Autophosphorylation of IKK2 at hitherto uncharacterized sites.

(A) NEMO is known to remain bound to the IKK-complex pre- and post-stimulation, helps in activation as well as substrate recognition to channelize IKK-activity towards IκBα. However, it is not clear how IKK specifically phosphorylates the Ser32&36 of IκBα. (B) Domain organisation of FL IKK2 depicting functional kinase domain (KD), ubiquitin-like domain (ULD), and scaffold dimerization domain (SDD) based on the X-ray structures. Known sites of Ser phosphorylation and NEMO interacting regions at the extreme C-termini are highlighted. Serine and tyrosine residues in the activation loop are shown, mutation of which to Glu renders IKK2 constitutively active. The conserved ATP-interacting Lys (at position 44) is also shown. (C) Autophosphorylation of FL IKK2WT upon incubation with radioactive ATP for different time periods in an in vitro kinase assay. (D) In vitro radioactive kinase assays performed to check the effect of NEMO on autophosphorylation of FL IKK2WT (left panel), and that of NEMO and IκBα on autophosphorylation and substrate phosphorylation (right panel) activity of FL IKK2WT. (E) Effect of Inhibitor VII on FL IKK2WT autophosphorylation and IκBα substrate phosphorylation at different inhibitor concentrations in a radioactive in vitro kinase assay. A schematic representation of the assay procedure is shown in Figure S1B. This assay was performed twice. (F) Radioactive kinase assay conducted to check auto- and substrate phosphorylation of full length and deletion constructs of constitutively active form of IKK2 harbouring phosphomimetic Ser177Glu and Ser181Glu mutations.

IKK2 possesses autocatalytic dual specificity.

(A) In vitro kinase assay, followed by immunoblotting with FL IKK2WT using antibodies against phosphor-Ser (177/181) and phospho-Tyr. (B) A different commercial source of phospho-Tyr antibody was used to detect the same. (C) Effect of Inhibitor VII on tyrosine autophosphorylation of FL IKK2WT. (D) Immunoblotting performed with phospho-Tyr antibody to check autophosphorylation of IKK2 mutant K44M at different time points compared to that of IKK2 WT. (E) Tyrosine autophosphorylation and substrate phosphorylation of GST (1-54) IκBα in presence of full length and deletion mutants of IKK2 harbouring phosphomimetic Ser177Glu and Ser181Glu mutations. (F) De novo auto-phosphorylation of IKK2 on tyrosine monitored by phospho-Ser and phospho-Tyr specific monoclonal antibodies. (G) Autophosphorylation and subsequent immunoblot analysis of FLIKK2 mutant S177A, S181A using antibodies against phospho-IKK2-Ser(177/181) and phospho-Tyr.

Autophosphorylation of IKK2 is critical to the specificity of IκBα phosphorylation.

(A) Immunoblotting performed with phospho-IKK2 Ser(177/181) antibody to check autophosphorylation of IKK2 mutant K44M at different time points compared to that of IKK2 WT. (B) Comparison of substrate and autophosphorylation activities of FL IKK2 WT and K44M mutant in an in vitro radioactive kinase assay performed with or without FL IκBα WT as the substrate. (C) Residue level specificity of phosphorylation by the FL IKK2 K44M was analysed using an antibody that specifically recognizes phospho-S32/36 of IκBα. (D) Differential sensitivity of specific vs. non-specific phosphorylation by IKK2 as assessed by the effect of Inhibitor VII on FL IKK2 WT and IKK2 K44M using IκBα WT and AA as substrates.

Tyrosine autophosphorylation is critical for IKK2 activity.

(A) Position of canonically important residues in the AL (purple ribbon) of IKK2-KD are shown in the context of its structure (PDB ID 4E3C; surface representation; KD is shown light green with respect to the SDD in teal). Autophosphorylated tyrosine residues identified in this study are shown in green. (B) Alignment of the activation segment sequences of different IKK family kinases. Conservation of Tyr at the DFG+1 (DLG in case of IKK1 and IKK2) is retained only in a stress response related plant kinase SnRK2, but not in other IKK homologues or any known dual specificity kinases, e.g., DYRK and GSK3β (both contain Ser at that position). Tyr188 (204 in PKA) is conserved. (C) Radioactive in vitro kinase assay performed with IKK2 Y169F and Y188F mutants using GST (1-54) IκBα WT and AA as substrates. (D) Radioactive in vitro kinase assay performed with IKK2 Y169F mutant with a range of GST (1-54) IκBα mutants where the substrate phosphorylation signal is abolished in case of S36 A/E IκBα. (E) Severe reduction in TNFα-induced IKK activity in ikk2-/- MEF-3T3 cells reconstituted with mutant (Y169F and Y188F) IKK2s compared to wild-type. Kinase assay was performed with IKK immunoprecipitated (IP-ed) with anti-NEMO antibody from whole cell extract. (F) AL-serine phosphorylation status of IKK2 WT and Y169F was assessed in reconstituted MEFs post-stimulation with TNF-α after immunoprecipitating the kinase with monoclonal anti-HA antibody.

Structural analyses of IKK2 autophosphorylation.

(A) Molecular dynamics (MD) simulations of all three differently phosphorylated states of IKK2 was performed. 200ns trajectory of energy for each state is shown in golden yellow, blue and copper red representing UnP-IKK2, p-IKK2 and P-IKK2, respectively. Same color coding has been followed throughout this figure and the corresponding supplementary figure. (B) 200ns trajectory of RMSD for each state is shown. (C) Residues forming canonical R-(labelled in blue) & C-spines (labelled in dark gray) representative of an active conformation in p-IKK2 are shown. (D) Superposition of structures representing the differently phosphorylated states post 200ns MD simulation. Relevant sections, e.g., activation loop, αC-helix, Gly-rich loop areas are highlighted in the right panel. (E) ATP-bound p-IKK2 structure is depicted. In the right panel, relevant residues are highlighted. Y169 is found in a position poised for autophosphorylation. (F) ATP completes the desired continuum of R-& C-spine observed in active kinase conformations. (G) P-IKK2 cannot accommodate ATP in its cleft, and αC-helix is displaced. (H) M is moved away from the R-spine, failing to form canonically active conformation of R-& C-spines. (I & J) ADP-bound p-IKK2 and P-IKK2 states are shown, respectively. Both p-IKK2 and P-IKK2 states can accommodate ADP in their respective clefts.

Freshly autophosphorylated IKK2 relays phosphates to IκBα.

(A) Schematic representation of the experiment performed to monitor the path traversed by phosphate(s) from phospho-IKK2 to substrate by autoradiography detection method. (B) Purified autophosphorylated (radiolabelled) IKK2 transfers its phosphate to IκBα substrate in absence of any nucleotide, and transfer efficiency is enhanced upon addition of ADP or ATP. (C) Schematic representation of the experiment performed to monitor the path traversed by phosphate(s) from phospho-IKK2 to substrate by immunoblotting detection method. (D) Elution profile of the size exclusion chromatography run of phospho-IKK2 to get rid of excess unlabelled ATP performed in Superdex200 10/30 increase. (E) Purified autophosphorylated (cold) IKK2 transfers its phosphate to IκBα substrate with enhanced transfer efficiency upon addition of ADP or ATP monitored by immunoblotting using specific antibodies as indicated in the figure.

Specificity and fidelity of phosphotransfer by IKK2 to IκBα.

(A) AMP-PNP can support efficient phosphotransfer. Also, phosphotransfer is observed in IκBα WT but not in S32,36Ala double mutant. Domain organization and position of relevant S/T/Y residues of IκBα are shown above the autoradiograph. (B) A detailed scheme of reactions showing IKK2-activation and subsequent phosphorylation of the signal responsive S32,36 on IκBα by phosphotransfer in presence of ADP. (C) A cartoon representation of the proposed model of NF-κB activation by IKK2 employing phosphotransfer to IκBα.