Linking the kinome and phosphorylome--a comprehensive review of approaches to find kinase targets

Protein phosphorylation is associated with most cell signaling and developmental processes in eukaryotes. Despite the vast extent of the phosphoproteome within the cell, connecting specific kinases with relevant targets remains a significant experimental frontier. The challenge of linking kinases and their substrates reflects the complexity of kinase function. For example, kinases tend to exert their biological effects through supernumerary, redundant phosphorylation, often on multiple protein complex components. Although these types of phosphorylation events are biologically significant, those kinases responsible are often difficult to identify. Recent methods for global analysis of protein phosphorylation promise to substantially accelerate efforts to map the dynamic phosphorylome. Here, we review both conventional methods to identify kinase targets and more comprehensive genomic and proteomic approaches to connect the kinome and phosphorylome.     

Construction of human activity‐based phosphorylation networks

The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)‐based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase‐substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high‐quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high‐resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B‐cell receptor signaling. Overall, these studies provide global insights into kinase‐mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.     

Kinomics: methods for deciphering the kinome

Phosphorylation by protein kinases is the most widespread and well-studied signaling mechanism in eukaryotic cells. Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes. Cataloging and understanding protein phosphorylation is no easy task: many kinases may be expressed in a cell, and one-third of all intracellular proteins may be phosphorylated, representing as many as 20,000 distinct phosphoprotein states. Defining the kinase complement of the human genome, the kinome, has provided an excellent starting point for understanding the scale of the problem. The kinome consists of 518 kinases, and every active protein kinase phosphorylates a distinct set of substrates in a regulated manner. Deciphering the complex network of phosphorylation-based signaling is necessary for a thorough and therapeutically applicable understanding of the functioning of a cell in physiological and pathological states. We review contemporary techniques for identifying physiological substrates of the protein kinases and studying phosphorylation in living cells.     

An atlas of human kinase regulation

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision‐making has been limited by the small number of simultaneously monitored phospho‐regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co‐regulation along the conditions predicts kinase–complex and kinase–substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation‐based signaling and the necessary context to better understand kinase‐driven decision‐making.     

Experimental and computational tools useful for (re)construction of dynamic kinase–substrate networks

The explosion of site‐ and context‐specific in vivo phosphorylation events presents a potentially rich source of biological knowledge and calls for novel data analysis and modeling paradigms. Perhaps the most immediate challenge is delineating detected phosphorylation sites to their effector kinases. This is important for (re)constructing transient kinase–substrate interaction networks that are essential for mechanistic understanding of cellular behaviors and therapeutic intervention, but has largely eluded high‐throughput protein‐interaction studies due to their transient nature and strong dependencies on cellular context. Here, we surveyed some of the computational approaches developed to dissect phosphorylation data detected in systematic proteomic experiments and reviewed some experimental and computational approaches used to map phosphorylation sites to their effector kinases in efforts aimed at reconstructing biological signaling networks.     

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

Leucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.     

Identification of the phosphorylation targets of symbiotic receptor‐like kinases using a high‐throughput multiplexed assay for kinase specificity

Detecting the phosphorylation substrates of multiple kinases in a single experiment is a challenge, and new techniques are being developed to overcome this challenge. Here, we used a multiplexed assay for kinase specificity (MAKS) to identify the substrates directly and to map the phosphorylation site(s) of plant symbiotic receptor‐like kinases. The symbiotic receptor‐like kinases nodulation receptor‐like kinase (NORK) and lysin motif domain‐containing receptor‐like kinase 3 (LYK3) are indispensable for the establishment of root nodule symbiosis. Although some interacting proteins have been identified for these symbiotic receptor‐like kinases, very little is known about their phosphorylation substrates. Using this high‐throughput approach, we identified several other potential phosphorylation targets for both these symbiotic receptor‐like kinases. In particular, we also discovered the phosphorylation of LYK3 by NORK itself, which was also confirmed by pairwise kinase assays. Motif analysis of potential targets for these kinases revealed that the acidic motif xxxsDxxx was common to both of them. In summary, this high‐throughput technique catalogs the potential phosphorylation substrates of multiple kinases in a single efficient experiment, the biological characterization of which should provide a better understanding of phosphorylation signaling cascade in symbiosis.     

Analytical strategies for phosphoproteomics

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho‐specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.     

The sea urchin kinome: a first look

This paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism.     

Chemical genetic identification of a lectin receptor kinase that transduces immune responses and interferes with abscisic acid signaling

Insight into how plants simultaneously cope with multiple stresses, for example, when challenged with biotic stress from pathogen infection and abiotic stress from drought, is important both for understanding evolutionary trade‐offs and optimizing crop responses to these stresses. Mechanisms by which initial plant immune signaling antagonizes abscisic acid (ABA) signal transduction require further investigation. Using a chemical genetics approach, the small molecule [5‐(3,4‐dichlorophenyl)furan‐2‐yl]‐piperidine‐1‐ylmethanethione (DFPM) has previously been identified due to its ability to suppress ABA signaling via plant immune signaling components. Here, we have used forward chemical genetics screening to identify DFPM‐insensitive loci by monitoring the activity of ABA‐inducible pRAB18::GFP in the presence of DFPM and ABA. The ability of DFPM to attenuate ABA signaling was reduced in rda mutants (resistant to DFPM inhibition of ABA signaling). One of the mutants, rda2, was mapped and is defective in a gene encoding a lectin receptor kinase. RDA2 functions in DFPM‐mediated inhibition of ABA‐mediated reporter expression. RDA2 is required for DFPM‐mediated activation of immune signaling, including phosphorylation of mitogen‐activated protein kinase (MAPK) 3 (MPK3) and MPK6, and induction of immunity marker genes. Our study identifies a previously uncharacterized receptor kinase gene that is important for DFPM‐mediated immune signaling and inhibition of ABA signaling. We demonstrate that the lectin receptor kinase RDA2 is essential for perceiving the DFPM signal and activating MAPKs, and that MKK4 and MKK5 are required for DFPM interference with ABA signal transduction.     

酪氨酸磷酸化蛋白质组学技术研究进展及其在生物医学研究中的应用

在酪氨酸磷酸化蛋白质组学的研究过程中,酪氨酸磷酸化位点的富集是最重要的一步。目前常用的富集方法是抗体亲和富集或SH2 superbinder富集。此外,通过质谱与生物信息学等技术,可实现大规模酪氨酸磷酸化位点的鉴定。对酪氨酸磷酸化蛋白质组学进行深度覆盖研究,揭示癌症发生发展过程中失调的激酶,将有助于深入理解癌症的发生发展过程;且由于75%的致癌基因是酪氨酸激酶基因,酪氨酸激酶抑制剂作为抗癌药物受到了越来越多的关注。应用酪氨酸磷酸化蛋白质组学技术,可以鉴定与癌症等重大疾病相关的酪氨酸激酶,从而帮助找到酪氨酸激酶抑制剂。总之,酪氨酸磷酸化蛋白质组学技术可以在酪氨酸激酶鉴定、酪氨酸激酶抑制剂研究及酪氨酸磷酸化信号通路研究等生物医学领域中得到很好的应用。     

Peptide arrays for kinome analysis: new opportunities and remaining challenges

Phosphorylation is the predominant mechanism of post-translational modification for regulation of protein function. With central roles in virtually every cellular process, and strong linkages with many diseases, there is a considerable interest in defining, and ultimately controlling, kinase activities. Investigations of human cellular phosphorylation events, which includes over 500 different kinases and tens of thousands of phosphorylation targets, represent a daunting challenge for proteomic researchers and cell biologists alike. As such, there is a priority to develop tools that enable the evaluation of cellular phosphorylation events in a high-throughput, and biologically relevant, fashion. Towards this objective, two distinct, but functionally related, experimental approaches have emerged; phosphoproteome investigations, which focus on the sub-population of proteins which undergo phosphorylation and kinome analysis, which considers the activities of the kinase enzymes mediating these phosphorylation events. Within kinome analysis, peptide arrays have demonstrated considerable potential as a cost-effective, high-throughput approach for defining phosphorylation-mediated signal transduction activity. In particular, a number of recent advances in the application of peptide arrays for kinome analysis have enabled researchers to tackle increasingly complex biological problems in a wider range of species. In this review, recent advances in kinomic analysis utilizing peptides arrays including several of the biological questions studied by our group, as well as outstanding challenges still facing this technology, are discussed.     

Large-scale Proteomics Analysis of the Human Kinome

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis.Reversible protein phosphorylation represents the most common type of post-translational modification (PTM)¹ in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4–6). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7, 8). Protein kinases can not only modulate each other''s functions and activities through site-specific phosphorylation events, but often also undergo site-specific autophosphorylation once they get activated (9). Thus, the comprehensive assessment of kinase-derived phosphopeptides can provide important insights into the regulation of these key players in phosphorylation-controlled signaling.Regulatory enzymes such as protein kinases are often expressed at low cellular levels. This can impede their detection by LC-MS in highly complex peptide mixtures derived from total cell or tissue extracts. These analytical challenges are further aggravated in phosphoproteomic experiments due to the fact that many phosphopeptide species result from sub-stoichiometric phosphorylation events (10). Consequently, phosphopeptide isolation methods have proven to be essential. Among others, techniques such as immobilized metal affinity chromatography or enrichment by means of titanium dioxide (TiO₂)-coated beads have found widespread use in MS-based phosphoproteomics (11–13). In addition, to reduce initial sample complexity, either protein fractionation by gel electrophoresis or peptide separation by strong cation exchange chromatography is typically included in contemporary phosphoproteomics workflows (14–16). These separation techniques in combination with LC-MS on state-of-the-art mass spectrometers enabled the identification of thousands of phosphorylation sites from total cellular extracts (15, 17, 18). Despite these impressive advances, such large-scale efforts require considerable instrument time, and the current methodology is still not comprehensive across the full dynamic range of the entire phosphoproteome. This creates the rationale for sub-proteome analyses to achieve high coverage and analytical sensitivity, which is particularly relevant for members of the protein kinase enzyme family.To date, the only pre-fractionation techniques permitting the enrichment of more than a few protein kinases are affinity capture methods relying on immobilized and kinase-selective small molecule inhibitors (19–21). We and others have demonstrated that combinations of such kinase inhibitor resins efficiently pre-fractionate kinases for subsequent phosphorylation analysis (7, 22, 23). Ideally, capture molecules for kinase proteomics have two properties. First, they should exhibit high non-selectivity within the kinase superfamily. Second, they should efficiently discriminate between protein kinases and other classes of cellular proteins under the biochemical conditions of the pre-fractionation procedure.In our efforts to characterize affinity reagents fulfilling these criteria, we quantitatively compared a selection of immobilized pyrido[2,3-d]pyrimidine-based inhibitors with respect to their proteome-wide kinase binding properties. Based on this assessment, an affinity matrix displaying the small molecule VI16832 was used as an enrichment tool for the comparative expression analysis of protein kinases in different cancer cell lines. The highly efficient VI16832 affinity resin further enabled a large-scale phosphoproteomics survey resulting in the identification and confident assignment of about 1200 phosphorylation sites on more than 200 distinct protein kinases.     

High-throughput screening of the yeast kinome: identification of human serine/threonine protein kinases that phosphorylate the hepatitis C virus NS5A protein

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione S-transferase-tagged protein kinases purified from Saccharomyces cerevisiae to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Kinases and Pseudokinases: Lessons from RAF

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be “frozen” by introducing mutations at their hydrophobic spines.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.