h. IKKβ immunoprecipitates were blotted for K63-linkage specific polyubiquitin (top panel) or K48-linkage specific polyubiquitin (third panel). Membranes were reprobed for IKKβ (2nd and 4th panels).

Figure 3.

IKKβ Lys171 Activation of STAT3. (A) HEK293T cells expressing IKKβ derivatives were treated with 10 ng/ml TNFα or 10 ng/ml IL-6 for 10 min. Lysates were immunoblotted for Phospho-STAT3 (Tyr705) (top), STAT3 (middle panel) and IKKβ (bottom). (B) HEK293T cells expressing IKKβ mutants were treated with 10 ng/ml of TNFα for 5, 15, 45 and 60 min. Lysates were immunoblotted for Phospho-STAT3 (Tyr705) (top), STAT3 (2nd panel), Phospho-IκBα (Ser32/36) (3rd panel), IκBα (4th panel), and IKKβ (bottom). Phosphorylation of STAT3 was quantitated relative to the K171E mutant at time zero, +/− sem. (bottom panel). (C) HEK293T cells expressing IKKβ WT, K171E and S177E/S181E were treated with +/− 10 ng/ml TNFα and +/− 10 μM MG132 for 12 h. Lysates were immunoblotted as in (B). (D) Lysates from HEK293T cells expressing IKKβ derivatives were immunoblotted as in (A). STAT3 phosphorylation was quantitated relative to the Lys171Thr mutant, +/− sem. (bottom).

Figure 4.

Identification and Biological Function of IKKβ ubiquitination Sites. (A) Representative spectrum of peptides showing the relative intensity of selected b and y ions for the major sites of IKKβ ubiquitination. Note that not all identified ions are labeled due to space constraints. (B) HEK293T cells expressing IKKβ Lys to Arg mutants were immunoprecipitated and immunoblotted as in Figure 2A. (C) Lysates from HEK293T cells expressing IKKβ Lys to Arg mutants were immunoblotted as in Figure 1(C).

Figure 5.

Inhibition of K63-linked ubiquitination Blocks IKKβ Lys171 Signaling. (A) Lysates from HEK293T cells expressing IKKβ WT and K171E were treated with 2 μM and 5 μM NSC697923 for 2 h and immunoblotted for Phospho-IKKα/β (top), IKKβ (middle panel) and β-tubulin (bottom). (B) Duplicate samples from (A) were immunoblotted for Phospho-STAT3 (Tyr705) (top) and STAT3 (bottom). (C) Lysates from HEK293T cells expressing IKKβ K171E treated with +/− 5 μM NSC697923 for 2 h and +/− 10 ng/ml IL-6 for 10 min were immunoblotted as in (B). (D) Lysates from HEK293T cells expressing IKKβ Lys to Arg mutants were immunoblotted as in (B).