The Ste5 scaffold directs mating signaling by catalytically unlocking the Fus3 MAP kinase for activation.

The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3's activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the k(cat) of Ste7-->Fus3 phosphorylation but has no effect on Ste7-->Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway and not by other pathways that also utilize Ste7.

Discovery and Characterization of Linear Motif Mediated Protein-Protein Complexes.

There are myriads of protein-protein complexes that form within the cell. In addition to classical binding events between globular domains, many protein-protein interactions involve short disordered protein regions. The latter contain so-called linear motifs binding specifically to ordered protein domain surfaces. Linear binding motifs are classified based on their consensus sequence, where only a few amino acids are conserved. In this chapter we will review experimental and in silico techniques that can be used for the discovery and characterization of linear motif mediated protein-protein complexes involved in cellular signaling, protein level and gene expression regulation.

Disordered Protein Kinase Regions in Regulation of Kinase Domain Cores.

Since publication of the crystal structure of protein kinase (PK)A three decades ago, a structural portrait of the conserved kinase core has been drawn. The next challenge is to elucidate structures of full-length kinases and to address the intrinsically disordered regions (IDRs) that typically flank the core as well as the small linear motifs (SLiMs) that are embedded within the IDRs. It is increasingly apparent that unstructured regions integrate the kinase catalytic chassis into multienzyme-based regulatory networks. The extracellular signal-regulated kinase-ribosomal S6 PK-phosphoinositide-dependent kinase (ERK-RSK-PDK) complex is an excellent example to demonstrate how IDRs and SLiMs govern communication between four different kinase catalytic cores to mediate activation and how in molecular terms these promote the formation of kinase heterodimers in a context dependent fashion.

Phosphorylation-Assisted Luciferase Complementation Assay Designed to Monitor Kinase Activity and Kinase-Domain-Mediated Protein-Protein Binding.

Protein kinases are key regulators of cell signaling and have been important therapeutic targets for three decades. ATP-competitive drugs directly inhibit the activity of kinases but these enzymes work as part of complex protein networks in which protein-protein interactions (often referred to as kinase docking) may govern a more complex activation pattern. Kinase docking is indispensable for many signaling disease-relevant Ser/Thr kinases and it is mediated by a dedicated surface groove on the kinase domain which is distinct from the substrate-binding pocket. Thus, interfering with kinase docking provides an alternative strategy to control kinases. We describe activity sensors developed for p90 ribosomal S6 kinase (RSK) and mitogen-activated protein kinases (MAPKs: ERK, p38, and JNK) whose substrate phosphorylation is known to depend on kinase-docking-groove-mediated protein-protein binding. The in vitro assays were based on fragment complementation of the NanoBit luciferase, which is facilitated upon substrate motif phosphorylation. The new phosphorylation-assisted luciferase complementation (PhALC) sensors are highly selective and the PhALC assay is a useful tool for the quantitative analysis of kinase activity or kinase docking, and even for high-throughput screening of academic compound collections.

Systematic Discovery of FBXW7-Binding Phosphodegrons Highlights Mitogen-Activated Protein Kinases as Important Regulators of Intracellular Protein Levels.

A FBXW7 is an F-box E3 ubiquitin-ligase affecting cell growth by controlling protein degradation. Mechanistically, its effect on its substrates depends on the phosphorylation of degron motifs, but the abundance of these phosphodegrons has not been systematically explored. We used a ratiometric protein degradation assay geared towards the identification of FBXW7-binding degron motifs phosphorylated by mitogen-activated protein kinases (MAPKs). Most of the known FBXW7 targets are localized in the nucleus and function as transcription factors. Here, in addition to more transcription affecting factors (ETV5, KLF4, SP5, JAZF1, and ZMIZ1 CAMTA2), we identified phosphodegrons located in proteins involved in chromatin regulation (ARID4B, KMT2E, KMT2D, and KAT6B) or cytoskeletal regulation (MAP2, Myozenin-2, SMTL2, and AKAP11), and some other proteins with miscellaneous functions (EIF4G3, CDT1, and CCAR2). We show that the protein level of full-length ARID4B, ETV5, JAZF1, and ZMIZ1 are affected by different MAPKs since their FBXW7-mediated degradation was diminished in the presence of MAPK-specific inhibitors. Our results suggest that MAPK and FBXW7 partnership plays an important cellular role by directly affecting the level of key regulatory proteins. The data also suggest that the p38α-controlled phosphodegron in JAZF1 may be responsible for the pathological regulation of the cancer-related JAZF1-SUZ12 fusion construct implicated in endometrial stromal sarcoma.

Characterization of the Intramolecular Interactions and Regulatory Mechanisms of the Scaffold Protein Tks4.

The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called "tandem SH3") and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

Ndr/Lats Kinases Bind Specific Mob-Family Coactivators through a Conserved and Modular Interface.

Ndr/Lats kinases bind Mob coactivator proteins to form complexes that are essential and evolutionarily conserved components of "Hippo" signaling pathways, which control cell proliferation and morphogenesis in eukaryotes. All Ndr/Lats kinases have a characteristic N-terminal regulatory (NTR) region that binds a specific Mob cofactor: Lats kinases associate with Mob1 proteins, and Ndr kinases associate with Mob2 proteins. To better understand the significance of the association of Mob protein with Ndr/Lats kinases and selective binding of Ndr and Lats to distinct Mob cofactors, we determined crystal structures of Saccharomyces cerevisiae Cbk1NTR-Mob2 and Dbf2NTR-Mob1 and experimentally assessed determinants of Mob cofactor binding and specificity. This allowed a significant improvement in the previously determined structure of Cbk1 kinase bound to Mob2, presently the only crystallographic model of a full length Ndr/Lats kinase complexed with a Mob cofactor. Our analysis indicates that the Ndr/LatsNTR-Mob interface provides a distinctive kinase regulation mechanism, in which the Mob cofactor organizes the Ndr/Lats NTR to interact with the AGC kinase C-terminal hydrophobic motif (HM), which is involved in allosteric regulation. The Mob-organized NTR appears to mediate association of the HM with an allosteric site on the N-terminal kinase lobe. We also found that Cbk1 and Dbf2 associated specifically with Mob2 and Mob1, respectively. Alteration of residues in the Cbk1 NTR allows association of the noncognate Mob cofactor, indicating that cofactor specificity is restricted by discrete sites rather than being broadly distributed. Overall, our analysis provides a new picture of the functional role of Mob association and indicates that the Ndr/LatsNTR-Mob interface is largely a common structural platform that mediates kinase-cofactor binding.

Theileria highjacks JNK2 into a complex with the macroschizont GPI (GlycosylPhosphatidylInositol)-anchored surface protein p104.

Constitutive c-Jun N-terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI-(GlycosylPhosphatidylInositol)-anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase-A (PKA)-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages.

The eukaryotic linear motif resource - 2018 update.

Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome.

Meiotic DNA double-strand breaks and chromosome asynapsis in mice are monitored by distinct HORMAD2-independent and -dependent mechanisms.

Meiotic crossover formation involves the repair of programmed DNA double-strand breaks (DSBs) and synaptonemal complex (SC) formation. Completion of these processes must precede the meiotic divisions in order to avoid chromosome abnormalities in gametes. Enduring key questions in meiosis have been how meiotic progression and crossover formation are coordinated, whether inappropriate asynapsis is monitored, and whether asynapsis elicits prophase arrest via mechanisms that are distinct from the surveillance of unrepaired DNA DSBs. We disrupted the meiosis-specific mouse HORMAD2 (Hop1, Rev7, and Mad2 domain 2) protein, which preferentially associates with unsynapsed chromosome axes. We show that HORMAD2 is required for the accumulation of the checkpoint kinase ATR along unsynapsed axes, but not at DNA DSBs or on DNA DSB-associated chromatin loops. Consistent with the hypothesis that ATR activity on chromatin plays important roles in the quality control of meiotic prophase, HORMAD2 is required for the elimination of the asynaptic Spo11(-/-), but not the asynaptic and DSB repair-defective Dmc1(-/-) oocytes. Our observations strongly suggest that HORMAD2-dependent recruitment of ATR to unsynapsed chromosome axes constitutes a mechanism for the surveillance of asynapsis. Thus, we provide convincing evidence for the existence of a distinct asynapsis surveillance mechanism that safeguards the ploidy of the mammalian germline.

Ubiquitin-dependent regulation of MEKK2/3-MEK5-ERK5 signaling module by XIAP and cIAP1.

Mitogen-activated protein kinases (MAPKs) are highly conserved protein kinase modules, and they control fundamental cellular processes. While the activation of MAPKs has been well studied, little is known on the mechanisms driving their inactivation. Here we uncover a role for ubiquitination in the inactivation of a MAPK module. Extracellular-signal-regulated kinase 5 (ERK5) is a unique, conserved member of the MAPK family and is activated in response to various stimuli through a three-tier cascade constituting MEK5 and MEKK2/3. We reveal an unexpected role for Inhibitors of Apoptosis Proteins (IAPs) in the inactivation of ERK5 pathway in a bimodal manner involving direct interaction and ubiquitination. XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3 which directly impede MEK5-ERK5 interaction in a trimeric complex leading to ERK5 inactivation. Consistently, loss of XIAP or cIAP1 by various strategies leads to hyperactivation of ERK5 in normal and tumorigenic cells. Loss of XIAP promotes differentiation of human primary skeletal myoblasts to myocytes in a MEKK2/3-ERK5-dependent manner. Our results reveal a novel, obligatory role for IAPs and ubiquitination in the physical and functional disassembly of ERK5-MAPK module and human muscle cell differentiation.

The Structure of an NDR/LATS Kinase-Mob Complex Reveals a Novel Kinase-Coactivator System and Substrate Docking Mechanism.

Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. "Hippo" pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with "Mob" coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1-Mob2, to our knowledge the first of an NDR/LATS kinase-Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1's regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides.

Structural assembly of the signaling competent ERK2-RSK1 heterodimeric protein kinase complex.

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase-kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 "docking" groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they "readjust," whereas generic kinase domain surface contacts bring them into a catalytically competent state.

Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

Systematic discovery of linear binding motifs targeting an ancient protein interaction surface on MAP kinases.

Mitogen-activated protein kinases (MAPK) are broadly used regulators of cellular signaling. However, how these enzymes can be involved in such a broad spectrum of physiological functions is not understood. Systematic discovery of MAPK networks both experimentally and in silico has been hindered because MAPKs bind to other proteins with low affinity and mostly in less-characterized disordered regions. We used a structurally consistent model on kinase-docking motif interactions to facilitate the discovery of short functional sites in the structurally flexible and functionally under-explored part of the human proteome and applied experimental tools specifically tailored to detect low-affinity protein-protein interactions for their validation in vitro and in cell-based assays. The combined computational and experimental approach enabled the identification of many novel MAPK-docking motifs that were elusive for other large-scale protein-protein interaction screens. The analysis produced an extensive list of independently evolved linear binding motifs from a functionally diverse set of proteins. These all target, with characteristic binding specificity, an ancient protein interaction surface on evolutionarily related but physiologically clearly distinct three MAPKs (JNK, ERK, and p38). This inventory of human protein kinase binding sites was compared with that of other organisms to examine how kinase-mediated partnerships evolved over time. The analysis suggests that most human MAPK-binding motifs are surprisingly new evolutionarily inventions and newly found links highlight (previously hidden) roles of MAPKs. We propose that short MAPK-binding stretches are created in disordered protein segments through a variety of ways and they represent a major resource for ancient signaling enzymes to acquire new regulatory roles.

Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity.

Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair.

Signaling pathways depend on regulatory protein-protein interactions; controlling these interactions in cells has important applications for reengineering biological functions. As many regulatory proteins are modular, considerable progress in engineering signaling circuits has been made by recombining commonly occurring domains. Our ability to predictably engineer cellular functions, however, is constrained by complex crosstalk observed in naturally occurring domains. Here we demonstrate a strategy for improving and simplifying protein network engineering: using computational design to create orthogonal (non-crossreacting) protein-protein interfaces. We validated the design of the interface between a key signaling protein, the GTPase Cdc42, and its activator, Intersectin, biochemically and by solving the crystal structure of the engineered complex. The designed GTPase (orthoCdc42) is activated exclusively by its engineered cognate partner (orthoIntersectin), but maintains the ability to interface with other GTPase signaling circuit components in vitro. In mammalian cells, orthoCdc42 activity can be regulated by orthoIntersectin, but not wild-type Intersectin, showing that the designed interaction can trigger complex processes. Computational design of protein interfaces thus promises to provide specific components that facilitate the predictable engineering of cellular functions.

Structural mechanism for the specific assembly and activation of the extracellular signal regulated kinase 5 (ERK5) module.

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.

Protein-peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase.

Linear motifs normally bind with only medium binding affinity (Kd of ∼0.1-10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein-peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein-peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted' crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein-peptide complexes.