Introduction

Protein kinases bestow novel identity to their substrates by adding phosphate moieties at the site of modification that often play key regulatory roles in a diverse array of signaling events. For example, inflammatory or pathogenic signals trigger precisely regulated induction of the transcription factor NF-κB in metazoans(Hoffmann and Baltimore, 2006) that depends upon the upstream activation of the Inhibitor of κB Kinase-complex (IKK-complex), comprising two catalytic subunits, IKK1 (also known as IKKα) and IKK2 (or IKKβ), and an essential regulatory scaffolding protein, NEMO (IKKγ)(DiDonato et al., 1997; Rothwarf et al., 1998; Zandi et al., 1997). IKK2 activity is maintained at a very low basal level in resting cells(Ghosh and Karin, 2002; Hacker and Karin, 2006; Hinz and Scheidereit, 2014; Liu et al., 2012). Upon activation, IKK2 phosphorylates the N-terminus of IκBα (Inhibitor of κBα) protein specifically at two Serine residues (S32 and S36) marking it for ubiquitinylation-mediated proteasomal degradation (Figure 1A). Signal-responsive phosphorylation of IκBα refers to the phosphorylation of S32 and S36 by the IKK-activity. Degradation of IκBα liberates NF-κB to execute its gene expression program (Hayden and Ghosh, 2008; Hinz and Scheidereit, 2014; Karin and Ben-Neriah, 2000; Scheidereit, 2006). Mutation of both S32 and S36 to alanine residues converts IκBα into a non-phosphorylatable super-repressor of NF-κB(Brown et al., 1995; Lin et al., 1995). Furthermore, IKK possesses exquisite specificity for these two serines, and it fails to phosphorylate IκBα when S32 and S36 are mutated even to another phosphorylatable residue, threonine(DiDonato et al., 1997).

Figure 1:

IKK2 is an essential Ser/Thr kinase that ensures the activation of a particular signaling cascade -NF-κB, with high fidelity despite its reported pleiotropy in other contexts(Antonia et al., 2021; Schröfelbauer et al., 2012; Schrofelbauer and Hoffmann, 2011). IKK2 is a multidomain protein consisting of a kinase domain (KD) followed by a Ubiquitin-like domain (ULD) and the scaffold dimerization domain (SDD) (Figure 1B). IKK2 toggles between inactive and active states based on the phosphorylation status of two serine residues (S177 and S181; S176 and S180 in the case of IKK1) within its activation loop (activation loop) in the KD(Huse and Kuriyan, 2002; Liu et al., 2013; Polley et al., 2013; Xu et al., 2011). Replacement of these two serines with phospho-mimetic glutamate residues (S177E, S181E; henceforth EE) renders the kinase constitutively active. IKK2 is reported to be primed through phosphorylation at S177 by the upstream kinase TAK1 that leads to auto-phosphorylation at S181, thus rendering the kinase fully active in cells(Zhang et al., 2014). However, oligomerization upon association with NEMO, in the presence of linear or Lys-63 linked poly-ubiquitin (Ub) chains or at a high concentration of IKK2, also enables trans auto-phosphorylation at S177 and S181(Chen, 2012; Du et al., 2022; Ea et al., 2006; Polley et al., 2013). It was also shown that NEMO not only helps in the activation of the IKK-complex, but also ensures that the IKK-activity is well directed towards IκBα in the inhibitor bound transcriptionally inactive NF-κB (NF-κB: IκBα complex)(Schröfelbauer et al., 2012). Activated IKK2 phosphorylates IκBα specifically at residues S32/S36, for which the C-terminal SDD of IKK2 (residue range 645-756) is critical – a shorter construct of IKK2 lacking the NBD and SDD displayed CK2-like activity that did not retain the exquisite specificity of IKK2 to phosphorylate the S32/36 of IκBα (Shaul et al., 2008). Other sites of IκBα e.g., the residues in the C-terminal PEST region that contains proline (P), glutamic acid (E), serine (S) and threonine (T) residues, but not S32/S36, can be phosphorylated by various kinases(Barroga et al., 1995; Tergaonkar et al., 2003). The role of IκBα-phosphorylation at sites other than the signal responsive S32/S36 phosphorylation in NF-κB activation and signaling is elusive. These findings underscore the ability of IKK2 to function specifically in order to guarantee rapid NF-κB activation in canonical signaling. It is however, still not fully understood how IKK2 achieves its exquisite specificity towards S32/S36 of IκBα to mark it for proteasomal degradation that liberates active NF-κB dimers.

Here we report an in-depth analysis of the catalytic features of IKK2 that revealed that IKK2 undergoes multisite autophosphorylation and can phosphorylate its own tyrosine residue(s) in addition to its own serines or the serines of its substrate IκBα, i.e., IKK2 possesses dual specificity. This phosphorylation at tyrosine residue(s) is autocatalytic. This phosphorylation at tyrosine residues is dependent upon the prior activation of IKK2 via activation loop S177/S181 phosphorylation, and is critical to phosphorylation of signal responsive S32/S36 of IκBα. Mutation of Y169 of IKK2 to phospho-ablative phenylalanine severely compromised phosphorylation of IκBα at S32. Mutation of Y188 to phenylalanine rendered the kinase practically inactive in vitro. IKK2 activity towards signal responsive S32/S36 of IκBα was severely compromised in MEF cells reconstituted with these two mutant IKK2s in response to treatment with TNFα. We also observed that mutation of the conserved ATP-interacting residue K44 in IKK2 to methionine leads to a loss of tyrosine as well as S177,S181 auto-phosphorylation activity of the kinase along with its ability to phosphorylate S32/S36 of IκBα, thereby clearly suggesting a critical role of this Lysine residue in regulating IKK2’s kinase activity for tyrosine as well as serine phosphorylations. Interestingly, IKK2 K44M retained non-specific kinase activity referring to phosphorylation of IκBα at residues other than S32 and S36. Furthermore, we observed that, after auto-phosphorylation at tyrosine, IKK2 can phosphorylate IκBα in the absence of exogenous supply of ATP, as long as ADP is present in the reaction. Overall, our results reveal unique, and likely transient, autophosphorylated intermediates of IKK2 that appear to be critical in relaying phosphoryl group(s) specifically to S32/S36 of IκBα rather than following the conventional mode of phosphate group transfer directly from ATP to the substrate seen in eukaryotic protein kinases. IKK2 thus ensures fidelity of NF-κB activation. Recent reports in other kinases indicate that multisite (auto)phosphorylation is a major determinant in regulating function of respective kinases and regulation exerted by those kinases. Our study adds another layer of regulation and critical nature of such autophosphorylation that plays a significant biological role in metazoans.

Results

IKK2 undergoes auto-phosphorylation at uncharacterized sites

Signal-induced phosphorylation of activation loop Ser177/Ser181 marks the general activation of IKK2 (Figure 1B). Hyperphosphorylation of several other serines located within the flexible C-terminal region of IKK2, spanning amino acid 701 to the end (Figure 1B), has been reported to down-regulate IKK2 activity in cells (Delhase et al., 1999). Furthermore, ectopically overexpressed IKK2 was found to be capable of autophosphorylating these two serines in the activation loop in trans even in the absence of an activating physiological signal {Polley, 2013 #27}. Indeed, recombinantly purified FL IKK2WT is capable of autophosphorylation in vitro when incubated with γ³²P-ATP that increases with the time of incubation (Figure 1C), possibly at multiple sites as indicated by the spread-out of slower migrating phospho-IKK2 band. We also observed that autophosphorylation of IKK2 occurred irrespective of the presence of its cognate binding partner NEMO and bonafide substrate IκBα (Figure 1D). An in vitro kinase assay using a kinase inactivating mutant, IKK2 K44M along with the WT kinase using γ³²P-ATP as the phosphor-donor confirmed that the above-mentioned phosphorylation of IKK2 was indeed autophosphorylation and not due to an unrelated kinase copurified with IKK2 as IKK2 K44M lacked both IκBα (substrate) and autophosphorylation activities (Figure S1A). Lys44 is the ATP-binding invariant lysine residue in IKK2 that is conserved in STYKs, mutation to which to Met is expected to greatly reduce or eliminate the kinase activity. These observations were further supported by another in vitro radioactive kinase assay performed in the presence or absence of an IKK-specific ATP-competitive inhibitor, Calbiochem Inhibitor VII (Figure 1E), that inhibits specific phosphorylation of S32/36 on IκBα (Figure S1B). In both cases kinase and kinase-substrate reactions were incubated for 30 minutes with the inhibitor prior to adding the phosphor-donor, γ³²P-ATP or cold ATP. Purification status of recombinant FL IKK2 WT and FL IKK2 K44M proteins used in this study are shown in Figure S1C, along with LC MS/MS study performed on the FL IKK2 K44M (Figure S1D).

Next, we used different versions of the IKK2 protein where all previously known phosphorylation sites were lacking: Ser177 and 181 were mutated to phosphor-mimetic Glu (EE; this version of IKK2 is known to be constitutively active (Zandi et al., 1998)), and the C-terminal serine-rich region was truncated to different extents. The shortest construct, Δ664EE that entirely lacked the flexible C-terminus (670-756) was further subjected to limited proteolysis using trypsin to eliminate residues, if any, from the flexible ends of the enzymes and this trypsin-treated IKK2 was purified prior to in vitro kinase assays. Each of these constructs was active in phosphorylating itself and the substrate IκBα (Figure 1F). The occurrence of autophosphorylation despite the absence of activation loop serines and serines within the C-terminus strongly suggested the presence of hitherto uncharacterized sites of autophosphorylation in IKK2.

Auto-phosphorylation of IKK2 reveals its dual specificity

IKK2 is known as a bonafide Ser/Thr kinase that undergoes autophosphorylation. In the previous section, we found that IKK2’s autophosphorylation activity might extend beyond the previously characterized site(s). We asked if it is a dual specificity kinase. To this end, we unexpectedly observed de novo auto-phosphorylation of IKK2 on tyrosine residues (Figure 2A, middle panel) along with the anticipated auto-phosphorylation of activation loop serines (Figure 2A, upper panel) when IKK2 was incubated with Mg²⁺-ATP for various time periods. Increase in autophosphorylation of activation loop-S177/181 with time indicates inhomogeneous and incomplete phosphorylation of activation loop-S177/181 in the recombinant IKK2 when purified from Sf9 cells. To circumvent the specificity-bias of the pan-phosphotyrosine antibody used in Figure 2A, we used another phospho-tyrosine specific antibody, which confirmed that detection of de novo phosphotyrosine on IKK2 was independent of the source of the antibody used (Figure 2B). The tyrosine auto-phosphorylation was abolished in the presence of an IKK-specific inhibitor, Inhibitor VII (Figure 2C), and the IKK2-K44M mutant (kinase inactivating) did not display any tyrosine autophosphorylation when incubated with ATP unlike WT IKK2 (Figure 2D). IKK2 autophosphorylation assay performed in presence of different concentration of Urea revealed that its tyrosine-autophosphorylation was abolished at urea concentration beyond 1M (Figure S2A). A more sensitive assay with γ³²P-ATP as the phospho-donor also indicated that urea concentrations above 1M abolished both pan-auto-phosphorylation and substrate (IκBα) phosphorylation of IKK2 (Figure S2B). These results confirmed that IKK2 is a dual specificity kinase that not only phosphorylates Ser on substrates, but also phosphorylates its own Ser and Tyr residues. The fact that activities of IKK2 towards S32 and S36 of IκBα and its own tyrosine residue(s) were affected similarly by various kinase inhibitors, both general (AMPPNP and Staurosporine) and IKK-specific (MLN-120B, TPCA, Inhibitor VII) (Figure S2C), confirm that both substrate phosphorylation and autophosphorylation activities of IKK2 are carried out by the same active site. We further observed that the C-terminal NEMO binding domain (NBD) was not required for autocatalytic dual specificity of IKK2 as various truncated versions of IKK2 (lacking NBD) were capable of phosphorylating itself on tyrosine residue(s), and phosphorylating IκBα at signal-responsive S32 and S36 (Figure 2E). Assessment of phospho-Ser and Tyr on a constitutively active NBD-deficient IKK2 (IKK2^Δ664EE) monitored by monoclonal phospho-serine and phospho-tyrosine antibodies revealed de novo tyrosine autophosphorylation of the kinase while de novo serine autophosphorylation (if any) was not discernible in this assay condition (Figure 2F). The phospho-ablative S177A, S181A (AA; non-phosphorylatable activation loop) mutant of IKK2 that was incapable of phosphorylating S32/36 of IκBα (Figure S2D), also lacked tyrosine autophosphorylation activity (Figure 2G) in in vitro kinase assay suggesting that activation loop S177/S181 serine phosphorylation is essential in conducting dual specificity of IKK2.

Figure 2:

Dual-specific autophosphorylation is critical for IKK2’s function

Previous reports suggested that phosphorylation of IKK2 on Y169, Y188 and/or Y199 was critical for its function(Darwech et al., 2010; Meyer et al., 2013). Our mass spectrometric analyses identified multiple tyrosine residues to be phosphorylated, e.g., Y169, Y188, Y325 (Figure S3A). To glean into the structural plausibility of tyrosine phosphorylation within the activation loop, we compared available structural models of human IKK2 (PDB ID: 4KIK, 4E3C) (Liu et al., 2013; Polley et al., 2013). In the active state conformers, the position and conformation of Tyr169 appears to be well-poised for accepting a phosphate from ATP (Figure S3B) intramolecularly. A superposition of IKK2 with a pseudo-substrate-bound PKA shows that the hydroxyl of Tyr169 in IKK2 projects toward the γ-phosphate of ATP similarly to the serine hydroxyl on the PKA inhibitor pseudo-substrate(Knighton et al., 1991; Nolen et al., 2004) (Figure S3B & C). Interestingly, Y169 is the DFG+1 (DLG in case of IKK2 and IKK1) residue while Y188 (equivalent to Y204 of PKA) is located at the end of activation loop prior to the conserved APE motif (Figure 3A & B). It was previously established that the DFG+1 position is crucial for defining the substrate specificity of a kinase(Chen et al., 2014). Y188 counterpart Y204 of PKA is reported to allosterically modulate PKA activity by altering its conformational entropy(Ahuja et al., 2017; Yang et al., 2005). In order to understand the functional consequence of dual-specific autophosphorylation of IKK2, we further focused our attention to Y169 and Y188 as they are located in the activation loop of the kinase along with the previously described K44M mutant.

Figure 3:

Firstly, we wanted to check the effect of autophosphorylation on IKK2 activity by using the kinase inactivating K44M mutant that was found to be incapable of tyrosine autophosphorylation (Figure 2D) and fails to undergo autophosphorylation of the activation loop serines (Figure S3D). Interestingly, IKK2-K44M showed a very weak auto-phosphorylation activity compared to WT IKK2 but phosphorylated full-length WT IκBα robustly albeit much less efficiently than WT IKK2 in a radioactive in vitro kinase assay (Figure 3C). However, IKK2-K44M was unable to specifically phosphorylate S32/S36 of IκBα (Figure 3D). These seemingly contrasting results possibly reflects phosphorylation of the PEST region of FL IκBα in the radioactive kinase assays as they report pan-phosphorylation of a substrate (Figure 3C & D). In addition, IKK2-K44M phosphorylated FL IκBαAA more efficiently than FL IκBα WT unlike WT IKK2 that phosphorylated FL IκBα WT much more efficiently than FL IκBαAA (Figure S3E). It indicates that IKK2-K44M retained non-specific kinase activity to some extent while lost functionally relevant specificity towards the S32, S36 of IκBα. We subjected 20ug of FL IKK2 K44M protein used in the above studies to LC-MS/MS analysis, which did not detect any significant contaminating kinase from Sf9 cells. It is noteworthy that an equivalent K to M mutation in Erk2 also did not abolish the basal activity, but rather retained ∼5 % of it(Robbins et al., 1993).

Next, we wanted to understand the functional relevance of tyrosine autophosphorylation using two tyrosine-phosphoablative mutants of IKK2 (FL IKK2-Y188F and FL IKK2-Y169F), both of which showed severe reduction in activity in an in vitro kinase assay (Figure 3E). Since the DFG+1 residue in kinases (Y169 in IKK2) is known to dictate its specificity, we created different single mutants of IκBα where S32 and S36 were independently altered to either alanine (phosphoablative) or glutamic acid (phosphomimetic) residues. FL IKK2-Y169F displayed drastically reduced level of kinase activity towards S36A/E mutants of IκBα while that for the S32A/E mutants were minimally affected compared to the WT substrate (Figure 3F). Reduction of phosphorylation S36A/E by the WT IKK2 was less severe compared to that by the Y169F mutant kinase (Figure S3F). The above observations suggest that the presence of Tyr (and possibly its phosphorylation) is critically important for signal-responsive phosphorylation of IκBα.

Next, we reconstituted IKK2 knock-out mouse embryonic fibroblast (ikk2^-/- MEF) cells with WT, Y169F and Y188F mutants of IKK2, and measured signal-induced activation of IKK2 upon TNF-α treatment. IKK2 activity is significantly reduced in Y169F and nearly abolished in Y188F (Figure 3G). Phosphorylation of activation loop serines of the less active Y169F mutant, nonetheless, was only marginally defective (Figure S3G) suggesting that activation loop phosphorylation is necessary but not sufficient for IKK2 to become fully active. Together, these results hint at an unreported communication between the phosphorylation of Y169 of IKK2 and that of S32 of IκBα.

Structural analyses of IKK2 autophosphorylation

To get a more comprehensive picture, it is necessary to analyze at least three phosphorylated states of IKK2: unphosphorylated (UnP-IKK2), phosphorylated at S177 and S181 (p-IKK2), and phosphorylated at Y169, S177 and S181 (P-IKK2). We took advantage of computational approaches using molecular dynamic (MD) simulations and flexible molecular docking (see methods). MD simulation was performed for 200ns for each of these three states of IKK2, unphosphorylated and phosphorylated. Phosphorylation at S177, S181 increased the stability of the kinase, which was further increased by the phosphorylation at Y169 as evidenced by a gradual decrease in the total energy of system (Figure 4A). A striking enhancement in stability of IKK2 upon this auto-phosphorylation is reflected in the change of Tm from ∼ 40 °C to ∼ 50 °C in differential scanning calorimetry (DSC) scans in the presence of ADP and ATP (Figure S4A). This behavior supports our observation in MD simulation, and tallies with increased solubility of IKK2 observed after auto-phosphorylation that was essential for its successful crystallization (Polley et al., 2013).

Figure 4:

Interestingly, RMSD values increased with more phosphorylation (Figure 4B) despite phosphorylation-induced stabilization, P-IKK2 showed the highest RMSD followed by p-IKK2 and UnP-IKK2, indicating increased internal motion in the kinase as a result of phosphorylation. It may be noted that, the changes in total energy and RMSD are consistent throughout the course of simulation though the magnitude is small. We found that, phosphorylation at S177, S181 and S177, S181, Y169 induced distinct structural alteration in the KD when compared with the unphosphorylated version (Figure 4C, models shown separately in Figure S4B). Careful analyses of the trajectory of RMSF values for these three states revealed that, different regions of the kinase domain had different RMSF values in different phosphorylated states (Figure S4C). We observed that, while the glycine-rich loop and the αC-helix in p-IKK2 were situated in a manner reminiscent of an active kinase, those in UnP and P-IKK2 were placed very differently (Figure 4C). In fact, helix αC was found to be distorted in P-IKK2, that is unlikely to support canonical phosphotransfer from ATP. Activation segment/loop (activation loop) in these three differently phosphorylated states adopted conformations distinct from each other: activation loop in the UnP model was splayed out and moved closer to the tip of the C-lobe, whereas activation loop in p-IKK2 and P-IKK2 moved inwards and adopted conformations closer to the N-lobe (Figure 4C). The formation of the proper R-and C-spines in p-IKK2 confirmed its active form (Figure 4D). p-IKK2 KD also exhibited other canonical features of an active kinase, viz., a salt bridge between K44 and E61 (K72, E91 in PKA, respectively), ‘DFG in’ (DLG here) conformation. In addition, dynamic cross-correlation matrix (DCCM) or contact map of each structure suggests that specific phosphorylation events render the kinase display distinct allosteric changes at locations far from the phosphorylation sites themselves (Figure S4D).

Next, we docked ATP onto the these three phosphorylated-state structures using LeDock and GOLD followed by rescoring with AutoDock Vina using the 0 ns (starting, S) and 200ns (end, E) structures from MD simulation (see methods). It was observed that ATP binding to P-IKK2 is relatively more unfavorable (docking score in positive range) both in the starting and end structures (Figure S4E), whereas UnP and p-IKK2 binds to ATP favorably. Interestingly, each of the phospho-/unphosphorylated IKK2 species binds to ADP favorably except for the P-IKK2 in the starting conformation (Figure S4E). To look into these complexes more carefully, we extracted 50 intermediate structures from a 10 ns MD simulation and calculated binding free energies (ΔG) of these intermediate structures using the MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) method. Clearly, ATP-binding is highly unfavourable for the P-IKK2 population that has high relative preference for ADP, while p-IKK2 shows comparable preference for both (Figure S4F). Among the docked structures, ATP and its phosphate groups exhibited a pose (ΔG = -10.64 kcal/mol) (Figure S4G) very similar to that in the PKA structure (PDB ID: 1ATP), and the terminal phosphate was in close proximity to Y169-OH making autophosphorylation of Y169 highly plausible (Figure 4E). Further analyses of the ATP-bound p-IKK2 structure confirm that presence of ATP helped the R and C-spine form the desired continuum seen in the active form of a kinase (Figure 4F). Superposition of the ATP from the p-IKK2 on P-IKK2 indicated that the narrowing of canonical ATP-binding pocket in P-IKK2 may lead to rejection of ATP from the canonical binding pocket emanating from severe clashes between the glycine-rich loop and ATP (Figure 4G). However, this does not exclude the possibility of P-IKK2 employing alternative binding modes to interact with ATP. Furthermore, the R-spine in P-IKK2 was not continuous (Figure 4H), wherein M65 (L95 in PKA) of the four conserved residues were located at a position far away from the rest of three residues that constitute the R-spine, and from the glycine rich loop.

ATP binding to p-IKK2 leads to autophosphorylation at Y169 generating P-IKK2 in an ADP bound state. Interestingly, the superposition of ADP (from the docked complex of p-IKK2 and ADP, ΔG = -9.88 kCal/mol, Figure 4I) onto the P-IKK2 structure confirmed that while P-IKK2 was unable to accommodate ATP in a manner similar to that in other known experimentally derived kinase structures, it could comfortably accommodate ADP into the ATP-binding cleft (Figure 4J). It may be noted that, p-IKK2 has comparable affinities for ATP and ADP, like other kinases (Becher et al., 2013), as indicated by their respective binding energy/docking score values. Worth noting, cellular concentration of ATP and ADP plays important role in the outcome of this process where ATP concentration is much higher than ADP. We speculate that ADP-bound P-IKK2 is an intermediate step where presence of as many phosphate groups in the activation loop and ADP in such a close proximity persuades the kinase to adopt a conformation that can withstand this high density of negative charges albeit risking the integrity of an active fold, and making some of those de novo phosphate groups transient in nature to return back to the p-IKK2 state to readily bind to ATP that is abound in the reaction.

These structural observations suggest that it is possible for the residues within the flexible activation segment to undergo auto-phosphorylation and possibly render the kinase with a novel phospho-transfer activity distinct from the conventional transfer of γ-phosphate from ATP to the substrate.

Freshly auto-phosphorylated IKK2 phosphorylates IκBα even in absence of ATP

We observed phosphorylation of tyrosine residue(s) only upon fresh ATP-treatment, indicating that tyrosines of purified IKK2 loses its phosphate group during the course of purification (Eckhart et al., 1979). It may be noted that, we did not observe any density of phosphates on any of these residues in the human IKK2 crystal structure derived from ATP-treated protein sample (Polley et al., 2013). Of note, human IKK2 crystals grown with autophosphorylated IKK2 (PDB ID 4E3C) took about a month at 18-20°C before they were subjected to X-ray diffraction. This suggested an intriguing possibility that the phosphate linked to Tyr residue(s) may serve as a temporary phosphate-sink and could eventually be transferred to substrate IκBα. To test this possibility, we first allowed purified IKK2 to undergo auto-phosphorylation by incubating the excess kinase with limiting amounts of radiolabeled γ-³²P-ATP followed by two passes through desalting spin columns (40 kDa MW cut-off) to remove unreacted ATP. Nucleotide-free auto-phosphorylated IKK2 (P-IKK2) was incubated with IκBα either in absence of nucleotide or with newly added unlabeled nucleotides, and phosphorylation status of the substrate was subsequently monitored by autoradiography after resolving the reaction mixture on an SDS-PAGE (Figure 5A). Of note, such an experimental scheme essentially captures a single turnover phosphorylation of the substrate IκBα. Transfer of radiolabeled phosphate to IκBα was observed and the transfer was significantly enhanced in the presence of cold ADP (Figure 5B). Phosphorylation of IκBα did not significantly decrease in the presence of a high concentration of cold ATP. That addition of excess cold ATP did not reduce the extent of IκBα phosphorylation-signal on the autoradiograph strongly suggests the transfer of ³²P-phosphate from ³²P-IKK2 to IκBα (Figure 5B) by a relay mechanism. This transfer perhaps occurs through a phosphoenzyme intermediate with or without involving ADP.

Figure 5:

We undertook a different strategy to further confirm transfer of phosphate from the kinase (and not ATP) to the substrate. We auto-phosphorylated IKK2^FL-WT with excess cold ATP and removed the unreacted ATP from the reaction mixture using size-exclusion chromatography on a Superdex200 column (Figure 5C). P-IKK2 eluted at fractions corresponding to MW between 670 and 158 kDa, i.e., much larger than free ATP (Figure 5D). This P-IKK2 protein was incubated with FL IκBα either in presence or absence of ADP for different time periods. A similar reaction in presence of ATP instead of ADP was performed as a positive control. Phosphorylation of S32 and S36 (detected by the phospho-serine antibody specific to S32 and S36 of IκBα) was efficient in presence of 10 and 50 μM ADP but absent when ADP was absent in the reaction mixture. Phosphorylation of IκBα in the presence of 50 μM ATP was significantly more robust as anticipated (Figure 5E). The minor detection of IκBα phosphorylation in absence of ADP in the radioactive assay and lack of it in the cold immunoblotting assay (compare lanes marked with vertical arrows in Figures 5B and 5F) may indicate higher sensitivity of the radioactive assay. The need of ADP in phosphotransfer prompted us to consider whether the ADP was contaminated with a trace amount of ATP. ESI-MS analysis of 50 μM ADP did not detect any peak corresponding to ATP (Figure S5A). Dependence of IκBα phosphorylation on ADP could raise another possibility if microscopic reversibility generated ATP from ADP in the active site of the kinase. To this end, we employed TLC-based analysis to detect generation/presence of ATP in a similar setting as described in Figure 5A & B which revealed that no detectable ATP was present in the system, i.e., no detectable ATP was retained in the protein after passing it through the spin column twice, nor did any γP³²-ATP was generated when incubated with cold ADP (Figure S5B).

Phosphotransfer from P-IKK2 is critical to fidelity and specificity of IκBα phosphorylation

We next tested if phosphotransfer is restricted only to the N-terminal signal responsive S32/S36 or to serines/threonines of the PEST domain as well. Radioactive kinase assay as described earlier was performed with full-length WT and mutant S32A, S36A (AA) IκBα as the phosphate recipient. Phosphate group(s) of P-IKK2 was not transferred to full-length IκBα AA which retains the serines and threonines within its PEST domain (Figure 6A), indicating phosphotransfer only to the N-terminal serines of IκBα that are S32 and S36. We also tested the effect of AMPPNP, an unhydrolyzable analogue of ATP, on the phosphotransfer, and found no inhibitory effect irrespective of the presence of ADP. This suggests adenine nucleotides poising the kinase for phosphotransfer (Figure 6A). A recent report proposed that IKK2 phosphorylates IκBα at S32 followed by S36 following a single binding event and that phosphorylation of S32 increases the phosphorylation rate of S36(Stephenson et al., 2022). Based on our results and other evidence, we propose that IKK2 phosphorylated at S177/S181 undergoes autophosphorylation on tyrosine residues in its activation loop area that serves as a phosphoenzyme intermediate to transfer phosphate group from IKK2 to S32 that enhances the rate of second site (S36) phosphorylation (Figure 6B). For the second site phosphorylation ATP serves as the phosphate donor.

Figure 6:

Discussion

We report several intriguing properties of IKK2 that contributes to its substrate phosphorylation specificity and efficiency: 1) dual specificity, involving autophosphorylation of several tyrosine residues of IKK2, in addition to known auto-phosphorylation at activation loop serines; 2) phosphate relay to the substrate IκBα via Tyr169 of auto-phosphorylated IKK2 (P-IKK2); and 3) loss of substrate specificity, despite retained catalytic activity, upon disruption of a universally conserved salt bridge mediated by Lys44.

Dual specificity of IKK2

Our present analyses of IKK2, however, revealed its surprising property wherein IKK2 phosphorylated at activation loop-serines (S177/S181) could be further auto-phosphorylated by ATP at multiple other residues yielding another form (P-IKK2) (Figure 6B). This marks IKK2 as an autocatalytic dual-specificity kinase rather than a prototype S/T kinase. This multisite phosphorylated form of IKK2 (P-IKK2) could transfer phosphate(s) to substrate IκBα in presence of ADP without requiring exogenous supply of fresh ATP (Figure 5B & E), hinting at an unusual property of IKK2 acting possibly as a phosphate-sink.

The phospho relay mechanism

Through various experimental approaches, we demonstrated that S32 of IκBα is phosphorylated via a relay mechanism. The transfer of phosphate from Tyr169 to Ser32 of IκBα represents an intriguing and unprecedented mechanism among metazoan protein kinases. We propose a plausible explanation for process: The hydroxyl group of Tyr169 is optimally positioned to readily accept the γ-phosphate from ATP at the expense of minimum energy in presence or absence of the substrate IκBα. The energy required to transfer the phosphate from Tyr169 to Ser32 is lower than that needed for a direct transfer of the same phosphate from ATP to Ser32. Thus, this relay pathway – from ATP to Tyr169 to Ser32 – might offer a more energetically favorable pathway than a direct transfer from ATP to the substrate. Another intriguing feature of phosphorelay in IKK2 is IKK2’s dependence on ADP (Figure 5B & E), which the generic kinase inhibitor AMPPNP (unhydrolyzable ATP analogue) failed to inhibit (Figure 6A). Taken together, we surmise that presence of ADP or AMP-PNP helps IKK2 attain a conformational state poised for phosphorylating S32/36 of IκBα even in absence of fresh exogenous ATP. Similar phenomenon was reported for IRE1, where ADP was found to be a more potent stimulator of its ribonuclease activity and that AMPPNP could also potentiate this activity in IRE1(Lee et al., 2008; Sidrauski and Walter, 1997). We speculate that Ser32 cannot be efficiently phosphorylated by p-IKK2:ATP due to a deliberate chemical mismatch of the hydroxyl group of Ser32 within the active site. However, it aligns well with the P-IKK2:ADP intermediate. To validate this model, further structural studies and MD simulations studies are necessary. Indirect supporting evidence, as described below, underscores the existence of a unique phospho-transfer mechanism.

Tyr169 is the only residue surrounding the active site that can be involved in this relay. As phosphorylation of S32, S36 of IκBα is observed with auto-phosphorylated IKK2 EE, transferred phosphates are not from S177 or S181. Y169 is uniquely located at the DFG+1 (DLG in IKK2) position of the magnesium binding loop. This residue is exclusive to IKK1 and IKK2 and is absent even in close mammalian homologues, IKKε and TBK1. Notably, neither IKKε nor TBK1 can phosphorylate IκBα at S32 and S36, either in vitro or in vivo(Larabi et al., 2013; Tu et al., 2013) supporting the assertion that the phospho-transfer is a genuine and specific event. Recent findings suggest that the DFG+1 residue plays a critical in distinguishing Ser versus Thr residues (Chen et al., 2014). These observations highlight the dual role of Y169 in both substrate selectivity and catalytic activity. This dual selectivity – at residue level (serine versus threonine) and the catalytic level (mediated by relay Tyr169) underpins the exquisite specificity of IκBα for the IKK. Additional mechanisms, such as conformational changes associated with Tyr169 phosphorylation may further enhance the efficiency of the relay by properly positioning Ser32 for phospho-transfer. It is worthwhile to note that, a phospho-resistant mutation at S32 in IκBα to Ile (S32I) is associated with ectodermal dysplasia and T-cell immunodeficiency in patients(Courtois et al., 2003; Mooster et al., 2015). To the best of our knowledge, it is the first report, apart from MHCK of Dictyostelium discoideum (an atypical EPK), to suggest that a metazoan EPK confirms its specificity through the formation of a phosphoenzyme intermediate(Ye et al., 2010).

Tyrosine residues within the activation loop of IKK2 have been previously reported to undergo signal-induced phosphorylation in cells. Although these modifications were proposed to be mediated by a tyrosine kinase, no specific tyrosine kinase has been identified to date(Meyer et al., 2013; Otero et al., 2008; Rieke et al., 2011). Notably, several members of the Ser/Thr kinase family are known to undergo tyrosine phosphorylation, employing diverse mechanisms for autophosphorylation (Bhattacharyya et al., 2006; Ge et al., 2002; Lochhead, 2009; Lochhead et al., 2006, 2005; Tigno-Aranjuez et al., 2010). Regardless of the mechanism responsible for Tyr169 phosphorylation, the phosphate attached to Tyr169 is uniquely primed for transfer to Ser32 of IκBα.

Allostery in phosphorylation

Several other residues, including tyrosines distributed throughout the kinase molecule, also undergo phosphorylation. While most of these phosphorylations play a role in global stabilization of the kinase, Tyr188 plays a critical role in substrate phosphorylation. Mutation of Tyr188—a conserved tyrosine residue—to a non-phosphorylatable phenylalanine completely abolished IKK2 kinase activity both in vitro and in MEF cells following TNF treatment (Figure 3E & G). The structural placement of Tyr188 suggests that its mutation may allosterically disrupt kinase activity, making its precise role in phosphotransfer unclear. Autophosphorylation of kinase domains is a well-documented regulatory mechanism in other kinases as well. For example, autophosphorylation at Thr220 significantly impacts both the activity and substrate specificity of CK1 (Cullati et al., 2022).

Signaling specificity and digital activation

The strength of the input signal, along with the extent of IκBα degradation and NF-κB activation, is closely associated with the amplitude, duration, and kinetics of IKK activity (Behar and Hoffmann, 2013; Cheong et al., 2006). The digital (all-or-none) activation profile of NF-κB appears to arise from the rapid activation and inactivation dynamics of IKK2. While IKK phosphorylates multiple substrates in vivo, the swift activation of NF-κB is predominantly governed by IκBα, with IκBβ and IκBε playing lesser roles. This digital activation profile may be intrinsically linked to the phosphate relay process. We propose that Tyr169 serves as both a selection mechanism and a mediator of the rapid phosphorylation kinetics.

Interestingly, the regulatory system of IKK2 may be somewhat analogous to the two-component histidine kinase-effector systems in prokaryotes, which exhibit threshold activation responses (Bhate et al., 2015; Lamarche et al., 2008; Laub and Goulian, 2007). It is noteworthy that phosphate transfer directly from pHis is energetically far more favorable than from pTyr, except in enzymatic processes. However, Tyr169 might be special case, behaving similar to active site His in histidine kinases.

The conserved salt bridge is specificity determinant not activity

Our study highlights the role of K44 in IKK2 autophosphorylation, which is crucial for the specific phosphorylation of Ser32 and Ser36 of IκBα. Interestingly, while the K44 mutant retains non-specific kinase activity, its phosphorylation specificity is compromised. Notably, a monomeric form of IKK2, lacking the NBD and a large portion of the dimeric interface in the SDD, maintains specificity for the N-terminal serines of IκBα (Hauenstein et al., 2014). However, a truncated construct containing only the KD and ULD loses this specificity, behaving like CK2 and phosphorylating the PEST domain (Shaul et al., 2008). Further studies are needed to determine whether a specific structural state of IKK2, possibly influenced by alternative modifications or interactions with other proteins, enables non-specific substrate phosphorylation.

Conclusion

In all, it is intriguing how the gateway to the activation of NF-κB is regulated through unique phosphorylation events in IKK2. It appears that a multi-layered fail-safe mechanism is devised to tightly control NF-κB activation. In absence of upstream signals, the IKK-complex is unable to phosphorylate Ser 32,36 of IκBα efficiently thereby keeping check on the aberrant and untimely activation of NF-κB. Upon encountering proinflammatory cues, cells need to activate NF-κB immediately, efficiently and specifically, i.e., IKK needs to be activated and phosphorylate Ser 32,36 of IκBα. NEMO warrants IKK-activation and ensures that the IKK-complex specifically chooses IκBα from a large pool of substrates of IKK2. IKK2 is designed in such a manner that it phosphorylates itself at activation loop-tyrosine when activated, such that phosphate group(s) can be relayed directly to Ser32,36 of IκBα with great fidelity, thus leaving little chance of a misconstrued signaling event thereby confirming NF-κB activation. Our discovery might also present the beginning of a new aspect in eukaryotic cell-signaling by EPKs and provide a foundation for mechanistic studies of an intriguing phospho-transfer reaction that helps accomplish the desired specificity.

Methods summary

Protein purification, auto-phosphorylation assay, immunoprecipitation coupled kinase assay, phosphotransfer assay and other experiments have been described in details in the supplementary document.

Acknowledgements

We thank Tony Hunter (Salk Institute) and Kaushik Biswas (BI) for commenting on the work. Authors also thank members of Polley lab (BI) and Ghosh Lab (UCSD) for their continuous support. This work was supported by an Intermediate Fellowship from the DBT Wellcome Trust India Alliance (IA/I/15/1/501852) & intramural funding from Bose Institute to SP, NIH grant to GG (AI163327) and American Cancer Society grant RSG-08-287-01-GMC to TH. Biochemistry research at SDSU is supported in part by the California Metabolic Research Foundation. PB was supported by the graduate research fellowship from Bose Institute. SC acknowledges CSIR-IICB for infrastructure support. AC acknowledges ICMR for post-doctoral Research Associateship [BMI/11(55)2022].

Additional information

Author contribution

SP, GG and TB designed experiments; PB, SP, TB & GG performed experiments. Computational analyses were performed by AC & SC. SP, GG, TB, PB, TH, SC and AC analyzed the results. SP wrote the manuscript with the help of PB, TB and AC in preparing figures and images. GG, TB, PB and TH edited/modified the manuscript. All authors commented on the manuscript.

Additional files

Materials and methods.