Unmasking determinants of specificity in the human kinome.

Protein kinases control cellular responses to environmental cues by swift and accurate signal processing. Breakdowns in this high-fidelity capability are a driving force in cancer and other diseases. Thus, our limited understanding of which amino acids in the kinase domain encode substrate specificity, the so-called determinants of specificity (DoS), constitutes a major obstacle in cancer signaling. Here, we systematically discover several DoS and experimentally validate three of them, named the αC1, αC3, and APE-7 residues. We demonstrate that DoS form sparse networks of non-conserved residues spanning distant regions. Our results reveal a likely role for inter-residue allostery in specificity and an evolutionary decoupling of kinase activity and specificity, which appear loaded on independent groups of residues. Finally, we uncover similar properties driving SH2 domain specificity and demonstrate how the identification of DoS can be utilized to elucidate a greater understanding of the role of signaling networks in cancer (Creixell et al., 2015 [this issue of Cell]).

Kinome-wide decoding of network-attacking mutations rewiring cancer signaling.

Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks.

The intrinsic substrate specificity of the human tyrosine kinome.

Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.

An atlas of substrate specificities for the human serine/threonine kinome.

Protein phosphorylation is one of the most widespread post-translational modifications in biology1,2. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes3,4. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible3. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.

Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting.

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.

Linear motif specificity in signaling through p38α and ERK2 mitogen-activated protein kinases.

Mitogen-activated protein kinase (MAPK) cascades are essential for eukaryotic cells to integrate and respond to diverse stimuli. Maintaining specificity in signaling through MAPK networks is key to coupling distinct inputs to appropriate cellular responses. Docking sites-short linear motifs found in MAPK substrates, regulators, and scaffolds-can promote signaling specificity through selective interactions, but how they do so remains unresolved. Here, we screened a proteomic library for sequences interacting with the MAPKs extracellular signal-regulated kinase 2 (ERK2) and p38α, identifying selective and promiscuous docking motifs. Sequences specific for p38α had high net charge and lysine content, and selective binding depended on a pair of acidic residues unique to the p38α docking interface. Finally, we validated a set of full-length proteins harboring docking sites selected in our screens to be authentic MAPK interactors and substrates. This study identifies features that help define MAPK signaling networks and explains how specific docking motifs promote signaling integrity.

Structural Basis for Noncanonical Substrate Recognition of Cofilin/ADF Proteins by LIM Kinases.

Cofilin/actin-depolymerizing factor (ADF) proteins are critical nodes that relay signals from protein kinase cascades to the actin cytoskeleton, in particular through site-specific phosphorylation at residue Ser3. This is important for regulation of the roles of cofilin in severing and stabilizing actin filaments. Consequently, cofilin/ADF Ser3 phosphorylation is tightly controlled as an almost exclusive substrate for LIM kinases. Here we determine the LIMK1:cofilin-1 co-crystal structure. We find an interface that is distinct from canonical kinase-substrate interactions. We validate this previously unobserved mechanism for high-fidelity kinase-substrate recognition by in vitro kinase assays, examination of cofilin phosphorylation in mammalian cells, and functional analysis in S. cerevisiae. The interface is conserved across all LIM kinases. Remarkably, we also observe both pre- and postphosphotransfer states in the same crystal lattice. This study therefore provides a molecular understanding of how kinase-substrate recognition acts as a gatekeeper to regulate actin cytoskeletal dynamics.

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

Small Molecule Inhibition of the Autophagy Kinase ULK1 and Identification of ULK1 Substrates.

Many tumors become addicted to autophagy for survival, suggesting inhibition of autophagy as a potential broadly applicable cancer therapy. ULK1/Atg1 is the only serine/threonine kinase in the core autophagy pathway and thus represents an excellent drug target. Despite recent advances in the understanding of ULK1 activation by nutrient deprivation, how ULK1 promotes autophagy remains poorly understood. Here, we screened degenerate peptide libraries to deduce the optimal ULK1 substrate motif and discovered 15 phosphorylation sites in core autophagy proteins that were verified as in vivo ULK1 targets. We utilized these ULK1 substrates to perform a cell-based screen to identify and characterize a potent ULK1 small molecule inhibitor. The compound SBI-0206965 is a highly selective ULK1 kinase inhibitor in vitro and suppressed ULK1-mediated phosphorylation events in cells, regulating autophagy and cell survival. SBI-0206965 greatly synergized with mechanistic target of rapamycin (mTOR) inhibitors to kill tumor cells, providing a strong rationale for their combined use in the clinic.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Homing in: Mechanisms of Substrate Targeting by Protein Kinases.

Protein phosphorylation is the most common reversible post-translational modification in eukaryotes. Humans have over 500 protein kinases, of which more than a dozen are established targets for anticancer drugs. All kinases share a structurally similar catalytic domain, yet each one is uniquely positioned within signaling networks controlling essentially all aspects of cell behavior. Kinases are distinguished from one another based on their modes of regulation and their substrate repertoires. Coupling specific inputs to the proper signaling outputs requires that kinases phosphorylate a limited number of sites to the exclusion of hundreds of thousands of off-target phosphorylation sites. Here, we review recent progress in understanding mechanisms of kinase substrate specificity and how they function to shape cellular signaling networks.

Comprehensive profiling of the STE20 kinase family defines features essential for selective substrate targeting and signaling output.

Specificity within protein kinase signaling cascades is determined by direct and indirect interactions between kinases and their substrates. While the impact of localization and recruitment on kinase-substrate targeting can be readily assessed, evaluating the relative importance of direct phosphorylation site interactions remains challenging. In this study, we examine the STE20 family of protein serine-threonine kinases to investigate basic mechanisms of substrate targeting. We used peptide arrays to define the phosphorylation site specificity for the majority of STE20 kinases and categorized them into four distinct groups. Using structure-guided mutagenesis, we identified key specificity-determining residues within the kinase catalytic cleft, including an unappreciated role for the kinase ß3-αC loop region in controlling specificity. Exchanging key residues between the STE20 kinases p21-activated kinase 4 (PAK4) and Mammalian sterile 20 kinase 4 (MST4) largely interconverted their phosphorylation site preferences. In cells, a reprogrammed PAK4 mutant, engineered to recognize MST substrates, failed to phosphorylate PAK4 substrates or to mediate remodeling of the actin cytoskeleton. In contrast, this mutant could rescue signaling through the Hippo pathway in cells lacking multiple MST kinases. These observations formally demonstrate the importance of catalytic site specificity for directing protein kinase signal transduction pathways. Our findings further suggest that phosphorylation site specificity is both necessary and sufficient to mediate distinct signaling outputs of STE20 kinases and imply broad applicability to other kinase signaling systems.

An AMPK-independent signaling pathway downstream of the LKB1 tumor suppressor controls Snail1 and metastatic potential.

The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.

Identification of a major determinant for serine-threonine kinase phosphoacceptor specificity.

Eukaryotic protein kinases are generally classified as being either tyrosine or serine-threonine specific. Though not evident from inspection of their primary sequences, many serine-threonine kinases display a significant preference for serine or threonine as the phosphoacceptor residue. Here we show that a residue located in the kinase activation segment, which we term the "DFG+1" residue, acts as a major determinant for serine-threonine phosphorylation site specificity. Mutation of this residue was sufficient to switch the phosphorylation site preference for multiple kinases, including the serine-specific kinase PAK4 and the threonine-specific kinase MST4. Kinetic analysis of peptide substrate phosphorylation and crystal structures of PAK4-peptide complexes suggested that phosphoacceptor residue preference is not mediated by stronger binding of the favored substrate. Rather, favored kinase-phosphoacceptor combinations likely promote a conformation optimal for catalysis. Understanding the rules governing kinase phosphoacceptor preference allows kinases to be classified as serine or threonine specific based on their sequence.

Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase.

Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.

Recognition of physiological phosphorylation sites by p21-activated kinase 4.

Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues. The only well documented exception is LIM domain kinase 1 (LIMK1), which is phosphorylated on an activation loop threonine (Thr508) to promote its catalytic activity. To understand the molecular and kinetic basis for PAK4 substrate selectivity we compared its mode of recognition of LIMK1 (Thr508) with that of a known serine substrate, ß-catenin (Ser675). We determined X-ray crystal structures of PAK4 in complex with synthetic peptides corresponding to its phosphorylation sites in LIMK1 and ß-catenin to 1.9 Å and 2.2 Å resolution, respectively. We found that the PAK4 DFG + 1 residue, a key determinant of phosphoacceptor preference, adopts a sub-optimal orientation when bound to LIMK1 compared to ß-catenin. In peptide kinase activity assays, we find that phosphoacceptor identity impacts catalytic efficiency but does not affect the Km value for both phosphorylation sites. Although catalytic efficiency of wild-type LIMK1 and ß-catenin are equivalent, T508S mutation of LIMK1 creates a highly efficient substrate. These results suggest suboptimal phosphorylation of LIMK1 as a mechanism for controlling the dynamics of substrate phosphorylation by PAK4.

Substrate priming enhances phosphorylation by the budding yeast kinases Kin1 and Kin2.

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.

Exceptionally Selective Substrate Targeting by the Metalloprotease Anthrax Lethal Factor.

The zinc-dependent metalloprotease anthrax lethal factor (LF) is the enzymatic component of a toxin thought to have a major role in Bacillus anthracis infections. Like many bacterial toxins, LF is a secreted protein that functions within host cells. LF is a highly selective protease that cleaves a limited number of substrates in a site-specific manner, thereby impacting host signal transduction pathways. The major substrates of LF are mitogen-activated protein kinase kinases (MKKs), which lie in the middle of three-component phosphorylation cascades mediating numerous functions in a variety of cells and tissues. How LF targets its limited substrate repertoire has been an active area of investigation. LF recognizes a specific sequence motif surrounding the scissile bonds of substrate proteins. X-ray crystallography of the protease in complex with peptide substrates has revealed the structural basis of selectivity for the LF cleavage site motif. In addition to having interactions proximal to the cleavage site, LF binds directly to a more distal region in its substrates through a so-called exosite interaction. This exosite has been mapped to a surface within a non-catalytic domain of LF with previously unknown function. A putative LF-binding site has likewise been identified on the catalytic domains of MKKs. Here we review our current state of understanding of LF-substrate interactions and discuss the implications for the design and discovery of inhibitors that may have utility as anthrax therapeutics.

Control of serotonin transporter phosphorylation by conformational state.

Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na(+) and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na(+) and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.

Loss of TRIM33 causes resistance to BET bromodomain inhibitors through MYC- and TGF-ß-dependent mechanisms.

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.