Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC)

Identification of phosphorylated proteins remains a difficult task despite technological advances in protein purification methods and mass spectrometry. Here, we report identification of tyrosine-phosphorylated proteins by coupling stable isotope labeling with amino acids in cell culture (SILAC) to mass spectrometry. We labeled HeLa cells with stable isotopes of tyrosine, or, a combination of arginine and lysine to identify tyrosine phosphorylated proteins. This allowed identification of 118 proteins, of which only 45 proteins were previously described as tyrosine-phosphorylated proteins. A total of 42 in vivo tyrosine phosphorylation sites were mapped, including 34 novel ones. We validated the phosphorylation status of a subset of novel proteins including cytoskeleton associated protein 1, breast cancer anti-estrogen resistance 3, chromosome 3 open reading frame 6, WW binding protein 2, Nice-4 and RNA binding motif protein 4. Our strategy can be used to identify potential kinase substrates without prior knowledge of the signaling pathways and can also be applied to profiling to specific kinases in cells. Because of its sensitivity and general applicability, our approach will be useful for investigating signaling pathways in a global fashion and for using phosphoproteomics for functional annotation of genomes.     

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.     

Profiling Y561-dependent and -independent substrates of CSF-1R in epithelial cells

Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.     

Multiple in vivo tyrosine phosphorylation sites in EphB receptors

Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.     

Receptor tyrosine kinases.

A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.     

Tyrosine phosphoproteomics of fibroblast growth factor signaling: a role for insulin receptor substrate-4

Signal transduction by receptor tyrosine kinases is initiated by recruitment of a variety of signaling proteins to tyrosine-phosphorylated motifs in the activated receptors. Several signaling pathways are thus activated in parallel, the combination of which decides the cellular response. Here, we present a dual strategy for extensive mapping of tyrosine-phosphorylated proteins and probing of signal-dependent protein interactions of a signaling cascade. The approach relies on labeling of cells with "heavy" and "light" isotopic forms of Arg to distinguish two cell populations. First, tyrosine-phosphorylated proteins from stimulated ("heavy"-labeled) and control samples ("normal"-labeled) are isolated and subjected to high sensitivity Fourier transform ion cyclotron resonance mass spectrometry analysis. Next, phosphopeptides corresponding to tyrosine phosphorylation sites identified during the tyrosine phosphoproteomic analysis are used as baits to isolate phosphospecific protein binding partners, which are subsequently identified by mass spectrometry. We used this approach to identify 28 components of the signaling cascade induced by stimulation with the basic fibroblast growth factor. Insulin receptor substrate-4 was identified as a novel candidate in fibroblast growth factor receptor signaling, and we defined phosphorylation-dependent interactions with other components, such as adaptor protein Grb2, of the signaling cascade. Finally, we present evidence for a complex containing insulin receptor substrate-4 and ShcA in signaling by the fibroblast growth factor receptor.     

Differential expression of novel tyrosine kinase substrates during breast cancer development

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.     

Structural analysis of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the γ-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.     

Use of a chemical genetic technique to identify myosin IIb as a substrate of the Abl-related gene (Arg) tyrosine kinase

Abl family kinases have been implicated in the regulation of cell morphogenesis and migration, but the molecular mechanisms through which they operate are not fully elucidated. We applied the bump-hole technique, pioneered by Shokat and colleagues, to identify direct substrates of Abl and the Abl-related gene (Arg) kinases. This technique required the engineering of Abl/Arg to utilize an unnatural ATP analogue as a phospho-donor. Mutation of T334A and T361A in Abl and Arg, respectively, altered their nucleotide specificity and allowed them to utilize N6-benzyl-ATP as a phospho-donor. These mutations did not affect the catalytic activity or protein substrate specificity of Abl and Arg. An unexpected high level of background labeling necessitated further optimization of this approach. Dialysis, pretreatment with a broad-spectrum Ser/Thr kinase inhibitor, K-252a, and purification of phosphotyrosine-containing proteins allowed for definitive identification of putative substrates. Using mass spectrometry, we identified eight putative substrates. One of these putative substrates, myosin IIB, can be phosphorylated in vivo by Arg. Our results indicate that the bump-hole technique can be used to identify Abl family kinase substrates and suggests that myosin IIB may be regulated by tyrosine phosphorylation.     

Ligands and signaling through receptor-type tyrosine phosphatases

Virtually every aspect of cellular proliferation and differentiation is regulated by changes in tyrosine phosphorylation. Tyrosine phosphorylation, in turn, is controlled by the opposing activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs are often transmembrane proteins (receptor PTKs) whose enzymatic activities and signaling functions are tightly regulated by the binding of specific ligands. A variety of transmembrane PTPs has also been identified; these proteins are called receptor PTPs (RPTPs), but in most cases their roles as receptors are very poorly understood. This review discusses the evidence that RPTPs are actually receptors for extrinsic ligands, and the extent to which interactions with putative ligands are known or suspected to cause changes in enzymatic activity. Finally, some of the RPTP substrates believed to be physiologically important are described. The evidence gathered to date suggests that models derived from studies of receptor PTKs may be too simple to account for the diversity and complexity of mechanisms through which ligand binding controls RPTP function.     

Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics

The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.     

Physical and functional interactome atlas of human receptor tyrosine kinases

Much cell‐to‐cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP‐MS) to characterize stable binding partners and RTK–protein complexes, proximity‐dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase‐deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well‐studied RTKs, offers insights into the functions of less well‐studied RTKs, and highlights RTK‐RTK interactions and shared signaling pathways.     

Signal processing by protein tyrosine phosphorylation in plants

Protein phosphorylation is a reversible post-translational modification controlling many biological processes. Most phosphorylation occurs on serine and threonine, and to a less extend on tyrosine (Tyr). In animals, Tyr phosphorylation is crucial for the regulation of many responses such as growth or differentiation. Only recently with the development of mass spectrometry, it has been reported that Tyr phosphorylation is as important in plants as in animals. The genes encoding protein Tyr kinases and protein Tyr phosphatases have been identified in the Arabidopsis thaliana genome. Putative substrates of these enzymes, and thus Tyr-phosphorylated proteins have been reported by proteomic studies based on accurate mass spectrometry analysis of the phosphopeptides and phosphoproteins. Biochemical approaches, pharmacology and genetic manipulations have indicated that responses to stress and developmental processes involve changes in protein Tyr phosphorylation. The aim of this review is to present an update on Tyr phosphorylation in plants in order to better assess the role of this post-translational modification in plant physiology.Key words: protein tyrosine phosphorylation, kinases, phosphatases, proteomics, mass spectrometry, signaling     

A Proteomic Approach for Comprehensively Screening Substrates of Protein Kinases Such as Rho-Kinase

Background

Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored.

Methodology/Principal Findings

We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo.

Conclusions/Significance

This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.     

p21rasGAP association with Fyn, Lyn, and Yes in thrombin-activated platelets.

Activation of platelets by thrombin and other physiological agonists leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3606-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). To date, none of the tyrosine kinases that are involved in platelet activation, nor the substrates that are phosphorylated in response to agonists, have been identified. A "kinase trapping" strategy, designed to take advantage of the stability of known tyrosine kinase-substrate interactions, was employed to address both issues. p21rasGAP antibodies were used to examine the phosphorylated state of GAP in agonist-treated platelets and to isolate potential GAP-kinase complexes. We show that GAP and two proteins of 59 and 68 kDa are phosphorylated on tyrosine after thrombin stimulation and that three Src-related protein tyrosine kinases, Fyn, Lyn and Yes, are associated with GAP in complexes, detectable only after agonist stimulation. The thrombin-dependent detection of these kinases in GAP immunoprecipitates suggests that thrombin may either induce the formation of these complexes or activate kinases that are associated with GAP prior to, or following, agonist stimulation. This approach of "trapping" kinases bound to their substrates will be useful in identifying non-receptor tyrosine kinases involved in signaling pathways. Furthermore, although GAP phosphorylation has been previously implicated in growth factor signaling pathways, this is the first example of its involvement downstream from a G-protein-coupled receptor.     

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.Reversible tyrosine phosphorylation plays an important role in numerous cellular processes like growth, differentiation, and migration. Phosphotyrosine signaling is tightly controlled by the balanced action of protein-tyrosine kinases and protein-tyrosine phosphatases. Aberrant tyrosine phosphorylation has been suggested to be an underlying cause in multiple cancers (1). Therefore, the identification of tyrosine phosphorylated proteins and the investigation into their involvement in signaling pathways are important. Several groups have attempted to comprehensively study tyrosine phosphorylation by proteomics means (2–5). However, large scale identification of tyrosine phosphorylation sites by MS can be hindered by the low abundance of tyrosine phosphorylated proteins. Especially, signaling intermediates are usually low abundance proteins that show substoichiometric phosphorylation levels. In addition, the identification by mass spectrometry of phosphopeptides from a complex cellular lysate digest is often complicated by ion suppression effects due to a high background of non-phosphorylated peptides. Enrichment of tyrosine phosphorylated proteins or peptides prior to mass spectrometric detection is therefore essential. Traditionally, antibodies against phosphorylated tyrosine have been used to immunoprecipitate tyrosine phosphorylated proteins from cultured cells (2–4, 6–8). This phosphoprotein immunoaffinity purification method has for example been used to study the global dynamics of phosphotyrosine signaling events after EGF¹ stimulation using stable isotope labeling by amino acids in cell culture (SILAC) (2). This approach led to the identification of known and previously unidentified signaling proteins in the EGF receptor (EGFR) pathway, including their temporal activation profile after stimulation of the EGFR, providing crucial information for modeling signaling events in the cell. However, as the identification and quantification of these phosphorylated proteins in these studies were not necessarily based on tyrosine phosphorylated peptides but largely on non-phosphorylated peptides, little information is derived on the exact site(s) of tyrosine phosphorylation. Also, binding partners of tyrosine phosphorylated proteins, which themselves are not tyrosine phosphorylated, might be co-precipitated and impair the tyrosine phosphorylation quantification. Immunoaffinity purification of phosphotyrosine peptides, rather than proteins, using anti-phosphotyrosine antibodies (5, 9–16) significantly facilitates the identification of the site(s) of phosphorylation as it greatly alleviates most of the above mentioned problems because the tyrosine phosphorylated site can be directly identified and quantified.Accurate MS-based quantification is typically performed by stable isotope labeling. The isotopes can be incorporated metabolically during cell culture as in SILAC (17) or chemically as in an isobaric tag for relative and absolute quantitation (iTRAQ) (18) or stable isotope dimethyl labeling (19–21). Typically, the most precise quantification can be obtained by metabolic labeling as the different samples can be combined at the level of intact cells (22). However, metabolic labeling is somewhat limited to biological systems that can be grown in culture, and the medium may have an effect on the growth and development of the cells. iTRAQ has been used in conjunction with phosphotyrosine peptide immunoprecipitation (5). As the chemical labeling is performed before immunoprecipitation, the differentially labeled samples can be precipitated together, thereby neutralizing the potentially largest source of variation. However, as this phosphotyrosine peptide immunoprecipitation is typically performed on several hundreds of micrograms to milligrams of protein sample, iTRAQ provides in these cases a rather cost-prohibitive means.Here, we present an optimized immunoaffinity purification approach for the analysis of tyrosine phosphorylation combined with stable isotope dimethyl labeling (19–21, 23). We efficiently enriched and identified by MS 1112 unique phosphopeptides derived from 4 mg of starting protein material without any further affinity chromatographic enrichment whereby up to 80% of the peptides analyzed in the final LC run were phosphotyrosine peptides. We further advanced the method by introducing triplex stable isotope dimethyl labeling prior to immunoprecipitation. We quantified differences in tyrosine phosphorylation upon pervanadate treatment or EGF stimulation to detect site-specific changes in tyrosine phosphorylation. 128 unique phosphotyrosine peptides were identified and quantified upon pervanadate treatment. By using an internal standard comprising both mock and pervanadate-treated samples, we could more confidently identify and quantify phosphorylation sites that are strongly regulated and on-off situations. Analysis of EGF-stimulated HeLa cells resulted in the quantification of 73 unique phosphotyrosine peptides. Most of the up-regulated phosphotyrosine peptides that were identified have been reported previously to be involved in the EGFR signaling pathway, validating our approach. However, for the first time, we found TFG to also become highly tyrosine phosphorylated upon EGF stimulation together with some tyrosine phosphorylation sites on for example IRS2, SgK269, and DLG3 that have not been firmly established earlier to be involved in EGFR signaling.In general, we show that the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale chemical stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in immunoprecipitation and LC-MS as samples can be combined before immunoprecipitation and the necessity of performing additional enrichment is removed by an optimization of the protocol. With only a single LC-MS run, already a rather complete qualitative and quantitative picture of a signaling event can be generated.     

On the cross-regulation of protein tyrosine phosphatases and receptor tyrosine kinases in intracellular signaling

Intracellular signaling proteins are very often regulated by site-specific phosphorylation. For example, growth factor receptors in eukaryotic cells contain intrinsic tyrosine kinase activity and use inter- and intra-molecular interactions to recruit and orient potential protein substrates for phosphorylation. Equally important in determining the magnitude and kinetics of such a response is protein dephosphorylation, catalysed by phosphatase enzymes. A growing body of evidence indicates that certain protein tyrosine phosphatases (PTPs), like tyrosine kinases, are affected by intermolecular interactions that alter the specific activity or localization of their catalytic domains. Using a detailed kinetic modeling framework, we theoretically explore the regulation of PTPs through their association with receptor tyrosine kinases, as noted for the Src homology 2-domain-containing PTPs, SHP-1 and -2. Receptor-PTP binding, in turn, is expected to influence the phosphorylation pattern of those receptors and proteins they associate with, and we show how PTPs might serve to co- or counter-regulate parallel pathways in a signaling network.     

Annexin VII and annexin XI are tyrosine phosphorylated in peroxovanadate-treated dogs and in platelet-derived growth factor-treated rat vascular smooth muscle cells

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitro phosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases.     

Dynamics of the Tec‐family tyrosine kinase SH3 domains

The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains.