Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection.

Picornaviridae represent a large family of single-stranded positive RNA viruses of which different members can infect both humans and animals. These include the enteroviruses (e.g., poliovirus, coxsackievirus, and rhinoviruses) as well as the cardioviruses (e.g., encephalomyocarditis virus). Picornaviruses have evolved to interact with, use, and/or evade cellular host systems to create the optimal environment for replication and spreading. It is known that viruses modify kinase activity during infection, but a proteome-wide overview of the (de)regulation of cellular kinases during picornavirus infection is lacking. To study the kinase activity landscape during picornavirus infection, we here applied dedicated targeted mass spectrometry-based assays covering ∼40% of the human kinome. Our data show that upon infection, kinases of the MAPK pathways become activated (e.g., ERK1/2, RSK1/2, JNK1/2/3, and p38), while kinases involved in regulating the cell cycle (e.g., CDK1/2, GWL, and DYRK3) become inactivated. Additionally, we observed the activation of CHK2, an important kinase involved in the DNA damage response. Using pharmacological kinase inhibitors, we demonstrate that several of these activated kinases are essential for the replication of encephalomyocarditis virus. Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

Phase separation underlies signaling activation of oncogenic NTRK fusions.

NTRK (neurotrophic tyrosine receptor kinase) gene fusions that encode chimeric proteins exhibiting constitutive activity of tropomyosin receptor kinases (TRK), are oncogenic drivers in multiple cancer types. However, the underlying mechanisms in oncogenesis that involve various N-terminal fusion partners of NTRK fusions remain elusive. Here, we show that NTRK fusion proteins form liquid-like condensates driven by their N-terminal fusion partners. The kinase reactions are accelerated in these condensates where the complexes for downstream signaling activation are also concentrated. Our work demonstrates that the phase separation driven by NTRK fusions is not only critical for TRK activation, but the condensates formed through phase separation serve as organizational hubs for oncogenic signaling.

Molecular basis for cellular retinoic acid-binding protein 1 in modulating CaMKII activation.

Introduction: Cellular retinoic acid (RA)-binding protein 1 (CRABP1) is a highly conserved protein comprised of an anti-parallel, beta-barrel, and a helix-turn-helix segment outside this barrel. Functionally, CRABP1 is thought to bind and sequester cytosolic RA. Recently, CRABP1 has been established as a major mediator of rapid, non-genomic activity of RA in the cytosol, referred to as "non-canonical" activity. Previously, we have reported that CRABP1 interacts with and dampens the activation of calcium-calmodulin (Ca2+-CaM)-dependent kinase 2 (CaMKII), a major effector of Ca2+ signaling. Through biophysical, molecular, and cellular assays, we, herein, elucidate the molecular and structural mechanisms underlying the action of CRABP1 in dampening CaMKII activation. Results: We identify an interaction surface on CRABP1 for CaMKII binding, located on the beta-sheet surface of the barrel, and an allosteric region within the helix segment outside the barrel, where both are important for interacting with CaMKII. Molecular studies reveal that CRABP1 preferentially associates with the inactive form of CaMKII, thereby dampening CaMKII activation. Alanine mutagenesis of residues implicated in the CaMKII interaction results in either a loss of this preference or a shift of CRABP1 from associating with the inactive CaMKII to associating with the active CaMKII, which corresponds to changes in CRABP1's effect in modulating CaMKII activation. Conclusions: This is the first study to elucidate the molecular and structural basis for CRABP1's function in modulating CaMKII activation. These results further shed insights into CRABP1's functional involvement in multiple signaling pathways, as well as its extremely high sequence conservation across species and over evolution.

Mode of autophosphorylation in bacteriophytochromes RpBphP2 and RpBphP3.

Phytochromes are red-light photoreceptors that regulate a wide range of physiological processes in plants, fungi and bacteria. Canonical bacteriophytochromes are photosensory histidine kinases that undergo light-dependent autophosphorylation, thereby regulating cellular responses to red light via two-component signaling pathways. However, the molecular mechanism of kinase activation remains elusive for bacteriophytochromes. In particular, the directionality of autophosphorylation is still an open question in these dimeric photoreceptor kinases. In this work, we perform histidine kinase assays on two tandem bacteriophytochromes RpBphP2 and RpBphP3 from the photosynthetic bacterium Rhodopseudomonas palustris. By examining the kinase activities of full-length bacteriophytochromes and two loss-of-function mutants under different light conditions, we demonstrate that RpBphP2 and RpBphP3 undergo light-dependent trans-phosphorylation between protomers in both homodimeric and heterodimeric forms. We have further determined the crystal structure of the histidine kinase domains of RpBphP2 at 3.19 Å resolution. Based on structural comparisons and homology modeling, we also present a model to account for the actions of trans-autophosphorylation in bacteriophytochromes.

Genetic complementation screening and molecular docking give new insight on phosphorylation-dependent Mastl kinase activation.

Mastl is a mitotic kinase that is essential for error-free chromosome segregation. It is an atypical member of AGC kinase family, possessing a unique non-conserved middle region. The mechanism of Mastl activation has been studied extensively in vitro. Phosphorylation of several residues were identified to be crucial for activation. These sites correspond to T193 and T206 in the activation loop and S861 in the C-terminal tail of mouse Mastl. To date, the significance of these phosphosites was not confirmed in intact mammalian cells. Here, we utilize a genetic complementation approach to determine the essentials of mammalian Mastl kinase activation. We used tamoxifen-inducible conditional knockout mouse embryonic fibroblasts to delete endogenous Mastl and screened various mutants for their ability to complement its loss. S861A mutant was able to complement endogenous Mastl loss. In parallel, we performed computational molecular docking studies to evaluate the significance of this residue for kinase activation. Our in-depth sequence and structure analysis revealed that Mastl pS861 does not belong to a conformational state, where the phosphoresidue contributes to C-tail docking. C-tail of Mastl is relatively short and it lacks a hydrophobic (HF) motif that would otherwise help its anchoring over N-lobe, required for the final steps of kinase activation. Our results show that phosphorylation of Mastl C-tail turn motif (S861) is dispensable for kinase function in cellulo.Communicated by Ramaswamy H. Sarma.

Regulation of Stress-Activated Kinases in Response to Tacaribe Virus Infection and Its Implications for Viral Replication.

Arenaviruses include important zoonotic pathogens that cause hemorrhagic fever (e.g., Junín virus; JUNV) as well as other viruses that are closely related but apathogenic (e.g., Tacaribe virus; TCRV). We have found that, while TCRV and JUNV differ in their ability to induce apoptosis in infected cells, due to active inhibition of caspase activation by the JUNV nucleoprotein, both viruses trigger similar upstream pro-apoptotic signaling events, including the activation/phosphorylation of p53. In the case of TCRV, the pro-apoptotic factor Bad is also phosphorylated (leading to its inactivation). These events clearly implicate upstream kinases in regulating the induction of apoptosis. Consistent with this, here we show activation in TCRV-infected cells of the stress-activated protein kinases p38 and JNK, which are known to regulate p53 activation, as well as the downstream kinase MK2 and transcription factor c-Jun. We also observed the early transient activation of Akt, but not Erk. Importantly, the chemical inhibition of Akt, p38, JNK and c-Jun all dramatically reduced viral growth, even though we have shown that inhibition of apoptosis itself does not. This indicates that kinase activation is crucial for viral infection, independent of its downstream role in apoptosis regulation, a finding that has the potential to shed further light on the determinants of arenavirus pathogenesis, as well as to inform future therapeutic approaches.

An auto-inhibited state of protein kinase G and implications for selective activation.

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iß that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iß in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iß AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iß auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

Kinase Activity of PAR1b, Which Mediates Nuclear Translocation of the BRCA1 Tumor Suppressor, Is Potentiated by Nucleic Acid-Mediated PAR1b Multimerization.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

An effective strategy for ligand-mediated pulldown of the HER2/HER3/NRG1ß heterocomplex and cryo-EM structure determination at low sample concentrations.

Obtaining high-resolution structures of Receptor Tyrosine Kinases that visualize extracellular, transmembrane and intracellular kinase regions simultaneously is an eagerly pursued but still unmet challenge of structural biology. The Human Epidermal Growth Factor Receptor 3 (HER3) that has a catalytically inactive kinase domain (pseudokinase) forms a potent signaling complex upon binding of growth factor neuregulin 1ß (NRG1ß) and upon dimerization with a close homolog, the HER2 receptor. The HER2/HER3/NRG1ß complex is often referred to as an oncogenic driver in breast cancer and is an attractive target for anti-cancer therapies. After overcoming significant hurdles in isolating sufficient amounts of the HER2/HER3/NRG1ß complex for structural studies by cryo-electron microscopy (cryo-EM), we recently obtained the first high-resolution structures of the extracellular portion of this complex. Here we describe a step-by-step protocol for obtaining a stable and homogenous HER2/HER3/NRG1ß complex for structural studies and our recommendation for collecting and processing cryo-EM data for this sample. We also show improved EM density for the transmembrane and kinase domains of the receptors, which continue to evade structural determination at high resolution. The discussed strategies are tunable and applicable to other membrane receptor complexes.

Heregulin Activity Assays for Residual Testing of Cell Therapy Products.

BACKGROUND: Heregulin is a ligand for the protooncogene product ErbB/HER that acts as  a key mitogenic factor for human Schwann cells (hSCs). Heregulin is required for sustained hSC growth in vitro but must be thoroughly removed before cell collection for transplantation due to potential safety concerns. The goal of this study was to develop simple cell-based assays to assess the effectiveness of heregulin addition to and removal from aliquots of hSC culture medium. These bioassays were based on the capacity of a ß1-heregulin peptide to elicit ErbB/HER receptor signaling in adherent ErbB2+/ErbB3+ cells. RESULTS: Western blotting was used to measure the activity of three different ß1-heregulin/ErbB-activated kinases (ErbB3/HER3, ERK/MAPK and Akt/PKB) using phospho-specific antibodies against key activating residues. The duration, dose-dependency and specificity of ß1-heregulin-initiated kinase phosphorylation were investigated, and controls were implemented for assay optimization and reproducibility to detect ß1-heregulin activity in the nanomolar range. Results from these assays showed that the culture medium from transplantable hSCs elicited no detectable activation of the aforementioned kinases in independent rounds of testing, indicating that the implemented measures can ensure that the final hSC product is devoid of bioactive ß1-heregulin molecules prior to transplantation. CONCLUSIONS: These assays may be valuable to detect impurities such as undefined soluble factors or factors for which other biochemical or biological assays are not yet available. Our workflow can be modified as necessary to determine the presence of ErbB/HER, ERK, and Akt activators other than ß1-heregulin using native samples, such as fresh isolates from cell- or tissue extracts in addition to culture medium.

The Ephb2 Receptor Uses Homotypic, Head-to-Tail Interactions within Its Ectodomain as an Autoinhibitory Control Mechanism.

The Eph receptor tyrosine kinases and their ephrin ligands direct axon pathfinding and neuronal cell migration, as well as mediate many other cell-cell communication events. Their dysfunctional signaling has been shown to lead to various diseases, including cancer. The Ephs and ephrins both localize to the plasma membrane and, upon cell-cell contact, form extensive signaling assemblies at the contact sites. The Ephs and the ephrins are divided into A and B subclasses based on their sequence conservation and affinities for each other. The molecular details of Eph-ephrin recognition have been previously revealed and it has been documented that ephrin binding induces higher-order Eph assemblies, which are essential for full biological activity, via multiple, distinct Eph-Eph interfaces. One Eph-Eph interface type is characterized by a homotypic, head-to-tail interaction between the ligand-binding and the fibronectin domains of two adjacent Eph molecules. While the previous Eph ectodomain structural studies were focused on A class receptors, we now report the crystal structure of the full ectodomain of EphB2, revealing distinct and unique head-to-tail receptor-receptor interactions. The EphB2 structure and structure-based mutagenesis document that EphB2 uses the head-to-tail interactions as a novel autoinhibitory control mechanism for regulating downstream signaling and that these interactions can be modulated by posttranslational modifications.

New Structural Perspectives in G Protein-Coupled Receptor-Mediated Src Family Kinase Activation.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.

TRAF6 Phosphorylation Prevents Its Autophagic Degradation and Re-Shapes LPS-Triggered Signaling Networks.

The ubiquitin E3 ligase TNF Receptor Associated Factor 6 (TRAF6) participates in a large number of different biological processes including innate immunity, differentiation and cell survival, raising the need to specify and shape the signaling output. Here, we identify a lipopolysaccharide (LPS)-dependent increase in TRAF6 association with the kinase IKKε (inhibitor of NF-κB kinase subunit ε) and IKKε-mediated TRAF6 phosphorylation at five residues. The reconstitution of TRAF6-deficient cells, with TRAF6 mutants representing phosphorylation-defective or phospho-mimetic TRAF6 variants, showed that the phospho-mimetic TRAF6 variant was largely protected from basal ubiquitin/proteasome-mediated degradation, and also from autophagy-mediated decay in autolysosomes induced by metabolic perturbation. In addition, phosphorylation of TRAF6 and its E3 ligase function differentially shape basal and LPS-triggered signaling networks, as revealed by phosphoproteome analysis. Changes in LPS-triggered phosphorylation networks of cells that had experienced autophagy are partially dependent on TRAF6 and its phosphorylation status, suggesting an involvement of this E3 ligase in the interplay between metabolic and inflammatory circuits.

Platelet-derived growth factor receptor beta activates Abl2 via direct binding and phosphorylation.

Abl family kinases are nonreceptor tyrosine kinases activated by diverse cellular stimuli that regulate cytoskeleton organization, morphogenesis, and adhesion. The catalytic activity of Abl family kinases is tightly regulated in cells by a complex set of intramolecular and intermolecular interactions and post-translational modifications. For example, the platelet-derived growth factor receptor beta (PDGFRß), important for cell proliferation and chemotaxis, is a potent activator of Abl family kinases. However, the molecular mechanism by which PDGFRß engages and activates Abl family kinases is not known. We show here that the Abl2 Src homology 2 domain directly binds to phosphotyrosine Y771 in the PDGFRß cytoplasmic domain. PDGFRß directly phosphorylates multiple novel sites on the N-terminal half of Abl2, including Y116, Y139, and Y161 within the Src homology 3 domain, and Y299, Y303, and Y310 on the kinase domain. Y116, Y161, Y272, and Y310 are all located at or near the Src homology 3/Src homology 2-kinase linker interface, which helps maintain Abl family kinases in an autoinhibited conformation. We also found that PDGFRß-mediated phosphorylation of Abl2 in vitro activates Abl2 kinase activity, but mutation of these four tyrosines (Y116, Y161, Y272, and Y310) to phenylalanine abrogated PDGFRß-mediated activation of Abl2. These findings reveal how PDGFRß engages and phosphorylates Abl2 leading to activation of the kinase, providing a framework to understand how growth factor receptors engage and activate Abl family kinases.

Computational studies of anaplastic lymphoma kinase mutations reveal common mechanisms of oncogenic activation.

Kinases play important roles in diverse cellular processes, including signaling, differentiation, proliferation, and metabolism. They are frequently mutated in cancer and are the targets of a large number of specific inhibitors. Surveys of cancer genome atlases reveal that kinase domains, which consist of 300 amino acids, can harbor numerous (150 to 200) single-point mutations across different patients in the same disease. This preponderance of mutations-some activating, some silent-in a known target protein make clinical decisions for enrolling patients in drug trials challenging since the relevance of the target and its drug sensitivity often depend on the mutational status in a given patient. We show through computational studies using molecular dynamics (MD) as well as enhanced sampling simulations that the experimentally determined activation status of a mutated kinase can be predicted effectively by identifying a hydrogen bonding fingerprint in the activation loop and the αC-helix regions, despite the fact that mutations in cancer patients occur throughout the kinase domain. In our study, we find that the predictive power of MD is superior to a purely data-driven machine learning model involving biochemical features that we implemented, even though MD utilized far fewer features (in fact, just one) in an unsupervised setting. Moreover, the MD results provide key insights into convergent mechanisms of activation, primarily involving differential stabilization of a hydrogen bond network that engages residues of the activation loop and αC-helix in the active-like conformation (in >70% of the mutations studied, regardless of the location of the mutation).

Methamphetamine Activates Trace Amine Associated Receptor 1 to Regulate Astrocyte Excitatory Amino Acid Transporter-2 via Differential CREB Phosphorylation During HIV-Associated Neurocognitive Disorders.

Methamphetamine (METH) use, referred to as methamphetamine use disorder (MUD), results in neurocognitive decline, a characteristic shared with HIV-associated neurocognitive disorders (HAND). MUD exacerbates HAND partly through glutamate dysregulation. Astrocyte excitatory amino acid transporter (EAAT)-2 is responsible for >90% of glutamate uptake from the synaptic environment and is significantly decreased with METH and HIV-1. Our previous work demonstrated astrocyte trace amine associated receptor (TAAR) 1 to be involved in EAAT-2 regulation. Astrocyte EAAT-2 is regulated at the transcriptional level by cAMP responsive element binding (CREB) protein and NF-κB, transcription factors activated by cAMP, calcium and IL-1ß. Second messengers, cAMP and calcium, are triggered by TAAR1 activation, which is upregulated by IL-1ß METH-mediated increases in these second messengers and signal transduction pathways have not been shown to directly decrease astrocyte EAAT-2. We propose CREB activation serves as a master regulator of EAAT-2 transcription, downstream of METH-induced TAAR1 activation. To investigate the temporal order of events culminating in CREB activation, genetically encoded calcium indicators, GCaMP6s, were used to visualize METH-induced calcium signaling in primary human astrocytes. RNA interference and pharmacological inhibitors targeting or blocking cAMP-dependent protein kinase A and calcium/calmodulin kinase II confirmed METH-induced regulation of EAAT-2 and resultant glutamate clearance. Furthermore, we investigated METH-mediated CREB phosphorylation at both serine 133 and 142, the co-activator and co-repressor forms, respectively. Overall, this work revealed METH-induced differential CREB phosphorylation is a critical regulator for EAAT-2 function and may thus serve as a mechanistic target for the attenuation of METH-induced excitotoxicity in the context of HAND.

Are redox changes a critical switch for mitotic progression?

Cell-cycle dependent redox changes result in increased protein oxidation in mitotic cells. We show that oxidative modifications of a conserved cysteine residue within Aurora A kinase (AURKA) can promote its activation during mitosis. Targeting redox-sensitive cysteine residues within AURKA may lead to the development of novel anti-cancer agents with improved clinical efficacy.

Catalytic Domain Plasticity of MKK7 Reveals Structural Mechanisms of Allosteric Activation and Diverse Targeting Opportunities.

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that affect its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small-molecule screening campaign yielded multiple scaffolds, including type II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase.

Locally-Induced CaMKII Translocation Requires Nucleotide Binding.

Calcium-calmodulin-dependent protein kinase (CaMKII) is a molecule involved in several cell processes including plasticity related to learning and memory. Activation of NMDA-type glutamate receptors results in translocation of CaMKII to synapses. However, there are at least two distinct mechanisms by which glutamate-dependent CaMKII translocation occurs: one well-studied process resulting from whole-cell glutamate stimulation and one resulting from brief, local glutamate application. Unlike the relatively fast CaMKII translocation seen following whole-cell glutamate delivery (seconds), local application results in CaMKII translocation that occurs gradually within 6-10 min. This locally-induced translocation of CaMKII requires L-type Ca2+ channel co-activation but does not rely on GluN2B receptor subunit expression, unlike translocation following whole-cell application of glutamate. The current study examined if nucleotide binding is necessary for locally-induced CaMKII translocation, similar to CaMKII translocation resulting from whole-cell glutamate application. Three different mechanisms of inhibition were employed: staurosporine (ATP inhibitor), CaMKII(281-302) peptide inhibitor and expression of the K42M mutation. Locally-induced CaMKII translocation was moderately suppressed in the presence of either the broad-spectrum kinase inhibitor staurosporine (100 nm) or the CaMKII(281-302) peptide inhibitor. However, expression of the catalytically dead K42M mutation that prevents ATP-binding to CaMKII, significantly inhibited locally-induced translocation. Thus, CaMKII translocation following brief, local glutamate application requires nucleotide binding, providing support for future research into the molecular mechanisms of this distinct form of CaMKII translocation.