IntroductionUbiquitination, a highly important and versatile reversible post-translational modification (PTM) in mammals, participates in almost all vital cellular processes, including protein degradation, DNA repair, gene transcription, autophagy, and innate immunity1,2,3,4,5,6. The ubiquitination of a target substrate is mainly achieved through the cooperative actions of three different enzymes: a ubiquitin (Ub)-activating E1 enzyme, a Ub-conjugating E2 enzyme and a Ub ligase E3 enzyme3,7,8. Since Ub can be modified by itself to form poly-Ub chains, there are totally eight different types of homotypic poly-ubiquitination (M1-, K6-, K11-, K27-, K29-, K33-, K48-, and K63-linked type), in addition to mono-ubiquitination9,10. M1-linked poly-ubiquitination (also named linear poly-ubiquitination) is a unique type of poly-ubiquitination, in which the poly-Ub chain is formed by Ub units covalently linked by peptide bonds in a head-to-tail manner11,12. The linear ubiquitin chain assembly complex (LUBAC) is the only currently known E3 ligase complex that mediates linear Ub chain formation, and is composed of three subunits: the catalytic subunit HOIP, and two regulatory subunits, Sharpin and HOIL-1L11,13,14,15,16,17. LUBAC has been demonstrated to play crucial roles in many cellular processes, including NF-κB and Wnt signaling pathways, autophagy, and mitosis11,16,18,19,20,21,22,23,24. Importantly, dysfunction of LUBAC due to the deficiency of any subunit of LUBAC causes immunodeficiency, inflammation or even death in mice or humans19,25,26,27,28. Given the critical roles played by LUBAC in various immune and inflammation-related signaling pathways, the activity of LUBAC has been precisely tuned temporally and spatially by other regulatory proteins29,30,31,32. However, many of the detailed molecular mechanisms underlying LUBAC regulation by these proteins are still not well understood.HOIP belongs to the RING-between-RING (RBR) type E3 ligase, characterized by an RBR module that includes RING1, IBR and RING2 domains (Fig. 1a). Previous elegant structural studies have well demonstrated that the RBR and LDD domains of HOIP pack together to form the catalytic core of HOIP for mediating the conjugation of linear Ub chains33,34. Specifically, the RING1 domain together with the RING2 and LDD domains of HOIP can recognize the E2 ~ Ub conjugate and catalyze the transfer of Ub from the E2~Ub conjugate to the catalytic Cys residue in the RING2 domain of HOIP33,34. Subsequently, Ub continues to translocate and form the linear Ub chain, a process mediated by a unique Ub-binding platform assembled by the RING2-LDD module of HOIP33,34. Interestingly, the RING1 and IBR region of HOIP also harbors an allosteric Ub-binding site, where Ub binding can dramatically increase the E3 activity of HOIP34. Notably, other RBR-type E3 ligases, such as Parkin and HOIL-1L, also contain similar allosteric Ub-binding sites35,36,37,38. Given the critical role of this allosteric Ub-binding site for RBR-type E3 ligases, whether the allosteric Ub-binding site of HOIP could be regulated by other types of PTM remains an open question.Fig. 1: Biochemical characterization of the interaction between HOIP and STK4.a Schematic diagram showing the domain organizations of HOIP, HOIL-1L, Sharpin and STK4. In the drawing, the inter-molecular interactions of HOIP, HOIL-1L and Sharpin are indicated by black two-way arrows, while the HOIP/STK4 interaction characterized in this study is highlighted by red two-way arrows. b The summarized mapping results of the binding regions between HOIP and STK4 based on our SEC-based analyses. c SEC-based analyses of the interaction between 20 μM HOIP RING2-LDD and 30 μM STK4 KD. The “SUM” stands for the theoretical sum of the SEC profiles of STK4 (residues 1–326) K59R and HOIP (residues 854–1072). This assay was performed using a Superdex 75 Increase 10/300 GL column (GE Healthcare). d ITC-based measurement of the binding affinity of HOIP RING2-LDD and STK4 KD. The Kd error is the fitted error obtained from the data analysis software when using the one-site binding model to fit the ITC data; DP, differential power, measured by the ITC machine; ΔH, heat change measured by the ITC machine. e Overlay plot of the sedimentation velocity data of HOIP RING2-LDD (blue), STK4 KD (red), and the mixture of HOIP RING2-LDD with STK4 KD in a molar ratio of 1:1 (green) or 1:5 (black); c(s) continuous sedimentation coefficient distribution; MW molecular weight. The relevant experiments depicted in this figure have been replicated three times.Full size imageKinase-mediated phosphorylation is one of the most fundamental and pervasive PTMs in biology, playing a critical role in regulating nearly all aspects of cellular processes, from metabolism to signal transduction, gene expression and cell growth39,40. Given the pervasive role of phosphorylation in regulating protein activity and interactions, it was expected that phosphorylation could influence the E3 ligase activity of LUBAC. Indeed, the E3 activity of HOIP was recently reported to be regulated by the kinase STK4 (also known as MST1)32. Particularly, STK4 can directly phosphorylate HOIP and inhibit its E3 activity, thereby attenuating the LUBAC-mediated activation of the NF-κB signaling pathway32. However, the detailed molecular mechanism underpinning the inhibition of HOIP’s E3 activity by STK4-mediated phosphorylation remains elusive.STK4, a member of the Group II germinal center kinases, plays vital roles in various biological processes in mammals, including cell growth, cell differentiation, stress response, and apoptosis41,42. Structurally, STK4 primarily contains an N-terminal conventional kinase domain (KD) and a C-terminal coiled-coil SARAH (Salvador/RASSF1/Hippo) domain (Fig. 1a), which enables STK4 to homodimerize or heterodimerize with other relevant SARAH domain-containing proteins43,44. As a serine/threonine kinase, STK4 specifically recognizes and phosphorylates dozens of protein substrates, including Mob1A/Mob1B, Lats1/Lats2, histone H2B, FOXO3 and HOIP, thereby regulating their relevant cellular functions32,41,45,46,47,48. However, due to the absence of complex structures, the precise mechanism by which STK4 specifically recognizes its substrates remains largely unknown.Here, we biochemically and structurally characterize the interaction between STK4 and LUBAC, discovering that STK4 directly binds to the RING2-LDD region of HOIP through its KD. We determine the crystal structure of the HOIP RING2-LDD in complex with STK4 KD, which not only elucidates the detailed molecular mechanism governing the specific association of STK4 with LUBAC, but also represents the first atomic structure, to our knowledge, showing how STK4 kinase recognizes its substrate. Additionally, based on our biochemical, mass spectrometry and structural analyses, we demonstrate that STK4 inhibits the E3 activity of HOIP by phosphorylating the T786 residue within HOIP’s allosteric Ub-binding site. In all, our findings uncover the molecular basis underlying the negative regulation of the E3 activity of HOIP by STK4 and unveil a unique regulatory mode for the RBR-type E3 ligase HOIP.ResultsSTK4 can specifically bind to the RING2-LDD region of HOIP through its KDTo elucidate how STK4 specifically regulates LUBAC activity, we first aimed to biochemically characterize the interaction of STK4 with LUBAC. Given that kinase-active STK4 exists in an inhomogeneous oligomer state44, we constructed the K59R mutation of STK4. This mutation, based on previous studies44,49, specifically abolishes the kinase activity of STK4, yielding kinase-dead full-length STK4 (residues 1–487) K59R proteins suitable for further biochemical characterization. Using Size Exclusion Chromatography (SEC)-based assays with purified STK4 (residues 1–487) K59R mutant, as well as relevant HOIL-1L and Sharpin proteins, we revealed that the STK4 (residues 1–487) K59R mutant was unable to interact with the full-length HOIL-1L and Sharpin (Supplementary Fig. S1). However, STK4 (residues 1–487) K59R directly bound to the HOIP (residues 480–1072) fragment, the HOIP (residues 480–1072)-containing truncated LUBAC complex, and the HOIP (residues 697–1072) fragment that includes the RING1-IBR-RING2-LDD module (Fig. 1b; Supplementary Figs. S2, S3a–e). Conversely, it did not bind to the HOIP (residues 1–480), HOIP (residues 480–639), or HOIP (residues 639–698) fragments (Fig. 1b; Supplementary Fig. S3f–h). Our isothermal titration calorimetry (ITC)-based analyses revealed that STK4 (residues 1–487) K59R directly binds to HOIP(residues 697–1072) with a dissociation constant (Kd) value of ∼102 μM (Supplementary Fig. S4a). Importantly, the HOIP (residues 854–1072) fragment, containing only the RING2 and LDD domains, also bound to STK4 (residues 1–487) K59R with a similar Kd value (Supplementary Fig. S4). These findings collectively indicated that the RING2-LDD region of HOIP is responsible for interacting with STK4.Subsequently, using SEC-based assays, we also mapped the HOIP-binding region of STK4. Our results revealed that the KD, rather than the SARAH domain, of STK4 directly interacts with HOIP (residues 697–1072) (Fig. 1b; Supplementary Fig. S5a, b). Further truncation analyses confirmed that STK4 (residues 1–326) K59R directly binds to the RING2-LDD module but not the RING1-IBR region of HOIP (Fig. 1c; Supplementary Fig. S5c). Concurrently, our quantitative ITC analyses confirmed that STK4 (residues 1–326) K59R directly binds to HOIP (residues 854–1072) (RING2-LDD) and HOIP (residues 697–1072) (RING1-IBR-RING2-LDD) with similar binding affinities (Fig. 1d; Supplementary Fig. S6a). Notably, the binding affinity of STK4 (residues 1–487) K59R with HOIP (residues 854–1072) is much weaker than that of the STK4 (residues 1–326) K59R/HOIP (residues 854–1072) interaction (Fig. 1d; Supplementary Fig. S4b), suggesting that other regions of STK4 can partially inhibit its KD from binding to HOIP, consistent with previous findings that full-length STK4 exists in an auto-inhibited state44. To facilitate our structural studies, we further narrowed down the KD region of STK4 for HOIP binding using ITC-based analyses. Our ITC results showed that STK4 (residues 11–326) K59R (hereafter referred to as STK4 KD), which lacks the N-terminal unconserved 10 residues of STK4 (residues 1–326) K59R (Supplementary Fig. S7), still interacts well with HOIP compared with STK4 (residues 1–326) K59R (Fig. 1d; Supplementary Fig. S6). Meanwhile, analytical ultracentrifugation-based analyses revealed that STK4 KD exists in a concentration-dependent monomer-dimer equilibrium (Supplementary Fig. S8), while HOIP (residues 854–1072) is a stable monomer in solution (Fig. 1e). Remarkably, STK4 KD can directly interact with HOIP(residues 854–1072) to form an unstable binary complex (Fig. 1e), likely due to the weak and dynamic nature of their interaction. In summary, our biochemical results clearly demonstrated that STK4 directly binds to HOIP through a specific interaction between STK4 KD and the HOIP RING2-LDD region.Overall structure of HOIP RING2-LDD in complex with STK4 KDTo uncover the mechanistic basis underlying the specific interaction between HOIP RING2-LDD and STK4 KD, we decided to solve the atomic structure of the HOIP (residues 854–1072)/STK4 KD complex using X-ray crystallography. However, conventional purification methods failed to yield a pure HOIP (residues 854–1072)/STK4 KD complex, likely due to the dynamic nature of the interaction between HOIP RING2-LDD and STK4 KD. Therefore, we only mixed HOIP (residues 854–1072) and STK4 KD in a 1:1 molar ratio, and conducted relevant crystal screening using this protein mixture. Fortunately, this approach successfully yielded good crystals that diffracted to a 2.8 Å resolution (Supplementary Table S1).Subsequently, we determined the crystal structure of this HOIP RING2-LDD/STK4 KD complex using the molecular replacement method with modified structures of HOIP RING2-LDD (PDB ID: 4LJO) and STK4 KD (PDB ID: 3COM) serving as search templates (Supplementary Table S1). In the final crystal structure, each asymmetric unit contains one STK4 KD dimer and two HOIP RING2-LDD molecules, forming a symmetrical butterfly-like hetero-tetramer (Fig. 2a, b). Intriguingly, within the HOIP RING2-LDD/STK4 KD complex, the two STK4 KD monomers, each adopting a canonical KD-fold consisting of an N-lobe and C-lobe (but lacking an ATP molecule due to the K59R mutation), directly pack with each other to form a symmetrical dimer through their N-lobes (Fig. 2a, b).Fig. 2: Structural analyses of the HOIP RING2-LDD in complex with STK4 KD.a Ribbon diagram showing the overall structure of the HOIP RING2-LDD/STK4 KD complex. b Surface representation of the HOIP RING2-LDD/STK4 KD complex. c The combined surface representation and the ribbon-stick model showing the hydrophobic interaction interface between HOIP RING2-LDD and STK4 KD. In this presentation, the STK4 KD is shown in the surface model, and the key residues of HOIP RING2-LDD involved in the interaction between them are shown in the stick-ball model. The hydrophobic amino acid residues in the surface model of STK4 KD are drawn in yellow, the positively charged residues in blue, the negatively charged residues in red, and the uncharged polar residues in gray. d Stereo view of the ribbon-stick model showing the detailed interactions between HOIP RING2-LDD and STK4 KD. In this drawing, the relevant side chains as well as backbone groups of the key binding interface residues are shown in the stick-ball mode, and the related hydrogen bonds and salt bridges involved in the binding are shown as dotted lines. e Summary of the binding affinities between HOIP RING2-LDD and STK4 (residues 1–326) K59R or their mutants measured by ITC-based analyses. f Mutagenesis-based Co-IP assay to confirm the interaction between HOIP and STK4 observed in the determined complex structure. IB, immunoblot. The experiments have been replicated three times.Full size imageThe overall structure of STK4 KD in the HOIP RING2-LDD/STK4 KD complex is highly similar to that of the previously solved active STK4 KD (PDB ID: 3COM) (Supplementary Fig. S9a). However, STK4 KD in our complex possesses additional N- and C-terminal extension structures. Furthermore, several residues but not the key “DFG” motif within the activation loop are missing in STK4 KD of the HOIP RING2-LDD/STK4 KD complex, likely due to the flexible conformation of the activation loop (Supplementary Fig. S9a). In parallel, the RING2 and LDD modules of each HOIP RING2-LDD pack together to form a unique architecture that is mainly composed of six α-helices and six β-strands, and coordinates four zinc ions (Fig. 2a). Specifically, through its RING2 region together with the β5 and β6 strands within the LDD domain, HOIP RING2-LDD specifically binds to the C-lobe of STK4 KD (Fig. 2a, b). Structural comparison analyses showed that the overall structure of HOIP RING2-LDD in the HOIP RING2-LDD/STK4 KD complex is similar to that of HOIP RING2-LDD in the HOIP RING2-LDD/mono-Ub complex (PDB ID: 4LJO)33, or the HOIP RBR-LDD/UBE2D2~Ub complex (PDB ID: 5EDV)34, except the N-terminal α1-helix region of HOIP RING2, which is directly involved in the binding with STK4 (Supplementary Fig. S9b, c).The molecular interface of the HOIP RING2-LDD/STK4 KD complexDetailed structure analyses uncovered that the binding interface between HOIP RING2-LDD and STK4 KD is primarily formed by residues from the α7-helix of STK4 KD, which accommodates HOIP residues located in the α1-helix and β2-strand of HOIP RING2, as well as the β5- and β6-strands of HOIP LDD through both hydrophobic and polar interactions (Fig. 2a–d; Supplementary Fig. S10). This interface buries a total area of ~553.8 Å (Fig. 2b). Specifically, the side chains of S877, Q974 and D983 of HOIP form hydrogen bonds with the side chains of STK4 T238 and R231, and the backbone amide group of STK4 H228, respectively. Moreover, the side chain of STK4 N239 couples with the backbone carbonyl group and amino group of HOIP S877 to form two highly specific hydrogen bonds (Fig. 2d). In addition, the hydrophobic side chain of HOIP M862 packs against the hydrophobic patch formed by the M230 and F234 residues of STK4, and the aliphatic side chain groups of HOIP Q865 and E866 form hydrophobic contacts with the hydrophobic side chain of the STK4 F234 residue (Fig. 2c, d). Concurrently, the hydrophobic side chain of HOIP A879 inserts into the hydrophobic pocket formed by the hydrophobic side chains of HOIP F234 and M235, as well as the aliphatic side chain groups of HOIP R231 (Fig. 2c, d). Moreover, an Arg-Glu pair (Arg231STK4-Glu983HOIP) of salt bridge further strengthens the HOIP RING2-LDD/STK4 KD interaction (Fig. 2d; Supplementary Fig. S10).In line with their important structural roles, all key interface residues of HOIP and STK4 are highly conserved throughout evolution (Supplementary Figs. S2, S7). Using SEC- and ITC-based analyses, we validated the specific interactions between HOIP RING2-LDD and STK4 KD observed in the complex structure. Consistent with our aforementioned structural data, the results showed that point mutations of key interface residues either from HOIP RING2-LDD or STK4 KD, such as the D983A and A879R mutations of HOIP, or the R231E and N239A mutations of STK4, significantly reduced or essentially abolished the specific interaction between HOIP RING2-LDD and STK4 KD (Fig. 2e; Supplementary Figs. S11, S12). In line with these in vitro biochemical results, our Co-IP experiments confirmed that point mutations of key interface residues, specifically the R231E and N239A mutations of STK4, largely attenuate the specific interaction of STK4 and HOIP in cells (Fig. 2f)The dimerization interface of STK4 KD in the HOIP RING2-LDD/STK4 KD complexConsistent with our aforementioned biochemical result that STK4 K59R can form a dimer in solution (Supplementary Fig. S8), further crystallographic symmetry analysis revealed that STK4 KD exists as a homo-dimer in the crystal structure of the HOIP RING2-LDD/STK4 KD complex, with a total dimerization surface area of ~1103 Å2 (Fig. 2a, b). Detailed structural analysis uncovered that the dimerization interface of STK4 KD is primarily formed by residues from the β1, β2, β3, β4, β5 strands, as well as the α2 helix (the conventional αC-helix in the kinase field) located in the N-lobe of STK4 KD through extensive hydrophobic and polar interactions (Supplementary Fig. S13a–c). In particular, a hydrophobic patch formed by the hydrophobic side chain groups of L67, I71, I74, Y88, F93, and L98 residues, together with a hydrophobic patch assembled by the hydrophobic side chain groups of L22, T23, V29, F30, H49, V56, Y89 and the aliphatic side chain groups of K24, E27 residues of STK4, pack against their counterparts in other STK4 to form two large hydrophobic interfaces of STK4 dimer (Supplementary Fig. S13a, b). In parallel, the dimer formation of STK4 is stabilized by several hydrogen bonds formed between the side chains of T23, E27, Q54, S75, and Q78 from one chain of the STK4 dimer and their corresponding counterparts in the other chain (Supplementary Fig. S13a, c).In line with this structural data, our analytical ultracentrifugation analyses showed that mutating the STK4 F93 residue, located at the dimerization interface of STK4 KD, to a polar Asn residue can attenuate the dimerization ability of STK4 KD in solution, especially under low concentration conditions (Supplementary Fig. S13d, e). Notably, a similar dimerization mode was also observed in the previously determined structure of STK3 KD in complex with a selective inhibitor50 (Supplementary Fig. S13f), suggesting a common dimerization mechanism shared by the KDs of STK4 and STK3. Based on a previous study32, STK4 can directly phosphorylate HOIP. To test whether the dimerization of STK4 KD domain could affect HOIP phosphorylation mediated by STK4, we performed relevant in vitro phosphorylation assays. Our results showed that the F93N mutant of either full-length STK4 or STK4 KD, which loses the dimerization ability of the KD domain, can still phosphorylate HOIP effectively, similar to wild-type (WT) STK4 (Supplementary Fig. S14). This data suggested that the dimerization of STK4 KD is not essential for the in vitro phosphorylation of HOIP mediated by STK4.The phosphorylation of HOIP S1066 does not affect the E3 activity of HOIP in vitroA recent study reported that STK4 can directly phosphorylate the S1066 residue of HOIP, thereby interfering with Ub recognition by HOIP LDD and inhibiting the E3 activity of LUBAC32. Unexpectedly, using in vitro linear ubiquitination assays, we revealed that the phosphomimic S1066E mutant of HOIP (residues 697–1072) exhibits a similar ability to catalyze the formation of linear ubiquitin chains as WT HOIP (residues 697–1072) (Supplementary Fig. S15a). Furthermore, our in vitro linear ubiquitination results using either WT or the S1066A mutant of HOIP (residues 697–1072) showed that STK4 can inhibit the E3 activities of both constructs (Supplementary Fig. S15b, c). This suggests that the phosphorylation of other HOIP sites, rather than S1066, is responsible for the STK4-imposed inhibition of HOIP’s E3 activity. Importantly, using SEC-based assays, we confirmed that the phosphomimic S1066E mutant of HOIP (residues 697–1072) can interact well with linear di-Ub (M1-2Ub) or tetra-Ub (M1-4Ub) (Supplementary Fig. S16), which contrasts with the previously reported result32. Based on these findings, we concluded that the phosphorylation of HOIP S1066 is unlikely to affect the E3 activity of HOIP.The phosphorylation of HOIP T786 is mainly responsible for the inhibition of the E3 activity of HOIP by STK4Our study revealed that STK4 KD can directly interact with the RING2-LDD of HOIP (Fig. 2). Interestingly, structural comparison analysis revealed that the binding of STK4 KD to the RING2-LDD of HOIP should hinder the binding of the donor Ub from the E2~Ub conjugate to HOIP due to the potential steric exclusion (Supplementary Fig. S17). However, using in vitro ubiquitination assays, we demonstrated that WT STK4 effectively inhibits the E3 activity of LUBAC, while kinase-dead K59R and T183A mutants of STK4 do not (Fig. 3a, b; Supplementary Fig. S18). Thus, the suppression of HOIP’s E3 activity by STK4 is primarily mediated by STK4’s kinase activity, rather than simply its binding.Fig. 3: Phosphorylation of HOIP T786 residue mediated by STK4.a, b In vitro linear ubiquitination assays showing the catalytic activity of the LUBAC complex can be blocked by the WT STK4 (a) but not the STK4 K59R mutant (b). The LUABC complex used in this assay consists of HOIP (residues 480–1072), the full-length HOIL-1L C460A mutant and the full-length Sharpin purified from E coil. c In vitro phosphorylation assays showing that HOIP (residues 697–1072) can be specially phosphorylated by the WT STK4 (residues 1–487) or STK4 (residues 1–326). The gels in the lower panels are normal SDS-PAGE gels, while those in the upper panels are phos-tag gels. In these panels, p-HOIP stands for phosphorylated HOIP (residues 697–1072). d This figure shows a higher-energy collisional dissociation (HCD) MS/MS spectrum recorded on the [M + 3H]3+ ion at m/z 376.1960 of the HOIP peptide KLtEGVLMR harboring one phosphorylated site (denoted by lowercase t). Predicted b- and y-type ions (not including all) are listed above and below the peptide sequence, respectively. Ions observed are labeled in the spectrum, revealing that HOIP T786 is phosphorylated. Notably, the KD of STK4 was used to phosphorylate HOIP (residues 697–1072) for the MS-based identification of phosphorylation sites on HOIP. e Cartoon combined with the stick-ball model of Ub binding to HOIP RING1-IBR (PDB ID: 5EDV) showing that the phosphorylation of HOIP T786 residue should affect the Ub binding to the allosteric site of HOIP RING1-IBR. Meanwhile, the T786, V789, L790 residues of HOIP and the L8, I44, V70 and H68 residues of ubiquitin are shown in the stick-ball mode. f In vitro linear ubiquitination assays showing the abilities of the WT HOIP (residues 697–1072) and the phosphomimic HOIP (residues 697–1072) T786E mutant to assemble linear ubiquitin chains. Notably, the T786E mutation of HOIP (residues 697–1072) largely reduces the E3 activity of HOIP. g In vitro ubiquitination assays in the presence of STK4 showing that the E3 activity of HOIP T786A mutant is unable to be suppressed by STK4-mediated phosphorylation. h, i In vitro ubiquitination assays using the WT HOIP (residues 697–1072) (h) the HOIP (residues 697–1072) T786A mutant (i) or their phosphorylated counterparts, showing that the E3 activity of HOIP is suppressed by STK4-mediated phosphorylation rather than STK4-binding. The relevant experiments depicted in this figure have been replicated three times.Full size imageTo identify the key phosphorylation site on HOIP responsible for STK4-mediated suppression of E3 activity of HOIP, we first performed relevant in vitro phosphorylation assays. The results showed that both full-length STK4 and STK4 (residues 1–326) can effectively phosphorylate the HOIP (residues 697–1072) fragment (Fig. 3c; Supplementary Fig. S19a). Subsequent mass spectrometry-based analysis confidently identified 12 phosphorylation sites on the HOIP (residues 697–1072) fragment, phosphorylated by either full-length STK4 or STK4 (residues 1–326): T715, T720, T731, T756, T764, S772, T786, T823, S840, T891, T955, and S1066 (Fig. 3d; Supplementary Fig. S19b, c). Notably, STK4 can also directly phosphorylate the HOIP (residues 480–1072) subunit within the LUBAC complex (Supplementary Fig. S19d). Mass spectrometry analyses confirmed that all 12 phosphorylation sites identified in isolated HOIP (residues 697–1072) were also present in the STK4-phosphorylated HOIP (residues 480–1072) subunit of the LUBAC complex (Supplementary Fig. S19e). Interestingly, careful structural analyses of the 12 phosphorylation sites of HOIP (residues 697–1072) revealed that the T786 site is located within the allosteric Ub-binding site of RING1 (Fig. 3e; Supplementary Fig. S19f). The binding of a Ub entity to this site is known to dramatically enhance the E3 activity of HOIP34. Our sequence alignment analysis revealed that the T731, T764, T786, S840, T891 and T955 residues of HOIP are highly conserved across different eukaryotic species (Supplementary Fig. S2). Therefore, we just focused on these six conserved residues for further characterization. Using in vitro ubiquitination assays with relevant phosphomimic mutants, we demonstrated that the S840E and T955E mutations of HOIP (residues 697–1072) had little effect on the E3 activity of HOIP, while the T731E, T764E, T786E and T891E mutations of HOIP (residues 697–1072) significantly reduced HOIP’s E3 activity for catalyzing linear ubiquitin chain formation (Fig. 3f; Supplementary Fig. S20). Thus, we preliminarily identified the HOIP T731, T764, T786 and T891 residues as potential key phosphorylation sites mediated by STK4.We then individually mutated these four residues to an unphosphorylable alanine residue. Using in vitro ubiquitination assays, we found that only the E3 activity of HOIP (residues 697–1072) T786A could not be effectively inhibited by STK4 (Fig. 3g; Supplementary Figs. S21, S22). It is worthwhile noting that the HOIP T731A and T786A mutants exhibited lower activities than their WT counterpart in our in vitro ubiquitination assays (Supplementary Figs. S21, S22a, b). Relevant structural analysis revealed that HOIP T731 is located within the α1-helix of HOIP RING1, and its hydroxyl side chain group forms a specific hydrogen bond with the backbone group of R727, stabilizing the formation of the RING1 α1-helix, which directly participates in the interaction with E2 in the HOIP RBR-LDD/UBE2D2~Ub/Ub complex structure (PDB ID: 5EDV) (Supplementary Fig. S23a). Thus, we speculated that the T731A mutation of HOIP might affect the interaction of HOIP RING1 with E2, thereby decreasing the enzymatic activity of HOIP for assembling linear ubiquitin chains. Indeed, our SEC-based analyses revealed that the T731A mutation of HOIP RING1 (residues 697–793) caused a detectable reduction of the interaction of HOIP RING1 with UBE2L3 (Supplementary Fig. S23b–d). Meanwhile, based on our structural analysis, the HOIP T786 residue directly participates in the interaction of HOIP RING1 with the allosteric ubiquitin (Fig. 3e). Therefore, the T786A mutation should impair the binding ability of HOIP for the allosteric ubiquitin, consequently affecting the activation of the linear ubiquitination activity of HOIP. In line with our biochemical data, structural modeling analysis showed that the RBR-LDD module of HOIP exhibits considerable conformational flexibility (Supplementary Fig. S24). This flexibility allows relative movement between its N-terminal RING1-IBR module and C-terminal RING2-LDD region, thereby permitting the T786 residue within HOIP RING1 to be accessible for STK4 KD-mediated phosphorylation (Supplementary Fig. S24).Finally, using purified phosphorylated HOIP (residues 697–1072) and HOIP (residues 697–1072) T786A mutant proteins (Supplementary Fig. S25), we demonstrated that the E3 activity of the phosphorylated HOIP (residues 697–1072), but not the phosphorylated HOIP (residues 697–1072) T786A mutant, was largely reduced compared with that of the un-phosphorylated HOIP counterpart (Fig. 3h, i). This directly revealed that STK4 primarily inhibits HOIP’s E3 activity through STK4-mediated phosphorylation of HOIP T786. In conclusion, our findings indicated that phosphorylation of HOIP T786, rather than S1066, is mainly responsible for the STK4-mediated inhibition of HOIP’s E3 activity.The T786 phosphorylation site of HOIP is critical for regulating the NF-κB signalingTo test whether the identified T786 phosphorylation site of HOIP is relevant in cells, we conducted several cellular assays to investigate its role in the NF-κB signaling. Consistent with previous studies14,15, co-expression of WT HOIP with HOIL-1L and Sharpin generated large amounts of M1-linked Ub chains in HeLa cells (Fig. 4a). Strikingly, co-expression of the phosphomimic HOIP T786E mutant, but not the HOIP S1066E mutant, with HOIL-1L and Sharpin, was essentially unable to promote the formation of the M1-linked Ub chains in HeLa cells (Fig. 4a).Fig. 4: STK4-mediated phosphorylation of HOIP T786 attenuates the intracellular NF-κB signaling and the transfer of ubiquitin from the E2~Ub conjugate to the catalytic cysteine of HOIP.a In vivo linear ubiquitination assay showing the abilities of the WT HOIP, the phosphomimetic T786E and S1066E mutants of HOIP to assemble linear Ub chains in HeLa cells when co-expressing with HOIL-1L and Sharpin. b NF-κB reporter dual-luciferase assays using overexpressed HOIL-1L and Sharpin without or with the WT HOIP, the HOIP T786E mutant or the HOIP S1066E mutant in HEK293T cells. Error bars denote the SEM between three replicates. An unpaired Student’s t-test analysis was used to define a statistically significant difference, and the stars indicate significant differences between the indicated bars (***P