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Members of the New Kinase Family 3 (NKF3), PEAK1/SgK269 and Pragmin/SgK223 pseudokinases, have emerged as important regulators of cell motility and cancer progression. Here, we demonstrate for the first time that C19orf35 (PEAK3), a newly identified member of the NKF3 family, is a kinase-like protein evolutionarily conserved across mammals and birds and a novel regulator of cell motility. In contrast to its family members, which promote cell elongation when overexpressed in cells, PEAK3 overexpression does not have an elongating effect on cell shape but instead is associated with loss of actin filaments. Through an unbiased search for PEAK3 binding partners, we identified several regulators of cell motility, including the adaptor protein CrkII. We show that by binding to CrkII, PEAK3 prevents the formation of CrkII-dependent membrane ruffling. This function of PEAK3 is reliant upon its dimerization, which is mediated through a split helical dimerization (SHED) domain conserved among all NKF3 family members. Disruption of the conserved DFG motif in the PEAK3 pseudokinase domain also interferes with its ability to dimerize and subsequently bind CrkII, suggesting that the conformation of the pseudokinase domain might play an important role in PEAK3 signaling. Hence, our data identify PEAK3 as an NKF3 family member with a unique role in cell motility driven by dimerization of its pseudokinase domain.

Pseudokinases are members of the protein kinase superfamily that are predicted to be catalytically inactive despite retaining the conserved kinase domain fold. Consequently, pseudokinases have evolved to signal exclusively through non-catalytic mechanisms, such as allostery and scaffolding. Though pseudokinases have been implicated in a wide range of diseases and are known to be involved in critical cellular processes, their lack of catalytic activity has made their functions challenging to study. Members of the Tribbles family of pseudokinases have recently emerged as key regulators of diverse signaling pathways, including the MAPK cascade, proteasome-dependent degradation, the TGF-β/BMP pathway, and the PI3K/Akt pathway. However, the molecular mechanisms underlying Tribbles-mediated signaling are poorly understood. One of the most highly conserved functions of Tribbles pseudokinases is their role in promoting ubiquitination of C/EBP transcription factors by the E3 ubiquitin ligase COP1. Dysregulation of this function has been implicated in acute myeloid leukemia, where overexpression of Trb1 or Trb2 is sufficient to induce leukemogenesis in mice through depletion of C/EBPα. Tribbles pseudokinases are thought to function as scaffolds to facilitate interactions between COP1 and substrates like C/EBPα, but the molecular basis for this function is only beginning to be uncovered. Here, we have investigated the molecular and structural determinants of regulation of COP1 by the pseudokinase Trb1. In doing so, we have uncovered a previously uncharacterized role for Trb1 in promoting COP1 nuclear localization through disrupting a conserved intramolecular regulatory interaction within COP1, and we have identified key structural elements that control COP1 localization. Collectively, these findings will deepen our understanding of the mechanisms underlying regulation of COP1 by Tribbles pseudokinases and broaden our knowledge of the diverse signaling functions of the Tribbles family.

Pseudoenzymes are present within many, but not all, known enzyme families and lack one or more conserved canonical amino acids that help define their catalytically active counterparts. Recent findings in the pseudokinase field confirm that evolutionary repurposing of the structurally defined bilobal protein kinase fold permits distinct biological functions to emerge, many of which rely on conformational switching, as opposed to canonical catalysis. In this analysis, we evaluate progress in evaluating several members of the ‘dark’ pseudokinome that are pertinent to help drive this expanding field. Initially, we discuss how adaptions in erythropoietin‐producing hepatocellular carcinoma (Eph) receptor tyrosine kinase domains resulted in two vertebrate pseudokinases, EphA10 and EphB6, in which co‐evolving sequences generate new motifs that are likely to be important for both nucleotide binding and catalysis‐independent signalling. Secondly, we discuss how conformationally flexible Tribbles pseudokinases, which have radiated in the complex vertebrates, control fundamental aspects of cell signalling that may be targetable with covalent small molecules. Finally, we show how species‐level adaptions in the duplicated canonical kinase protein serine kinase histone (PSKH)1 sequence have led to the appearance of the pseudokinase PSKH2, whose physiological role remains mysterious. In conclusion, we show how the patterns we discover are selectively conserved within specific pseudokinases, and that when they are modelled alongside closely related canonical kinases, many are found to be located in functionally important regions of the conserved kinase fold. Interrogation of these patterns will be useful for future evaluation of these, and other, members of the unstudied human kinome., Here, we evaluate several families of poorly studied 'dark' kinases and pseudokinases using a bioinformatic and structural analysis approach. Our investigation revealed several new features of two erythropoietin‐producing hepatocellular carinoma (Eph) psedudokinases: EphA10 and EphB6. Furthermore, we discuss newly identified features of the Tribbles pseudokinases as well as the unique pseudokinase protein serine kinase histone 2 (PSKH2).

The spectrum of kinase alterations displays distinct functional characteristics and requires kinase mutation‐oriented strategies for therapeutic interference. Besides phosphotransferase activity, protein abundance, and intermolecular interactions, particular patient‐mutations promote pathological kinase conformations. Despite major advances in identifying lead molecules targeting clinically relevant oncokinase functions, still many kinases are neglected and not part of drug discovery efforts. One explanation is attributed to challenges in tracking kinase activities. Chemical probes are needed to functionally annotate kinase functions, whose activities may not always depend on catalyzing phospho‐transfer. Such non‐catalytic kinase functions are related to transitions of full‐length kinase conformations. Recent findings underline that cell‐based reporter systems can be adapted to record conformation changes of kinases. Here, we discuss the possible applications of an extendable kinase conformation (KinCon) reporter toolbox for live‐cell recording of kinase states. KinCon is a genetically encoded bioluminescence‐based biosensor platform, which can be subjected for measurements of conformation dynamics of mutated kinases upon small molecule inhibitor exposure. We hypothesize that such biosensors can be utilized to delineate the molecular modus operandi for kinase and pseudokinase regulation. This should pave the path for full‐length kinase‐targeted drug discovery efforts aiming to identify single and combinatory kinase inhibitor therapies with increased specificity and efficacy.

International audience; Phosphorylation by protein kinases is a fundamental mechanism of signal transduction. Many kinase families contain one or several members that, although evolutionarily conserved , lack the residues required for catalytic activity. Studies combining structural, biochemical , and functional approaches revealed that these pseudokinases have crucial roles in vivo and may even represent attractive targets for pharmacological intervention. Pseudokinases mediate signal transduction by a diversity of mechanisms, including allo-steric regulation of their active counterparts, assembly of signaling hubs, or modulation of protein localization. One such pseudokinase, named Tra1 in yeast and transformation/ transcription domain-associated protein (TRRAP) in mammals, is the only member lacking all catalytic residues within the phosphatidylinositol 3-kinase related kinase (PIKK) family of kinases. PIKKs are related to the PI3K family of lipid kinases, but function as Serine/Threonine protein kinases and have pivotal roles in diverse processes such as DNA damage sensing and repair, metabolic control of cell growth, nonsense-mediated decay, or transcription initiation. Tra1/TRRAP is the largest subunit of two distinct tran-scriptional co-activator complexes, SAGA and NuA4/TIP60, which it recruits to promoters upon transcription factor binding. Here, we review our current knowledge on the Tra1/ TRRAP pseudokinase, focusing on its role as a scaffold for SAGA and NuA4/TIP60 complex assembly and recruitment to chromatin. We further discuss its evolutionary history within the PIKK family and highlight recent findings that reveal the importance of molecular chaperones in pseudokinase folding, function, and conservation.

Background Plants reprogram metabolism and development to rapidly adapt to biotic and abiotic stress. Protein kinases play a significant role in this process by phosphorylating protein substrates that activate or inactivate signaling cascades that regulate cellular and metabolic adaptations. Despite their importance in plant biology, a notably small fraction of the plant kinomes has been studied to date. Results In this report, we describe ZmDRIK1, a stress-responsive receptor-like pseudokinase whose expression is downregulated under water restriction. We show the structural features and molecular basis of the absence of ATP binding exhibited by ZmDRIK1. The ZmDRIK1 kinase domain lacks conserved amino acids that are essential for phosphorylation activity. The crystal structure of the ZmDRIK1 kinase domain revealed the presence of a spine formed by the side chain of the triad Leu240, Tyr363, and Leu375 that occludes the ATP binding pocket. Although ZmDRIK1 is unable to bind nucleotides, it does bind the small molecule ENMD-2076 which, in a cocrystal structure, revealed the potential to serve as a ZmDRIK1 inhibitor. Conclusion ZmDRIK1 is a novel receptor-like pseudokinase responsive to biotic and abiotic stress. The absence of ATP binding and consequently, the absence of phosphorylation activity, was proven by the crystal structure of the apo form of the protein kinase domain. The expression profiling of the gene encoding ZmDRIK1 suggests this kinase may play a role in downregulating the expression of stress responsive genes that are not necessary under normal conditions. Under biotic and abiotic stress, ZmDRIK1 is down-regulated to release the expression of these stress-responsive genes.

Significance Proteins are chemically modified after translation to regulate their functions. Protein kinases are enzymes that modify proteins with phosphate molecules. Using bioinformatics, we identified an unusual family of kinases (WNG kinases) that lack a structural motif, called the Gly-loop, which is absolutely required for the activity of all previously described kinases. Nevertheless, we found that the most conserved WNG kinase, WNG1, is catalytically active. The WNG kinases are only found in certain intracellular parasites, such as the human pathogen Toxoplasma gondii. We show that the founding member of the family, WNG1, phosphorylates parasite proteins that regulate membrane structure, and is therefore required for the proper biogenesis of the Toxoplasma parasitophorous vacuole, a structure essential for the parasite to cause disease., Apicomplexan parasites replicate within a protective organelle, called the parasitophorous vacuole (PV). The Toxoplasma gondii PV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network’s functions. Here, we identify the parasite-secreted kinase WNG1 (With-No-Gly-loop) as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, we show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, our results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.

Simple Summary Accumulating evidence supports a key function for Tribbles proteins in oncogenesis, both in leukemia and solid tumors. However, the exact role of these proteins is hard to define since in a context-dependent manner they can function as both oncogenes and tumor suppressors. Their complex role arises from the capacity to interact with a wide range of target molecules thereby acting as molecular scaffolds and signaling regulators of multiple pathways. This review focuses on one particular Tribbles family member, namely, TRIB3, addressing its gene and protein expression, as well as its role in cancer development and progression. Abstract The first Tribbles protein was identified as critical for the coordination of morphogenesis in Drosophila melanogaster. Three mammalian homologs were subsequently identified, with a structure similar to classic serine/threonine kinases, but lacking crucial amino acids for the catalytic activity. Thereby, the very weak ATP affinity classifies TRIB proteins as pseudokinases. In this review, we provide an overview of the regulation of TRIB3 gene expression at both transcriptional and post-translational levels. Despite the absence of kinase activity, TRIB3 interferes with a broad range of cellular processes through protein–protein interactions. In fact, TRIB3 acts as an adaptor/scaffold protein for many other proteins such as kinase-dependent proteins, transcription factors, ubiquitin ligases, or even components of the spliceosome machinery. We then state the contribution of TRIB3 to cancer development, progression, and metastasis. TRIB3 dysregulation can be associated with good or bad prognosis. Indeed, as TRIB3 interacts with and regulates the activity of many key signaling components, it can act as a tumor-suppressor or oncogene in a context-dependent manner.

Highlight This study shows that the pseudokinase domain of the CLV2 co-receptor CRN is actively involved in transmitting the CLV3 peptide signal in the Arabidopsis shoot, but not in the proximal root meristem., Stem cell maintenance in plants depends on the activity of small secreted signaling peptides of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) family, which, in the shoot, act through at least three kinds of receptor complexes, CLAVATA1 (CLV1) homomers, CLAVATA2 (CLV2) / CORYNE (CRN) heteromers, and CLV1/CLV2/CRN multimers. In the root, the CLV2/CRN receptor complexes function in the proximal meristem to transmit signals from the CLE peptide CLE40. While CLV1 consists of an extracellular receptor domain and an intracellular kinase domain, CLV2, a leucine-rich repeat (LRR) receptor-like protein, and CRN, a protein kinase, have to interact to form a receptor–kinase complex. The kinase domain of CRN has been reported to be catalytically inactive, and it is not yet known how the CLV2/CRN complex can relay the perceived signal into the cells, and whether the kinase domain is necessary for signal transduction at all. In this study we show that the kinase domain of CRN is actively involved in CLV3 signal transduction in the shoot apical meristem of Arabidopsis, but it is dispensable for CRN protein function in root meristem maintenance. Hence, we provide an example of a catalytically inactive pseudokinase that is involved in two homologous pathways, but functions in distinctively different ways in each of them.

While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance., eLife digest Around 20% of breast cancers are caused because cells have too many copies of a receptor protein called HER2 on their surface. HER2 is responsible for telling the cell to divide. Cells with too many of these receptors – and breast cancer cells can have up to 1000 times too many – divide uncontrollably. This causes the cancer to grow. Several successful anti-cancer drugs, such as Herceptin and Kadcyla, are used in the clinic to block the signals produced by HER2. Other drugs called kinase inhibitors prevent HER2 from building its faulty signals. However, a particular kinase inhibitor called lapatinib was not as successful in clinical trials as the medical community had hoped. Kinase inhibitors can have unexpected effects. While they can block specific signals in a cell, they can sometimes also cause new types of signals. Could this be one of the reasons behind the disappointing clinical trial results for lapatinib? By performing experiments on breast cancer cells grown in the laboratory, Claus, Patel et al. found that lapatinib can counterintuitively boost the growth of breast cancer cells. This occurs because lapatinib causes HER2 receptors to cluster together like a daisy chain along with another protein receptor of the same family, called HER3. These chains are primed to rapidly respond to a molecule called neuregulin, a growth factor that is commonly associated with breast cancer. The results presented by Claus, Patel et al. indicate that a particular subset of breast cancer patients – those whose cancer cells do not increase production of HER3 receptors – might better respond to lapatinib than others. The insights gained into what happens to HER2 when you try to block it should also influence the design of new drugs that target either HER2 or HER3.

Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (yc) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known. The pseudokinase domain (JAK homology 2, JH2) of JAK3 is of particular interest as approximately half of clinical JAK3 mutations cluster into it. In this study, we investigated the role of JH2s of JAK1 and JAK3 in IL-2R signaling and show that STAT5 activation requires both JH1 and JH2 of JAK1, while both JH1 and JH2 in JAK3 are specifically required for the cytokine-induction of cellular signaling. Characterization of recombinant JAK3 JH2 in thermal shift assay shows an unstable protein domain, which is strongly stabilized by ATP binding. Unexpectedly, nucleotide binding to JAK3 JH2 was found to be cation-independent. JAK3 JH2 showed higher nucleotide binding affinity in MANT-ATP and fluorescent polarization competition assays compared to the other JAK JH2s. Analysis of the functional role of ATP binding in JAK3 JH2 in cells and in zebrafish showed that disruption of ATP binding suppresses ligand-independent activation of clinical JAK3 gain-of-function mutations residing in either JH2 or JH1 but does not inhibit constitutive activation of oncogenic JAK1. ATP-binding site mutations in JAK3 JH2 do not, however, abrogate normal IL-2 signaling making them distinct from JH2 deletion or kinase-deficient JAK3. These findings underline the importance of JAK3 JH2 for cellular signaling in both ligand-dependent and in gain-of-function mutation-induced activation. Furthermore, they identify the JH2 ATP-binding site as a key regulatory region for oncogenic JAK3 signaling, and thus a potential target for therapeutic modulation.

The leucine-rich repeat receptor kinase (LRR-RK) BRASSINOSTEROID INSENSITIVE 1 (BRI1) requires a shape-complementary SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) co-receptor for brassinosteroid sensing and receptor activation1. Interface mutations that weaken the interaction between receptor and co-receptor in vitro reduce brassinosteroid signalling responses2. The SERK3 elongated (elg) allele3-5 maps to the complex interface and shows enhanced brassinosteroid signalling, but surprisingly no tighter binding to the BRI1 ectodomain in vitro. Here, we report that rather than promoting the interaction with BRI1, the elg mutation disrupts the ability of the co-receptor to interact with the ectodomains of BRI1-ASSOCIATED-KINASE1 INTERACTING KINASE (BIR) receptor pseudokinases, negative regulators of LRR-RK signalling6. A conserved lateral surface patch in BIR LRR domains is required for targeting SERK co-receptors and the elg allele maps to the core of the complex interface in a 1.25 A BIR3-SERK1 structure. Collectively, our structural, quantitative biochemical and genetic analyses suggest that brassinosteroid signalling complex formation is negatively regulated by BIR receptor ectodomains.

Janus-kinaasit (JAKit) ovat ei-reseptorisia tyrosiinikinaaseja, jotka välittävät yli 60 sytokiinin viestejä soluissa. Nämä viestit säätelevät lukuisia biologisia tapahtumia, kuten immuunijärjestelmän toimintaa, hematopoieesia, aineenvaihduntaa sekä kehitystä. JAKit ovat monidomeenisia proteeineja, joissa viestivää, aktiivista tyrosiinikinaasidomeenia (JH1) edeltää nk. pseudokinaasidomeeni (JH2). JH2 on ensisijaisen tärkeä JAK-aktiivisuuden säätelyssä. Domeenista onkin löydetty lukuisia kliinisesti merkittäviä mutaatioita, kuten autosomaalisia JAK2-mutaatioita, jotka aiheuttavat ligandiriippumatonta JAK-aktivaatiota ja siten johtavat hematopoieettisiin maligniteetteihin. Tässä työssä esitellyissä tutkimuksissa selvitettiin JH2:n sekä sen mutaatioiden toimintaa JAK-säätelyssä. Tutkimuksissa havaittiin, että kaikissa JAK JH2:ssa on toiminnallinen nukleotidinsitomistasku, joka kykenee sitomaan ATP:ta ja pienmolekyylisiä inhibiittoreita. Tärkeimpänä löydöksenä havaittiin, että ATP:n sitoutumisen estäminen JAK2 JH2:n ATP-sitomistaskuun alentaa ligandiriippumatonta JAK2-aktivaatiota. Nämä havainnot osoittavat, että JAK2 JH2 on mahdollinen lääkekohde kohdennettujen JAK-inhibiittoreiden kehittämiselle. Tällaiset inhibiittorit, jotka estäisivät kohdennetusti mutatoituneen (muttei villityyppisen) JAKin toimintaa, olisivat merkittävä parannus verrattuna nykyisiin JAK-estäjiin, jotka eivät kykene erottelemaan villityyppisen ja mutatoituneen JAKin välillä, eivätkä siten pysty parantamaan tautia. Tutkimuksissa kehitettiin yhteistyöprojektina myös molekyylimalli JH2:n toiminnasta JH1:n aktiivisuuden säätelijänä. JH2–JH1-malli selittää useimpien tunnettujen kliinisten JAK2-mutaatioiden toiminnan molekyylitasolla. Lisäksi suoritettiin systemaattinen analyysi JAK2-mutaatioista, jotka voivat estää tautia aiheuttavan JAK2-hyperaktivaation. Tämä analyysi tarkentaa ymmärrystämme JAKien toiminnasta sytokiinivälitteisessä sekä sytokiineista riippumattomassa JAK-aktivaatiossa ja samalla tunnistaa aiemmin tuntemattoman JAK2 JH2 rajapinnan, joka on välttämätön interferoni-γ signaloinnille. Janus kinases (JAKs) are non-receptor tyrosine kinases that mediate signalling of around sixty different cytokines governing various biological processes from the regulation of the immune system, to control of haematopoiesis, metabolism, and development. JAKs are multidomain proteins, in which the tyrosine kinase domain (JH1) is preceded by a pseudokinase domain (JH2). JH2 has critical regulatory functions and is a hotspot for many known oncogenic driver mutations. These mutations, which cause ligand-independent JAK activation, underlie various diseases—most notably haematopoietic malignancies caused by somatic JAK2 JH2 mutations. In the work presented here, we analysed the functions of JH2 and its mutations in the regulation of JAK activity. We found that all JAK JH2s have functional nucleotide-binding sites accessible to ATP and small molecule inhibitors. Most importantly, we found that disruption of ATP binding to JAK2 JH2 suppresses ligand-independent activation, thus identifying the JAK2 JH2 ATP-binding site as a potential drug target for the development of mutation-specific inhibitors. These inhibitors would be a distinct improvement over inhibitors of JAK2 currently used to treat MPNs, as current inhibitors do not distinguish between mutated and wild-type JAK2, and are unable to eradicate the disease. We also present a collaboration effort leading to a simulation-based model for JH2-mediated inhibition of JH1, thereby providing rationale for most known clinical JAK2 mutations. Moreover, we refine our understanding of ligand-mediated and ligand-independent activation of JAKs by presenting a systematic analysis of JAK2 mutations capable of inhibiting ligand-independent hyperactivation. We further identify a novel interface in JAK2 JH2, which is needed for heteromeric JAK2 activation in interferon-γ signalling.

Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed.

SummaryCCAAT-enhancer binding proteins (C/EBPs) are transcription factors that play a central role in the differentiation of myeloid cells and adipocytes. Tribbles pseudokinases govern levels of C/EBPs by recruiting them to the COP1 ubiquitin ligase for ubiquitination. Here, we present the first crystal structure of a Tribbles protein, which reveals a catalytically inactive TRIB1 pseudokinase domain with a unique adaptation in the αC helix. A second crystal structure and biophysical studies of TRIB1 with its C-terminal extension, which includes the COP1-binding motif, show that the C-terminal extension is sequestered at a site formed by the modified TRIB1 αC helix. In addition, we have identified and characterized the TRIB1 substrate-recognition sequence within C/EBPα, which is evolutionarily conserved in C/EBP transcription factors. Binding studies indicate that C/EBPα recruitment is weaker in the presence of the C-terminal COP1-binding motif, but the magnitude of this effect suggests that the two bind distinct rather directly overlapping binding sites.

Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation.

Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001, eLife digest Some proteins must be modified in order to work effectively. One common modification is to add a phosphate group to the protein, which is performed by enzymes called protein kinases. Although most of the protein kinases work on proteins inside the cell, it was discovered recently that a small group of kinases work within the ‘secretory pathway’ and modify proteins that are released (or secreted) out of cells. One such secretory pathway kinase—called Fam20C—phosphorylates a wide range of secreted proteins and helps to ensure the proper development of bones and teeth. Specifically, Fam20C and a closely related protein called Fam20A are important for forming enamel, the hardest substance in human body, which makes up the outer surface of teeth. However, the exact role of Fam20A is unknown. Cui et al. now show that Fam20A binds to Fam20C, and this increases the ability of Fam20C to phosphorylate the proteins that form the ‘matrix’ that guides the deposition of the enamel minerals. Furthermore, mutations in Fam20A lead to the inefficient phosphorylation of enamel matrix proteins by Fam20C, and prevent proper enamel formation. The results raise the possibility that similar mechanisms of secretory kinase activation may also be important in other biological processes where many secreted proteins need to be phosphorylated rapidly. DOI: http://dx.doi.org/10.7554/eLife.06120.002

Emerging evidence supports the dual function of kinase suppressor of Ras 1 (KSR1) as an active kinase and a scaffold, although it has been extensively referred as a pseudokinase, due to the absence of key residues in its catalytic domain [1, 2]. As a scaffolding protein, KSR1 orchestrates the assembly of the protein kinases RAF, mitogen activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK) in the canonical Ras-RAF-MAPKs pathway, in a Ras-dependent manner or upon growth factor treatment [1, 3]. Conversely, structural and biochemical studies reveal that KSR1 is capable of phosphorylating MEK and more importantly, the catalytic activity of KSR is markedly increased when BRAF or inhibitor-bound CRAF is introduced in the complexes [1, 4, 5]. Such findings add complexity to the ERK spatio-temporal pathway control and identify KSR1 as a modulator of these pathways. In light of its regulatory role in oncogenic Ras-RAF-MAPKs signaling, extensive efforts have attempted to establish KSR1 as an oncogene in Ras-dependent cancers. Indeed, KSR1 has been shown to contribute to oncogenesis in various forms of Ras-activated cancer, including skin, pancreatic and lung carcinomas [1]. First, absence of KSR1 inhibits tumor formation in different Ras-mediated mouse models (KSR1−/−), suggesting that KSR1 is required for Ras-transduced MAPK activated tumorigenesis. Moreover, depletion of KSR1 reduces tumor growth of K-Ras-dependent pancreatic and lung cancer xenografts in nude mice, further supporting KSR1 as an oncogene as well as a potential therapeutic target. However, our own studies reveal an interesting, and yet unexpected role of KSR1 in breast cancer, where Ras mutations are rare. Using tumor tissue microarrays in a large patient cohort with a long term follow-up, we observe that breast cancer patients with high KSR1 had better disease free- and overall survival, results also supported by Oncomine analyses, microarray data and genomic data from paired tumor and cell-free DNA (cfDNA) samples revealing loss of heterozygosity [6]. KSR1 expression is positively associated with breast cancer 1, early onset (BRCA1), BRCA1-associated ring domain 1 (BARD1) and checkpoint kinase 1 (Chk1) levels in breast cancer specimens. Intriguingly, phospho-profiling of major components of the canonical Ras-RAF-MAPKs pathway, including RAF, MEK and ERK, show no significant changes upon KSR1 overexpression or depletion. These results underline its role beyond coordinating MAPKs signaling and challenge its oncogenic function in breast cancer. Further experiments support a tumor suppressive role of KSR1. Of note, breast cancer cells overexpressing KSR1 form fewer and smaller size colonies compared to the parental ones, while an in vivo mouse model also demonstrates that the growth of xenograft tumors overexpressing KSR1 is inhibited. It appears that the tumor inhibitory effect of KSR1 is BRCA1-dependent as shown by in vitro 3D-matrigel and soft-agar assays. Consistent with the correlation between KSR1 and BRCA1 in clinical samples, in vitro assays demonstrate KSR1 overexpression increases BRCA1 protein levels by decreasing BRCA1 ubiquitination partly through elevating BARD1 protein abundance and the BRCA1-BARD1 interaction. These findings integrate KSR1 into a complicated signaling network involving BRCA1-BARD1 complex. Unquestionably, alternative mechanisms underlying BRCA1's up-regulation by KSR1 other than through BARD1 remain to be examined. Our current understanding of KSR1's function in breast cancer is far from complete (Figure ​(Figure1).1). Puzzlingly, in addition to executing its anti-tumor effects through BRCA1, KSR1 negatively regulates transcriptional activity of p53 by reducing p53 acetylation via modulation of deleted in breast cancer 1 (DBC1) phosphorylation [7]. How can KSR1 up-regulates BRCA1 and inhibits p53 activity at the same time? What determines its preferable binding partners and thus contributes to its inhibitory outcome? An interesting and simple theory might be the following: since DBC1 is a positive regulator of p53 activity and a repressor for BRCA1, it potentially positions KSR1 into a dynamic state connecting p53 and BRCA1. Ultimately, while BRCA1 compensates for the loss of p53 activity, KSR1 tilts the balance scale towards tumor suppression in cancer cells. Further investigation is much needed to address the underlying mechanisms. In addition, evidence regarding other aspects of its action are evolving: a very recent study characterizing the proteomic profile of KSR1-regulated signaling in response to genotoxic agents in breast cancer illustrates a broad functional network conferred by KSR1, highlighting its importance in the chemotherapy response [8].. Figure 1 KSR1-regulated signaling in cancer A better understanding of the molecular determinants of the distinctive behaviour of KSR1 in different types of cancer is essential to the optimal targeting of this dual functional kinase-scaffold protein. Our studies suggest the tumor suppressive action of KSR1 and its clinical relevance in patient stratification, placing KSR1 in the major oncoprotein pathways.

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90’s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.

Striated muscle tissues undergo adaptive remodelling in response to mechanical load. This process involves the myofilament titin and, specifically, its kinase domain (TK; titin kinase) that translates mechanical signals into regulatory pathways of gene expression in the myofibril. TK mechanosensing appears mediated by a C-terminal regulatory tail (CRD) that sterically inhibits its active site. Allegedly, stretch-induced unfolding of this tail during muscle function releases TK inhibition and leads to its catalytic activation. However, the cellular pathway of TK is poorly understood and substrates proposed to date remain controversial. TK's best-established substrate is Tcap, a small structural protein of the Z-disc believed to link TK to myofibrillogenesis. Here, we show that TK is a pseudokinase with undetectable levels of catalysis and, therefore, that Tcap is not its substrate. Inactivity is the result of two atypical residues in TK's active site, M34 and E147, that do not appear compatible with canonical kinase patterns. While not mediating stretch-dependent phospho-transfers, TK binds the E3 ubiquitin ligase MuRF1 that promotes sarcomeric ubiquitination in a stress-induced manner. Given previous evidence of MuRF2 interaction, we propose that the cellular role of TK is to act as a conformationally regulated scaffold that functionally couples the ubiquitin ligases MuRF1 and MuRF2, thereby coordinating muscle-specific ubiquitination pathways and myofibril trophicity. Finally, we suggest that an evolutionary dichotomy of kinases/pseudokinases has occurred in TK-like kinases, where invertebrate members are active enzymes but vertebrate counterparts perform their signalling function as pseudokinase scaffolds.

BACKGROUND: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and β. The roles of STRADα and STRADβ in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions. RESULTS: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization. CONCLUSIONS: We demonstrate a novel role for STRADβ in axogenesis and also show for the first time in vivo that STRADα, but not STRADβ, is responsible for LKB1 protein stability.

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Basal breast cancer cells feature high expression of the Src family kinase Lyn that has been implicated in the pathogenicity of this disease. In this study, we identified novel Lyn kinase substrates, the most prominent of which was the atypical kinase SgK269 (PEAK1). In breast cancer cells, SgK269 expression associated with the basal phenotype. In primary breast tumors, SgK269 overexpression was detected in a subset of basal, HER2-positive, and luminal cancers. In immortalized MCF-10A mammary epithelial cells, SgK269 promoted transition to a mesenchymal phenotype and increased cell motility and invasion. Growth of MCF-10A acini in three-dimensional (3D) culture was enhanced upon SgK269 overexpression, which induced an abnormal, multilobular acinar morphology and promoted extracellular signal–regulated kinase (Erk) and Stat3 activation. SgK269 Y635F, mutated at a major Lyn phosphorylation site, did not enhance acinar size or cellular invasion. We show that Y635 represents a Grb2-binding site that promotes both Stat3 and Erk activation in 3D culture. RNA interference–mediated attenuation of SgK269 in basal breast cancer cells promoted acquisition of epithelial characteristics and decreased anchorage-independent growth. Together, our results define a novel signaling pathway in basal breast cancer involving Lyn and SgK269 that offers clinical opportunities for therapeutic intervention. National Health and Medical Research Council of Australia, Cancer Council New South Wales (NSW) and Science Foundation Ireland (Grant No. 06/CE/B1129). DRC and DS were supported by Fellowships from Cancer Institute (CI) NSW. FH is supported by The Ministry of Education and Research, Bundesministerium für Bildung und Forschung (03Z1CN21). CMT is the recipient of a Research Scholarship from CINSW, and LZ an Australian Postgraduate Award. DGO is supported by a National Breast Cancer Foundation (NBCF) and Cure Cancer Australia Foundation Postdoctoral Fellowship. AS is supported by an Early Career Fellowship from the NBCF. Deposited by bulk import

The pathway that regulates asymmetric cell division in Caulobacter involves a signaling kinase whose catalytic output domain has been repurposed as an input sensor of the phosphorylation state of the response regulator – a reversal of the conventional direction of information flow; this allows wiring of simple linear signaling pathways into complex eukaryote-like networks., One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation., Author Summary Across all kingdoms of life the generation of cell-type diversity is the consequence of asymmetry at the point of cell division. The bacterium Caulobacter crescentus divides asymmetrically to produce daughter cells that have distinct morphology and behavior. As in eukaryotes, an unequal distribution of signaling proteins in daughter Caulobacter cells triggers the differential read-out of identical genomes. A critical interaction between two protein molecules – a protein kinase (DivL) and a response regulator (DivK) – is known to occur exclusively in one daughter cell and to thereby regulate differentiation. However, mapping the observed signaling interconnections that drive asymmetric division has been difficult to reconcile with traditional models of bacterial signaling. Here we determine how DivL detects and processes this DivK signal. Although DivL has an architecture that is typical of histidine kinases, which normally act by regulating the phosphorylation state of the appropriate response regulator, DivL's essential functions do not require kinase activity and DivL does not add or remove phosphate from DivK. Instead we find that DivL has converted its output kinase domain into an input sensor domain that specifically detects phosphorylated DivK, and we identify key features of DivL that underlie this specificity. This novel reassignment of sensory functions reverses the conventional kinase-to-response-regulator signaling flow and logically couples linear signaling pathways into complex eukaryote-like networks to regulate cell development.