Phosphoproteomic approaches to elucidate cellular signaling networks

Protein phosphorylation is crucial in the regulation of signaling pathways that control various biological responses. Recent progress in diverse methodologies to investigate protein phosphorylation in complex biological samples has resulted in more rapid, detailed and quantitative analyses of signaling networks. In particular, advances in mass spectrometry (MS) have enabled the identification and quantification of thousands of both known and novel phosphorylation sites. Initial MS-based information can be complemented with a variety of recently developed and improved phosphoproteomic techniques. These include multiplexed microbead or kinase activity assays, flow cytometry based single-cell analysis, protein microarrays and interaction studies. The combination of multiple approaches, coupled with phenotypic response measurements, computational modeling and biochemical manipulations, will ultimately reveal the mechanistic regulation of signaling networks.     

Proteomic analysis of phosphorylation in cancer

Constitutive activity of kinases is known to be crucial for a tumor to maintain its malignant phenotype, a phenomenon which is often referred to as oncogene addiction. The in-depth analysis of aberrant signaling pathways by the analysis of protein phosphorylation has become feasible through recent advances in proteomics technology. In this article we will review developments in the field of phosphoproteomics and its application in cancer research. The most widely used technologies for the generic enrichment of phosphopeptides are discussed as well as targeted approaches for the analysis of a specific subset of phosphopeptides. Validation experiments of phosphorylation sites using targeted mass spectrometry are also explained. Finally, we will highlight applications of phosphoproteomic technology in cancer research using cell lines and tissue.     

Signaling through focal adhesion kinase

Integrin receptor binding to extracellular matrix proteins generates intracellular signals via enhanced tyrosine phosphorylation events that are important for cell growth, survival, and migration. This review will focus on the functions of the focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) and its role in linking integrin receptors to intracellular signaling pathways. FAK associates with several different signaling proteins such as Src-family PTKs, p130^Cas, Shc, Grb2, PI 3-kinase, and paxillin. This enables FAK to function within a network of integrin-stimulated signaling pathways leading to the activation of targets such as the ERK and JNK/mitogen-activated protein kinase pathways. Focus will be placed on the structural domains and sites of FAK tyrosine phosphorylation important for FAK-mediated signaling events and how these sites are conserved in the FAK-related PTK, Pyk2. We will review what is known about FAK activation by integrin receptor-mediated events and also non-integrin stimuli. In addition, we discuss the emergence of a consensus FAK substrate phosphorylation sequence. Emphasis will also be placed on the role of FAK in generating cell survival signals and the cleavage of FAK during caspase-mediated apoptosis. An in-depth discussion will be presented of integrin-stimulated signaling events occurring in the FAK knockout fibroblasts (FAK⁻) and how these cells exhibit deficits in cell migration. FAK re-expression in the FAK⁻ cells confirms the role of this PTK in the regulation of cell morphology and in promoting cell migration events. In addition, these results reinforce the potential role for FAK in promoting an invasive phenotype in human tumors.     

Protein kinase C (PKC) isozyme-specific substrates and their design

Protein kinase C (PKC), a phospholipid-dependent serine/threonine kinase, appears to be involved in the signal transduction response to many hormones and growth factors; there are 11 different PKC isozymes. Because PKC isozymes directly and/or indirectly participate in signal transduction pathways of normal and transformed cells through phosphorylation of target proteins, it is critical to understand the diversity of the intracellular signaling pathways regulated by each PKC isozyme. Thus, PKC isozyme-specific substrates are useful to understand the characterization of the intracellular signaling pathways for each PKC isozyme. Consensus sequences and sequence information obtained from PKC target proteins are very important to design PKC isozyme-specific peptide substrates. Moreover, computational prediction programs of phosphorylation sites using a library of peptide substrates aid in the fast design of PKC isozyme-specific peptide substrates. Although a large number of target proteins and synthetic peptides for PKCs are known, only two peptide substrates (peptide 422–426 of murine elongation factor-1α and Alphatomega peptide) have been reported as PKC isozyme-specific peptide substrates. This discussion will review the literature concerning these native and synthetic PKC isozyme-specific peptide substrates and their design.     

Detection and characterization of 3D-signature phosphorylation site motifs and their contribution towards improved phosphorylation site prediction in proteins

Background  

Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites) has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites.     

Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways

Activation of T cells via the stimulation of the TCR plays a central role in the adaptive immunological response. Although much is known about TCR-stimulated signaling pathways, there are still gaps in our knowledge about the kinetics and sequence of events during early activation and about the in vivo specificity of kinases involved in these proximal signaling pathways. This information is important not only for understanding the activation of signaling pathways important for T cell function but also for the development of drug targets and computer-based molecular models. In this study, phospho-specific Abs directed toward individual sites on signaling proteins were used to investigate the early phosphorylation kinetics of proteins involved in proximal TCR-induced pathways. These studies indicate that linker for activation of T cells' tyrosines have substantially different phosphorylation kinetics and that Src homology 2 domain-containing leukocyte protein of 76 kDa has rapid, transient phosphorylation kinetics compared to other proteins. In additions, we provide evidence that ZAP-70 is the primary in vivo kinase for LAT tyrosine 191 and that Itk plays a role in the phosphorylation of tyrosine 783 on phospholipase C-gamma1. In total, these studies give new insight into the sequence, kinetics and specificity of early TCR-mediated signaling events that are vital for T cell activation.     

The Versatility of Helicobacter pylori CagA Effector Protein Functions: The Master Key Hypothesis

Several bacterial pathogens inject virulence proteins into host target cells that are substrates of eukaryotic tyrosine kinases. One of the key examples is the Helicobacter pylori CagA effector protein which is translocated by a type‐IV secretion system. Injected CagA becomes tyrosine‐phosphorylated on EPIYA sequence motifs by Src and Abl family kinases. CagA then binds to and activates/inactivates multiple signaling proteins in a phosphorylation‐dependent and phosphorylation‐independent manner. A recent proteomic screen systematically identified eukaryotic binding partners of the EPIYA phosphorylation sites of CagA and similar sites in other bacterial effectors by high‐resolution mass spectrometry. Individual phosphorylation sites recruited a surprisingly high number of interaction partners suggesting that each phosphorylation site can interfere with many downstream pathways. We now count 20 reported cellular binding partners of CagA, which represents the highest quantitiy among all yet known virulence‐associated effector proteins in the microbial world. This complexity generates a highly remarkable and puzzling scenario. In addition, the first crystal structure of CagA provided us with new information on the function of this important virulence determinant. Here we review the recent advances in characterizing the multiple binding signaling activities of CagA. Injected CagA can act as a ‘master key’ that evolved the ability to highjack multiple host cell signalling cascades, which include the induction of membrane dynamics, actin‐cytoskeletal rearrangements and the disruption of cell‐to‐cell junctions as well as proliferative, pro‐inflammatory and anti‐apoptotic nuclear responses. The discovery that different pathogens use this common strategy to subvert host cell functions suggests that more examples will emerge soon.     

Phosphorylation of the human full-length protein kinase Ciota

Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways.     

Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway.

Phosphorylation is one of the most common forms of protein modification. The most frequent targets for protein phosphorylation in eukaryotes are serine and threonine residues, although tyrosine residues also undergo phosphorylation. Many of the currently applied methods for the detection and localization of protein phosphorylation sites are mass spectrometry-based and are biased against the analysis of tyrosine-phosphorylated residues because of the stability and low reactivity of phosphotyrosines. To overcome this lack of sensitive methods for the detection of phosphotyrosine-containing peptides, we have recently developed a method that is not affected by the more predominant threonine or serine phosphorylation within cells. It is based on the specific detection of immonium ion of phosphotyrosine at 216.043 Da and does not require prior knowledge of the protein sequence. In this report, we describe the first application of this new method in a proteomic strategy. Using anti-phosphotyrosine antibodies for immunoprecipitation and one-dimensional gel electrophoresis, we have identified 10 proteins in the epidermal growth factor receptor signaling pathway, of which 8 have been shown previously to be involved in epidermal growth factor signaling. Most importantly, in addition to several known tyrosine phosphorylation sites, we have identified five novel sites on SHIP-2, Hrs, Cbl, STAM, and STAM2, most of which were not predicted to be phosphorylated. Because of its sensitivity and selectivity, this approach will be useful in proteomic approaches to study tyrosine phosphorylation in a number of signal transduction pathways.     

Characterization of phosphorylation sites on Tpl2 using IMAC enrichment and a linear ion trap mass spectrometer

Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Protein modifications present additional challenges because they occur at low stoichiometries and often change the analytical behavior of the molecule. For example, characterization of protein phosphorylation provides crucial information to signaling processes that are often associated with disease. Research into protein phosphorylation requires inter-disciplinary co-operation involving multiple investigators with expertise in diverse scientific fields. As such, techniques must be simple, effective, and incorporate multiple checkpoints that confirm the sample contains a phosphorylated protein in order to ensure resources are conserved. In this study, tumor progression locus 2 (Tpl2), which has been implicated in cell cycle regulation and has been shown to play a significant role in critical signal transduction pathways, was transfected into 293T cells, overexpressed and isolated from the cell lysate. Isolated proteins were separated via 1D gel electrophoresis, and their phosphorylation was confirmed using phosphospecific staining. The bands were excised and subjected to tryptic digestion and immobilized metal affinity chromatography (IMAC) prior to analysis by capillary-LC-MS/MS. Three phosphorylation sites were detected on Tpl2. One site had previously been reported in the literature but had not been characterized by mass spectrometric methods until this time; two additional novel sites of phosphorylation were detected.     

Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells

The mechanism by which stimulation of coreceptors such as CD28 contributes to full activation of TCR signaling pathways has been intensively studied, yet quantitative measurement of costimulation effects on functional TCR signaling networks has been lacking. In this study, phosphotyrosine networks triggered by CD3, CD28, or CD3 and CD28 costimulation were analyzed by site-specific quantitative phosphoproteomics, resulting in identification of 101 tyrosine and 3 threonine phosphorylation sites and quantification of 87 sites across four cell states. As expected, CD3 stimulation induced phosphorylation of CD3 chains and upstream components of TCR pathways such as Zap70, while CD28 stimulation induced phosphorylation of CD28, Vav-1, and other adaptor proteins including downstream of tyrosine kinase 1, Grb2-associated protein 2 (Grap2), and Wiskott-Aldrich syndrome protein. CD3 and CD28 costimulation induced a complex response including decreased threonine phosphorylation in the ERK1 and ERK2 activation loops and increased phosphorylation of selected tyrosine sites on ERK1/2, p38, phospholipase C-gamma, Src homology 2 domain-containing transforming protein 1, Grap2, and Vav-1, potentiating T cell activation. Hierarchical clustering and self-organizing maps were used to identify modules of coregulated phosphorylation sites within the network. Quantitative information in our study suggests quantitative and qualitative contribution by costimulation of CD28 on CD3-stimulated TCR signaling networks via enhanced phosphorylation of phospholipase C-gamma/Src homology 2 domain-containing transforming protein 1/Grap2/Vav-1 and their effects on downstream components including MAPKs.     

Protein kinase signaling networks in cancer

Protein kinases orchestrate the activation of signaling cascades in response to extracellular and intracellular stimuli to control cell growth, proliferation, and survival. The complexity of numerous intracellular signaling pathways is highlighted by the number of kinases encoded by the human genome (539) and the plethora of phosphorylation sites identified in phosphoproteomic studies. Perturbation of these signaling networks by mutations or abnormal protein expression underlies the cause of many diseases including cancer. Recent RNAi screens and cancer genomic sequencing studies have revealed that many more kinases than anticipated contribute to tumorigenesis and are potential targets for inhibitor drug development intervention. This review will highlight recent insights into known pathways essential for tumorigenesis and discuss exciting new pathways for therapeutic intervention.     

Global phosphoproteome of HT-29 human colon adenocarcinoma cells

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.     

Mass Spectrometric Identification of In Vivo Phosphorylation Sites of Differentially Expressed Proteins in Elongating Cotton Fiber Cells

Two-dimensional gel electrophoresis (2-DE)-based proteomics approach was applied to extensively explore the molecular basis of plant development and environmental adaptation. These proteomics analyses revealed thousands of differentially expressed proteins (DEPs) closely related to different biological processes. However, little attention has been paid to how peptide mass fingerprinting (PMF) data generated by the approach can be directly utilized for the determination of protein phosphorylation. Here, we used the software tool FindMod to predict the peptides that might carry the phosphorylation modification by examining their PMF data for mass differences between the empirical and theoretical peptides and then identified phosphorylation sites using MALDI TOF/TOF according to predicted peptide data from these DEP spots in the 2-D gels. As a result, a total of 48 phosphorylation sites of 40 DEPs were successfully identified among 235 known DEPs previously revealed in the 2-D gels of elongating cotton fiber cells. The 40 phosphorylated DEPs, including important enzymes such as enolase, transketolase and UDP-L-rhamnose synthase, are presumed to participate in the functional regulation of numerous metabolic pathways, suggesting the reverse phosphorylation of these proteins might play important roles in elongating cotton fibers. The results also indicated that some different isoforms of the identical DEP revealed in our 2-DE-based proteomics analysis could be annotated by phosphorylation events. Taken together, as the first report of large-scale identification of phosphorylation sites in elongating cotton fiber cells, our study provides not only an excellent example of directly identifying phosphorylation sites from known DEPs on 2-D gels but also provides a valuable resource for future functional studies of phosphorylated proteins in this field.     

Regulation of gap junctions by tyrosine protein kinases

Most of the gap junction proteins are regulated in part by post-translational phosphorylation. Phosphorylation has been shown to be important in gap junction assembly and turnover, and for channel function in the resting state. Connexin phosphorylation may be altered by the activation of intracellular signaling pathways in response to growth factors, tumor promoters, activated oncogenes, hormones and inflammatory mediators. In some instances altered phosphorylation has been associated with changes in connexin function and in other cases appears to be associated with changes in the levels of the connexin protein and/or mRNA. This review focuses on the role of tyrosine protein kinases in the regulation of gap junctions. The literature is most extensive for connexin43 and those studies are reviewed here. A great deal has been learned in recent years about how connexin43 is regulated by tyrosine kinase-dependent signaling pathways. These pathways are often complex and to some extent are cell type- and stimulus-dependent. Although considerable progress has been made in unraveling the cellular pathways that regulate connexin function, significant challenges remain to be addressed in identifying additional phosphorylation sites and determining the stoichiometries of the phosphorylation events that regulate connexin function and it's interaction with other cellular proteins.     

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.     

THANATOS: an integrative data resource of proteins and post-translational modifications in the regulation of autophagy

Macroautophagy/autophagy is a highly conserved process for degrading cytoplasmic contents, determines cell survival or death, and regulates the cellular homeostasis. Besides ATG proteins, numerous regulators together with various post-translational modifications (PTMs) are also involved in autophagy. In this work, we collected 4,237 experimentally identified proteins regulated in autophagy and cell death pathways from the literature. Then we computationally identified potential orthologs of known proteins, and developed a comprehensive database of The Autophagy, Necrosis, ApopTosis OrchestratorS (THANATOS, http://thanatos.biocuckoo.org), containing 191,543 proteins potentially associated with autophagy and cell death pathways in 164 eukaryotes. We performed an evolutionary analysis of ATG genes, and observed that ATGs required for the autophagosome formation are highly conserved across eukaryotes. Further analyses revealed that known cancer genes and drug targets were overrepresented in human autophagy proteins, which were significantly associated in a number of signaling pathways and human diseases. By reconstructing a human kinase-substrate phosphorylation network for ATG proteins, our results confirmed that phosphorylation play a critical role in regulating autophagy. In total, we mapped 65,015 known sites of 11 types of PTMs to collected proteins, and revealed that all types of PTM substrates were enriched in human autophagy. In addition, we observed multiple types of PTM regulators such as protein kinases and ubiquitin E3 ligases or adaptors were significantly associated with human autophagy, and again the results emphasized the importance of PTM regulations in autophagy. We anticipated THANATOS can be a useful resource for further studies.