Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle

Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.     

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.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphoproteomics in bacteria: towards a systemic understanding of bacterial phosphorylation networks

Bacteria use protein phosphorylation to regulate all kinds of physiological processes. Protein phosphorylation plays a role in several key steps of the infection process of bacterial pathogens, such as adhesion to the host, triggering and regulation of pathogenic functions as well as biochemical warfare; scrambling the host signaling cascades and impairing its defense mechanisms. Recent phosphoproteomic studies indicate that the bacterial protein phosphorylation networks could be more complex than initially expected, comprising promiscuous kinases that regulate several distinct cellular functions by phosphorylating different protein substrates. Recent advances in protein labeling with stable isotopes in the field of quantitative mass spectrometry phosphoproteomics will enable us to chart the global phosphorylation networks and to understand the implication of protein phosphorylation in cellular regulation on the systems scale. For the study of bacterial pathogens, in particular, this research avenue will enable us to dissect phosphorylation-related events during different stages of infection and stimulate our efforts to find inhibitors for key kinases and phosphatases implicated therein.     

Phosphorylation events catalyzed by major cell signaling proteins differ in response to thermal and osmotic stress among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels

Sharp environmental gradients encountered within the intertidal zone have driven the evolution of physiological adaptations that allow its inhabitants to maintain cellular function in the presence of fluctuating abiotic factors. These adaptations are mediated by gene-regulatory networks that, despite their inherent complexity, must remain evolvable and capable of responding to different selection pressures associated with specific ecological niches. Phosphorylation events catalyzed by cell-signaling enzymes represent a parsimonious mechanism to integrate new functional or regulatory properties into these gene-regulatory networks. In this study, proteins phosphorylated on consensus sequences for protein kinases A, B, and C; cyclin-dependent kinases; and mitogen-activated protein kinases, as well as the abundance of phosphorylated stress-activated protein kinase (phospho-SAPK/JNK), were quantified in order to ascertain whether phosphorylation events are divergent among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels that differ in their tolerance toward environmental stress. Abundances of phosphorylated substrate proteins for each of the major signaling proteins that were investigated, as well as the abundance of phospho-SAPK/JNK, differed both within and between species during thermal and osmotic stress. These data suggest that modulating protein function via phosphorylation may be an important mechanism to integrate novel properties into stress-regulatory networks. In turn, differential phosphorylation during environmental stress may contribute to species-specific tolerances toward abiotic stress, interspecies dynamics, and biogeographic patterns in Mytilus congeners.     

Characterization of the phosphorylation sites of Mycobacterium tuberculosis serine/threonine protein kinases, PknA, PknD, PknE, and PknH by mass spectrometry

In Mycobacterium tuberculosis (Mtb), regulatory phosphorylation of proteins at serine and/or threonine residues by serine/threonine protein kinases (STPKs) is an emerging theme connected with the involvement of these enzymes in virulence mechanisms. The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to identify the corresponding interaction networks. Detection of phosphorylated proteins as well as assignment of the phosphorylated sites in STPKs is a major challenge in proteomics since some of these enzymes might be interesting therapeutical targets. Using different strategies to identify phosphorylated residues, we report, in the present work, MS studies of the entire intracellular regions of recombinant protein kinases PknA, PknD, PknE, and PknH from Mtb. The on-target dephosphorylation/MALDI-TOF for identification of phosphorylated peptides was used in combination with LC-ESI/MS/MS for localization of phosphorylation sites. By doing so, seven and nine phosphorylated serine and/or threonine residues were identified as phosphorylation sites in the recombinant intracellular regions of PknA and PknH, respectively. The same technique led also to the identification of seven phosphorylation sites in each of the two recombinant kinases, PknD and PknE.     

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.     

Pseudokinases-remnants of evolution or key allosteric regulators?

Protein kinases provide a platform for the integration of signal transduction networks. A key feature of transmitting these cellular signals is the ability of protein kinases to activate one another by phosphorylation. A number of kinases are predicted by sequence homology to be incapable of phosphoryl group transfer due to degradation of their catalytic motifs. These are termed pseudokinases and because of the assumed lack of phosphoryltransfer activity their biological role in cellular transduction has been mysterious. Recent structure-function studies have uncovered the molecular determinants for protein kinase inactivity and have shed light to the biological functions and evolution of this enigmatic subset of the human kinome. Pseudokinases act as signal transducers by bringing together components of signalling networks, as well as allosteric activators of active protein kinases.     

Expression and characterization of MAP kinases in bacteria

Mitogen-activated protein kinases (MAPKs) are common signal transducers in all eukaryotic organisms. MAPKs are activated by protein kinase cascades consisting of MAPK kinases (MAP2Ks) and MAPK kinase kinases (MAP3Ks). Extracellular-signal regulated kinases 1 and 2 (ERK1/2) are the best characterized MAPKs. Like other MAPKs their activity is regulated by dual phosphorylation as well as dephosphorylation by a host of phosphoprotein phosphatases. The ability to phosphorylate or thiophosphorylate ERK2 in vitro, as described here, is valuable for use in downstream applications designed to investigate MAPK signaling networks.     

Kinase networks integrate profiles of N-methyl-D-aspartate receptor-mediated gene expression in hippocampus

The postsynaptic N-methyl-d-aspartate (NMDA) receptor activates multiple kinases and changes the phosphorylation of many postsynaptic proteins organized in signaling networks. Because the NMDA receptor is known to regulate gene expression, it is important to examine whether networks of kinases control signaling to gene expression. We examined the requirement of multiple kinases and NMDA receptor-interacting proteins for gene expression in mouse hippocampal slices. Protocols that induce long-term depression (LTD) and long-term potentiation (LTP) activated common kinases and overlapping gene expression profiles. Combinations of kinases were required for induction of each gene. Distinct combinations of kinases were required to up-regulate Arc, Npas4, Egr2, and Egr4 following either LTP or LTD protocols. Consistent with the combinatorial data, a mouse mutant model of the human cognition disease gene SAP102, which couples ERK kinase to the NMDA receptor, showed deregulated expression of specific genes. These data support a network model of postsynaptic integration where kinase signaling networks are recruited by differential synaptic activity and control both local synaptic events and activity-dependent gene expression.     

Systematic discovery of in vivo phosphorylation networks

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.     

Dissecting phosphorylation networks: lessons learned from yeast

Protein phosphorylation continues to be regarded as one of the most important post-translational modifications found in eukaryotes and has been implicated in key roles in the development of a number of human diseases. In order to elucidate roles for the 518 human kinases, phosphorylation has routinely been studied using the budding yeast Saccharomyces cerevisiae as a model system. In recent years, a number of technologies have emerged to globally map phosphorylation in yeast. In this article, we review these technologies and discuss how these phosphorylation mapping efforts have shed light on our understanding of kinase signaling pathways and eukaryotic proteomic networks in general.     

Impact of phosphoproteomics on studies of bacterial physiology

Protein phosphorylation on serine, threonine and tyrosine is recognized as a major tool of signal transduction in bacteria. However, progress in the field has been hampered by the lack of global and site-specific data on bacterial phosphoproteomes. Recent advances in mass spectrometry-based proteomics have encouraged bacteriologists to start using powerful gel-free approaches for global detection of phosphorylated proteins. These studies have generated large data sets of proteins phosphorylated on serine, threonine and tyrosine, with identified phosphorylation sites which represent an excellent starting point for in-depth physiological characterization of kinases and their substrates. The list of phosphorylated proteins inspired a number of physiological studies in which the identity of the phosphorylation site facilitated the elucidation of molecular mechanisms of signaling and regulation. Bacterial phosphoproteomics also provided interesting insights into the evolutionary aspects of protein phosphorylation. The field is rapidly embracing quantitative mass spectrometry strategies, comparing phosphoproteome dynamics in changing conditions and aiming to reconstruct the entire regulatory networks by linking kinases to their physiological substrates.     

Phosphorylation of Enoyl-Acyl Carrier Protein Reductase InhA Impacts Mycobacterial Growth and Survival

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.     

Directional and quantitative phosphorylation networks.

Directionality in protein signalling networks is due to modulated protein-protein interactions and is fundamental for proper signal progression and response to external and internal cues. This property is in part enabled by linear motifs embedding post-translational modification sites. These serve as recognition sites, guiding phosphorylation by kinases and subsequent binding of modular domains (e.g. SH2 and BRCT). Characterization of such modification-modulated interactions on a proteome-wide scale requires extensive computational and experimental analysis. Here, we review the latest advances in methods for unravelling phosphorylation-mediated cellular interaction networks. In particular, we will discuss how the combination of new quantitative mass-spectrometric technologies and computational algorithms together are enhancing mapping of these largely uncharted dynamic networks. By combining quantitative measurements of phosphorylation events with computational approaches, we argue that systems level models will help to decipher complex diseases through the ability to predict cellular systems trajectories.     

A versatile strategy to define the phosphorylation preferences of plant protein kinases and screen for putative substrates

Most signaling networks are regulated by reversible protein phosphorylation. The specificity of this regulation depends in part on the capacity of protein kinases to recognize and efficiently phosphorylate particular sequence motifs in their substrates. Sequenced plant genomes potentially encode over than 1000 protein kinases, representing 4% of the proteins, twice the proportion found in humans. This plethora of plant kinases requires the development of high-throughput strategies to identify their substrates. In this study, we have implemented a semi-degenerate peptide array screen to define the phosphorylation preferences of four kinases from Arabidopsis thaliana that are representative of the plant calcium-dependent protein kinase and Snf1-related kinase superfamily. We converted these quantitative data into position-specific scoring matrices to identify putative substrates of these kinases in silico in protein sequence databases. Our data show that these kinases display related but nevertheless distinct phosphorylation motif preferences, suggesting that they might share common targets but are likely to have specific substrates. Our analysis also reveals that a conserved motif found in the stress-related dehydrin protein family may be targeted by the SnRK2-10 kinase. Our results indicate that semi-degenerate peptide array screening is a versatile strategy that can be used on numerous plant kinases to facilitate identification of their substrates, and therefore represents a valuable tool to decipher phosphorylation-regulated signaling networks in plants.     

Immunoaffinity profiling of tyrosine phosphorylation in cancer cells

Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small numbers of peptides containing phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large numbers of tyrosine phosphorylation sites. Peptides containing phosphotyrosine are isolated directly from protease-digested cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.     

Kinome analysis of receptor-induced phosphorylation in human natural killer cells

Background

Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.

Results

A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.

Conclusions

The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.     

In silico studies of potential phosphoresidues in the human nucleophosmin/B23: its kinases and related biological processes

Human nucleophosmin/B23 is a phosphoprotein involved in ribosome biogenesis, centrosome duplication, cancer, and apoptosis. Its function, localization, and mobility within cells, are highly regulated by phosphorylation events. Up to 21 phosphosites of B23 have been experimentally verified even though the corresponding kinase is known only for seven of them. In this work, we predict the phosphorylation sites in human B23 using six kinase-specific servers (KinasePhos 2.0, PredPhospho, NetPhosK 1.0, PKC Scan, pkaPS, and MetaPredPS) plus DISPHOS 1.3, which is not kinase specific. The results were integrated with information regarding 3D structure and residue conservation of B23, as well as cellular localizations, cellular processes, signaling pathways and protein-protein interaction networks involving both B23 and each predicted kinase. Thus, all 40 potential phosphosites of B23 were predicted with significant score (>0.50) as substrates of at least one of 38 kinases. Thirteen of these residues are newly proposed showing high susceptibility of phosphorylation considering their solvent accessibility. Our results also suggest that the enzymes CDKs, PKC, CK2, PLK1, and PKA could phosphorylate B23 at higher number of sites than those previously reported. Furthermore, PDK, GSK3, ATM, MAPK, PKB, and CHK1 could mediate multisite phosphorylation of B23, although they have not been verified as kinases for this protein. Finally, we suggest that B23 phosphorylation is related to cellular processes such as apoptosis, cell survival, cell proliferation, and response to DNA damage stimulus, in which these kinases are involved. These predictions could contribute to a better understanding, as well as addressing further experimental studies, of B23 phosphorylation.