DFG Motif Research Articles

Allosteric inhibition of c-Met kinase in sub-microsecond molecular dynamics simulations induced by its inhibitor, tivantinib.

Protein dynamics in the allosteric regulation of enzymes is crucial for understanding the regulation mechanism of enzymes and designing of inhibitors. Kinases have a conserved Asp-Phe-Gly motif (DFG motif) whose conformation determines the activation state of the kinase; however, knowledge on conformational transition of the DFG motif from the active state to the inactive state ("DFG-flip") is quite limited. Here we report a DFG-flip of c-Met kinase in molecular dynamics (MD) simulations, induced by its allosteric inhibitor tivantinib. Our MD simulations showed that, with the assistance of tivantinib, c-Met may transit from the DFG-in state to the DFG-out state in a sub-microsecond time-scale. A unique binding mode of tivantinib to c-Met was identified to be the key intermediate for the ligand-induced DFG-flip. This study provides a detailed process of inhibitor-induced kinase allostery, as well as important insights into the DFG-flip mechanism and the design of allosteric inhibitors, not only of c-Met, but also of other kinases.

Biophysical changes of ATP binding pocket may explain loss of kinase activity in mutant DAPK3 in cancer: A molecular dynamic simulation analysis

DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42–Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3.

Cooperative Activity of BRAF F595L and Mutant HRAS in Histiocytic Sarcoma Provides New Insights into Oncogenic BRAF Signaling

Activating BRAF mutations, in particular V600E/K, drive many cancers, including a substantial proportion of systemic histiocytic disorders, and mutant BRAF-selective inhibitors are promising therapeutics for these diseases. Activating BRAF alleles are considered mutually exclusive with mutations in RAS family members, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif can coexist with oncogenic RAS and cooperate via transactivation of wildtype RAF proteins and paradoxical MEK/ERK activation. Due to the increasing use of global approaches to tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We used several in vitro experimental systems, including Braf-deficient murine embryonic fibroblasts expressing a regulatable HRAS oncogene, to determine the biochemical properties and cellular effects of a largely uncharacterized mutation, F595L, in the DFG motif of the BRAF activation segment that was identified by clinical exome sequencing in a patient with histiocytic sarcoma and multiorgan involvement and also occurs as somatic alteration in colorectal adenoma or carcinoma, non-small cell lung cancer, cholangiocarcinoma, urothelial cancer, melanoma, and neuroblastoma and as germline mutation in cardio-facio-cutaneous syndrome. In addition, we investigated the interaction between BRAF F595L and a concomitant HRAS Q61R allele, which was present in the same tumor cell clone and occurs as acquired alteration in multiple tumor types and as inherited variant in Costello syndrome. Unlike previously described DFG motif mutants, BRAF F595L is a gain-of-function variant with intermediate activity towards MEK that, in sharp contrast to BRAF V600E, requires an intact dimer interface for downstream signaling. Furthermore, BRAF F595L does not act paradoxically, but nevertheless cooperates with mutant HRAS to induce maximal activity of the MEK-ERK signaling pathway. Of immediate clinical relevance, BRAF F595L shows divergent responses to the mutant BRAF-selective inhibitors vemurafenib and dabrafenib, whereas signaling driven by BRAF F595L with and without mutant HRAS is efficiently blocked by the pan-RAF inhibitors sorafenib and AZ628 and the MEK inhibitor trametinib. Consistent with this, sorafenib treatment led to abrogation of aberrant MEK/ERK signaling in the index case with histiocytic sarcoma driven by BRAF F595L and HRAS Q61R. Mutation data from patients and cell lines, representing 18 different tumor entities, show that BRAF F595L as well as other BRAF mutants with intermediate signaling activity coincide with mutant RAS in at least 40% and 23% of cases, respectively. These data define a distinct class of activating BRAF mutations that cooperate with oncogenic RAS in a non-paradoxical fashion, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway, and underscore the value of comprehensive genomic profiling for uncovering the vulnerabilities of individual tumors. DisclosuresOff Label Use: Administration of sorafenib in a patient with histiocytic sarcoma.

Towards a Molecular Understanding of the Link between Imatinib Resistance and Kinase Conformational Dynamics.

Due to its inhibition of the Abl kinase domain in the BCR-ABL fusion protein, imatinib is strikingly effective in the initial stage of chronic myeloid leukemia with more than 90% of the patients showing complete remission. However, as in the case of most targeted anti-cancer therapies, the emergence of drug resistance is a serious concern. Several drug-resistant mutations affecting the catalytic domain of Abl and other tyrosine kinases are now known. But, despite their importance and the adverse effect that they have on the prognosis of the cancer patients harboring them, the molecular mechanism of these mutations is still debated. Here by using long molecular dynamics simulations and large-scale free energy calculations complemented by in vitro mutagenesis and microcalorimetry experiments, we model the effect of several widespread drug-resistant mutations of Abl. By comparing the conformational free energy landscape of the mutants with those of the wild-type tyrosine kinases we clarify their mode of action. It involves significant and complex changes in the inactive-to-active dynamics and entropy/enthalpy balance of two functional elements: the activation-loop and the conserved DFG motif. What is more the T315I gatekeeper mutant has a significant impact on the binding mechanism itself and on the binding kinetics.

7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent.

Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.

BRAF G596R mutation in a slow-progressing melanoma

More than 50% of melanomas harbor a single V600E point mutation in the kinase domain of the BRAF gene, resulting in constitutive activation of the MAP kinase pathway. The kinase loop in BRAF begins with Asp-Phe-Gly (DFG; codons 594-596). Mutations in this region are rare, and the effect on tumorigenesis or progression is unknown. We present a slow- progressing metastatic melanoma with a p.G596R (c.1786G.C) mutation in an 84-year-old female. This mutation has only been reported in two cases of melanoma in the current literature, with no clinical information available. Mutations in the DFG motif are considered low-activating through indirect binding and allosteric activation of the CRAF protein. Our patient has carried a clinical diagnosis of melanoma for over 25 years, suggesting that the G596R mutation may be associated with her indolent clinical course. It is unknown whether patients carrying this mutation will benefit from vemurafenib therapy. Our patient declined medical treatment.

Abstract 1362: Distinct binding mode of lenvatinib to VEGFR2 revealed by biochemical characterization

Abstract Background: Lenvatinib mesilate (lenvatinib) selectively inhibits vascular endothelial growth factor receptors (VEGFR1-3), and other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib is currently evaluated in several clinical trials, including two phase 3 clinical trials in patients with thyroid cancers and hepatocellular carcinoma. Recently, lenvatinib achieved the primary endpoint in a phase 3 clinical trial in patients with differentiated thyroid cancer. To gain insight into the origin of the potency of lenvatinib in VEGFR2 inhibition, crystal structure analysis of VEGFR2 complexed with lenvatinib and kinetics interaction analysis of lenvatinib against VEGFR2 were conducted in comparison with other VEGFR2 inhibitors. Experimental procedures: Recombinant human VEGFR2 was crystalized with lenvatinib or sorafenib, respectively. The resulting crystals of kinase-inhibitor complexes were subjected to x-ray structural analysis and the three dimensional structures were solved. The reporter displacement assay was conducted by using recombinant human VEGFR2 and a synthetic reporter probe. Reporter displacement was measured after addition of compounds and the kinetic parameters were determined. Results: In crystal structure analysis, both lenvatinib and sorafenib bind to the ATP-binding site and the neighboring allosteric region. However there was a notable difference in the conformation of the DFG motif in the activation loop. While DFG motif assumed a DFG-out conformation in the complex with sorafenib as reported, it was maintained in a DFG-in conformation in the complex with lenvatinib. In the reporter displacement assay, lenvatinib showed intermediate binding kinetics of those of sorafenib and sunitinib. Its association rate constant (kon) and dissociation rate constant (koff) values were 4.8 × 105 s−1 × M−1 (61 times of sorafenib, a representative type 2 inhibitor) and 9.9 × 10−4 s−1 (3.8 times), respectively. Sunitinib, a representative type 1 VEGFR2 inhibitor had a much faster binding kinetics and we could not determine kinetics parameters (kon>1.9 × 105, koff >57 × 10−4). Conclusions: Lenvatinib was suggested to have interaction with not only ATP region but also the neighboring allosteric region of VEGFR2, which is a typical feature of type 2 inhibitors. However, VEGFR2 adopted DFG-in conformation in complex with lenvatinib, which is a characteristic of type 1 inhibitors. Kinetics analysis revealed that lenvatinib had intermediate characteristics among these three types of VEGFR2 inhibitors. These results suggest lenvatinib is very distinct in its ability in binding with VEGFR2. Citation Format: Kiyoshi Okamoto, Megumi Ikemori Kawada, Anja Jestel, Konstanze von König, Yasuhiro Funahashi, Tomohiro Matsushima, Akihiko Tsuruoka, Atsushi Inoue, Junji Matsui. Distinct binding mode of lenvatinib to VEGFR2 revealed by biochemical characterization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1362. doi:10.1158/1538-7445.AM2015-1362

Integration of signaling in the kinome: Architecture and regulation of the αC Helix

Eukaryotic protein kinases have evolved to be highly regulated and dynamic molecular switches that are typically kept in an inactive state and then activated in response to extracellular signals. The hallmark signature of an active kinase is a hydrophobic spine called the regulatory (R) spine, which consists of four residues, two in the N-lobe and two in the C-lobe. RS1 is in the catalytic loop, RS2 is the Phe in the DFG motif, RS3 is at the C-terminus of the αC-Helix, and RS4 is at the beginning of β4. Assembly of the R-spine is typically facilitated by phosphorylation of the Activation Loop. The assembled R-spine brings together all of the functional motifs that are essential for transferring the phosphate from ATP to a tethered protein substrate. This includes the G-Loop, which anchors the ATP, the catalytic loop, the DFG motif fused to the Activation Loop, and the αC-Helix. We focus here on the properties of the αC-Helix showing 1) how residues communicate with different parts of the molecule, 2) how it is recruited to the active site as a consequence of assembling of the R-spine, and 3) how it is regulated by linkers/motifs/proteins that lie outside the conserved kinase core. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

In silico identification of novel kinase inhibitors by targeting B-Raf(v660e) from natural products database.

The Ras/Raf/MEK/ERK (MAPK) signaling pathway has gained much attention from scientific community for therapeutic intervention in the past decades, specifically in oncology. Notably, a most prevalent B-Raf(v600e) mutant in Raf kinase family exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, virtual screening is applied to identify its potential inhibitors. Following the 25 ns molecular dynamic (MD) simulations, ZINC38541768, ZINC38541767, and ZINC12496469 are identified as B-Raf(v600e) potential inhibitors in a DFG-in conformation. Furthermore, according to the molecular mechanics/generalized born surface area (MM/GBSA) method, these three small molecules exhibit similar and good binding affinity toward B-Raf(v600e) (-38.76kcalmol(-1), -42.60kcalmol(-1), and -39.04kcalmol(-1)). At the same time, several critical residues, such as I463, V471 in the P-loop, and DFG motif residue D594 within the A-loop, are also well clarified. All these results may not only indicate some future applications of inhibitors targeting B-Raf(v600e), but also benefit B-Raf(v600e) harboring cancer patients.

Computational study of the "DFG-flip" conformational transition in c-Abl and c-Src tyrosine kinases.

Protein tyrosine kinases are crucial to cellular signaling pathways regulating cell growth, proliferation, metabolism, differentiation, and migration. To maintain normal regulation of cellular signal transductions, the activities of tyrosine kinases are also highly regulated. The conformation of a three-residue motif Asp-Phe-Gly (DFG) near the N-terminus of the long “activation” loop covering the catalytic site is known to have a critical impact on the activity of c-Abl and c-Src tyrosine kinases. A conformational transition of the DFG motif can switch the enzyme from an active (DFG-in) to an inactive (DFG-out) state. In the present study, the string method with swarms-of-trajectories was used to computationally determine the reaction pathway connecting the two end-states, and umbrella sampling calculations were carried out to characterize the thermodynamic factors affecting the conformations of the DFG motif in c-Abl and c-Src kinases. According to the calculated free energy landscapes, the DFG-out conformation is clearly more favorable in the case of c-Abl than that of c-Src. The calculations also show that the protonation state of the aspartate residue in the DFG motif strongly affects the in/out conformational transition in c-Abl, although it has a much smaller impact in the case of c-Src due to local structural differences.

Conformational analysis of the DFG-out kinase motif and biochemical profiling of structurally validated type II inhibitors.

Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations that have also been referred to as DFG-out in the literature but that do not have a fully formed allosteric pocket. We have completed a structural analysis of almost 200 small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that nonclassical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two data sets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity.

Structural Analysis of the Binding of Type I, I1/2, and II Inhibitors to Eph Tyrosine Kinases.

We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out). The three structures with type I1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases.

Exploring protein kinase conformation using swarm-enhanced sampling molecular dynamics.

Protein plasticity, while often linked to biological function, also provides opportunities for rational design of selective and potent inhibitors of their function. The application of computational methods to the prediction of concealed protein concavities is challenging, as the motions involved can be significant and occur over long time scales. Here we introduce the swarm-enhanced sampling molecular dynamics (sesMD) method as a tool to improve sampling of conformational landscapes. In this approach, a swarm of replica simulations interact cooperatively via a set of pairwise potentials incorporating attractive and repulsive components. We apply the sesMD approach to explore the conformations of the DFG motif in the protein p38α mitogen-activated protein kinase. In contrast to multiple MD simulations, sesMD trajectories sample a range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between the DFG-in and DFG-out conformations are predicted to have druggable pockets of interest for structure-based ligand design.

Diverse functionalization of Aurora-A kinase at specified surface and buried sites by native chemical modification.

The ability to obtain a homogeneous sample of protein is invaluable when studying the effect of alterations such as post-translational modifications (PTMs). Selective functionalization of a protein to investigate the effect of PTMs on its structure or activity can be achieved by chemical modification of cysteine residues. We demonstrate here that one such technique, which involves conversion of cysteine to dehydroalanine followed by thiol nucleophile addition, is suitable for the site-specific installation of a wide range of chemical mimics of PTMs, including acetylated and dimethylated lysine, and other unnatural amino acids. These reactions, optimized for the clinically relevant kinase Aurora-A, readily proceed to completion as revealed by intact protein mass spectrometry. Moreover, these reactions proceed under non-denaturing conditions, which is desirable when working with large protein substrates. We have determined reactivity trends for a diverse range of thiol nucleophile addition reactions at two separate sites on Aurora-A, and we also highlight limitations when using thiol nucleophiles that contain basic functional groups. We show that chemical modification of cysteine residues is possible not only on a flexible surface-exposed loop, but also within a deep active site pocket at the conserved DFG motif, which reveals the potential use of this method in exploring enzyme function through modification of catalytic site residues.

Pyrrolo[3,2-b]quinoxaline derivatives as types I1/2 and II Eph tyrosine kinase inhibitors: structure-based design, synthesis, and in vivo validation.

The X-ray crystal structures of the catalytic domain of the EphA3 tyrosine kinase in complex with two type I inhibitors previously discovered in silico (compounds A and B) were used to design type I1/2 and II inhibitors. Chemical synthesis of about 25 derivatives culminated in the discovery of compounds 11d (type I1/2), 7b, and 7g (both of type II), which have low-nanomolar affinity for Eph kinases in vitro and a good selectivity profile on a panel of 453 human kinases (395 nonmutant). Surface plasmon resonance measurements show a very slow unbinding rate (1/115 min) for inhibitor 7m. Slow dissociation is consistent with a type II binding mode in which the hydrophobic moiety (trifluoromethyl-benzene) of the inhibitor is deeply buried in a cavity originating from the displacement of the Phe side chain of the so-called DFG motif as observed in the crystal structure of compound 7m. The inhibitor 11d displayed good in vivo efficacy in a human breast cancer xenograft.

Insight into the Structural Features of Pyrazolopyrimidine- and Pyrazolopyridine-based B-RafV600EKinase Inhibitors by Computational Explorations

Presently, both ligand-based and receptor-based 3D-QSAR modelings were performed on 107 pyrazolopyrimidine- and pyrazolopyridine-based inhibitors of B-Raf(V600E) kinase. The optimal model is successful to predict the inhibitors' activity with Q(2) of 0.504, R(2) ncv of 0.960, and R(2) pred of 0.872. Besides, the 3D contour maps explain well the structural requirements of the interaction between the ligand and the receptor. Furthermore, molecular docking and MD were also carried out to study the binding mode. Our findings are the following: (i) Bulky substituents at position 3, 10 and ring D improve the inhibitory activity, but impair the activity at position 5, 11, and 19. (ii) Electropositive groups at position 10, 13 and 20 and electronegative groups at position 2 increase the biological activity. (iii) Hydrophobic substituents at ring C are beneficial to improve the biological activity, while hydrophilic substituents at position 11 and ring D are good for the activity. (4) This scaffold of inhibitors may bind to the B-Raf kinase with an 'L' conformation and belong to type III binding mode, which is fixed by hydrophobic interaction and hydrogen bonds with residues from hinge region and DFG motif. These results may be a guidance to develop new B-Raf(V600E) kinase inhibitors.

The resistance tetrad: amino acid hotspots for kinome-wide exploitation of drug-resistant protein kinase alleles.

Acquired resistance to targeted kinase inhibitors is a well-documented clinical problem that is potentially fatal for patients to whom a suitable back-up is not available. However, protein kinase alleles that promote resistance to inhibitors can be exploited experimentally as gold-standards for "on"- and "off"-target validation strategies and constitute a powerful resource for assessing the ability of new or combined therapies to override resistance. Clinical resistance to kinase inhibitors is an evident in all tyrosine kinase-driven malignancies, where high rates of mutation drive tumor evolution toward the insidious drug-resistant (DR) state through a variety of mechanisms. Unfortunately, this problem is likely to intensify in the future as the number of target kinases, approved inhibitors, and clinical indications increase. To empower the analysis of resistance in kinases, we have validated a bioinformatic, structural, and cellular workflow for designing and evaluating resistance at key mutational hotspots among kinome members. In this chapter, we discuss how mutation of amino acids in the gatekeeper and hinge-loop regions (collectively termed the "resistance tetrad") and the DFG motif represent an effective approach for generating panels of DR kinase alleles for chemical genetics and biological target validation.

The Free Energy Contribution of SH3 and SH2 in c-Abl 1b Autoinhibition Mechanism via a Computational Structure-Based Model

We report on a computational analysis of the dynamical and structural arrangements induced by the presence of the regulatory domains SH2 and SH3 in the simulated construct of protein c-Abl 1b. Our approach is based on a hybrid structure-based all-atom model combined with the parallel tempering metadynamics method to enhance conformational sampling. The combined benefits of these two approaches allow the study of events whose time scale is well beyond the capabilities of standard all-atom molecular dynamics simulations. This method allowed to witness in a single unified picture the correlation and the associated free energy profiles among the hallmarks of the activation process of c-Abl: the activation loop opening, the DFG motif flip, the docking of the regulatory modules in the characteristic conformations associated to activation and autoinhibition, with the inclusion of the bending of helix alpha-I associated to myristate binding and alpha-C helix rotation as secondary contributing factors.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Trb3 regulates LR axis formation in zebrafish embryos.

Tribless family proteins are pseudokinases that lack DFG (Asp-Phe-Gly) motif in the functional kinase domain, regulating Akt and BMP pathways, insulin metabolism, hypoxia, and ubiquitination. This report concerns expression patterns and functional roles of trb3 in zebrafish embryonic development. trb3 is evolutionarily well-conserved and located on zebrafish chromosome 11. Spatiotemporal expression studies show that trb3 transcripts are abundant throughout embryogenesis, but confined to mesendodermal cells during the late blastula phase. Over-expression of trb3 ventralizes the embryos while a knockdown of trb3 using morpholino alters positioning of the heart, liver, and pancreatic buds as well as gut looping. Furthermore, constitutive activation of TGF-signaling with TARAM-A* (TGF-related type I receptor) significantly increases the level of trb3 transcripts during the late blastula phase. Over-expression of trb3 reduces the level of smurf1 transcripts, a member of TGF-signaling. We thus propose that Trb3 governs left-right (LR) axis patterning as a component of TGF-signaling in vertebrate embryonic development.