Phosphotyrosine-specific Antibody Research Articles

Tyrosine Phosphorylation of CARM1 Promotes Its Enzymatic Activity and Alters Its Target Specificity in Myeloid Neoplasms with Hyperactivated JAK2

Introduction Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the asymmetric dimethylation of arginine residues in histone and non-histone substrates, including transcription factors, RNA splicing factors, and the SWI/SNF chromatin remodeling complex. CARM1 has emerged as an important target in acute myeloid leukemia. We have previously shown that Janus kinase 2 (JAK2) affects the activity of epigenetic modifiers. A V617F mutation in JAK2 genes leads to its hyperactivation. This mutation is a common hallmark of the BCR/ABL1-negative myeloproliferative neoplasms, and also found in patients with acute myeloid leukemia and myelodysplastic syndrome. Here, we find that hyperactivated JAK2, mediated by biallelic V617F mutation, triggers CARM1 phosphorylation, increasing its methyltransferase activity and altering its target specificity. Methods We generated two phospho-tyrosine specific antibodies against CARM1 tyrosine-149 (Y139) and -334 (Y334), and two asymmetric dimethylation-arginine specific antibodies against RUNX1 arginine-223 (R223) and -319 (R319). We also generated isogenic HEL cells carrying homozygous CARM1 non-phosphorylatable mutation (Y149F or Y334F single, or Y149F/Y334F double mutation), using the CRISPR/Cas9 nuclease system. Results We first performed cell-free in vitro kinase assays using recombinant CARM1 protein and JAK2 kinase, and mass spectrometry analysis, which identified Y139 and Y334 as the sites of JAK2 phosphorylation in CARM1. We next compared the levels of CARM1-Y149 and -Y334 phosphorylation in 15 myeloid leukemia cell lines and normal human CD34+ cord blood cells. HEL and UKE-1 cells, that harbor bi-allelic JAK2-V617F mutation, showed the highest level of phosphorylated CARM1-Y149 and -Y334, while SET2 cells with mono-allelic JAK2-V617F mutation and the other cell lines showed abundant CARM1 protein but a lower relative amount of phosphorylated CARM1 protein. We found that the JAK2 activation, through transphosphorylation by JAK2 and TYK2, enhances tyrosine kinase activity against CARM1 using HEL cells with knockout of either JAK1 or TYK2. Having previously shown that CARM1 methylates RUNX1 at R223, in an immunoprecipitation assay, WT-CARM1 was able to pull down RUNX1, while the non-phosphorylatable CARM1 mutants were not. Using mass spectrometry and an in vitro methylation assay, we confirmed R223 and R319 as CARM1 target sites. We examined non-phosphorylatable CARM1 mutation knock-in HEL cells and found that mutation of either site reduces the asymmetric dimethylation of RUNX1 as well as known substrates (BAF155 and PABP1). We observed that CARM1 Y149F/Y334F mutant-expressing HEL cells reduced cell proliferation with G2/M cell cycle arrest and an increase in apoptosis. To identify the transcription networks regulated by CARM1 phosphorylation, we performed RNA-seq on WT-CARM1 and double CARM1 Y149F/Y334F mutant-expressing HEL cells. Gene set enrichment analysis identified that gene sets associated with G2/M cell cycle progression and anti-apoptosis were significantly downregulated in Y149F/Y334F mutation knock-in HEL cells. We next performed ChIP-seq analyses using antibodies against total RUNX1 and against asymmetrically dimethylated R319-RUNX1 in HEL cells expressing knock-in of CARM1 non-phosphorylatable mutation. CARM1 non-phosphorylatable mutation decreased the signals of dimethylated RUNX1-R319 within 5 kb of the transcription start sites for RUNX1 target genes, with less effect on total RUNX1. We next assessed the efficacy of the combination with CARM1 inhibitor (EPZ025654) and JAK2 inhibitor (ruxolitinib). Dual targeting inhibition of JAK2 and CARM1 showed a synergistic inhibition effect on HEL and UKE-1 cells (with abundant phosphor-CARM1), but only an additive effect on SET2 cells (with less amount of phosphor-CARM1). Conclusion CARM1 phosphorylation mediated by hyperactivated JAK2 regulates its methyltransferase activity and is required for the maximal proliferation of myeloid neoplasms. We also demonstrated that dual targeting of JAK2 and CARM1 is more effective than monotherapy in cell lines with phosphor-CARM1. Thus, our results reveal a novel role for CARM1 phosphorylation in myeloid neoplasms.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 μg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Vascular Endothelial Receptor Tyrosine Phosphatase: Identification of Novel Substrates Related to Junctions and a Ternary Complex with EPHB4 and TIE2*[S

Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. We and others have shown recently that VE-PTP regulates vascular integrity by dephosphorylating substrates that are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome on exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction (29%) is related to cell junctions. Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein on VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signaling.

Incorporation of Phosphorylated Tyrosine into Proteins: In Vitro Translation and Study of Phosphorylated IκB-α and Its Interaction with NF-κB.

Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site-directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitrobenzyl protection of tyrosine phosphate enabled its incorporation into DHFR and IκB-α using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in parallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNACUA by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into IκB-α using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-κB binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated IκB-α also mediated the exchange of exogenous DNA into an NF-κB-cellular DNA complex isolated from the nucleus of activated Jurkat cells.

Abstract 4552: Profiling signalling protein expression, modifications and interactions with multi-dimensional antibody microarrays

Abstract Antibody microarrays permit sensitive and semi-quantitative analysis of the expression, covalent modification and interactions of proteins in lysates of cells and tissues. At Kinexus, we have developed high content Kinex KAM microarrays that feature nearly 900 pan- and phosphosite-specific antibodies for monitoring protein kinases, phosphatases and other low abundance cell signalling proteins with combinations of different detection systems. One method involved capture of in vitro dye-labeled proteins (e.g. with Cy3) from lysates from cells subjected to diverse treatments. Another method involved the detection of changes in their total phosphorylation with biotinylated pIMAGO stain and an anti-biotin antibody that is labeled with a different dye (e.g. Cy5). Alteration in protein-tyrosine phosphorylation were monitored with a dye-labelled, generic phosphotyrosine-specific PYK antibody in a sandwich antibody microarray (SAM) format. The SAM technique was also used to explore the interactions of adapter, scaffolding and chaperone proteins with hundreds of potential target signal transduction proteins with dye-labeled reporter antibodies for these highly interactive proteins. We used several human cancer cell lines (e.g. A431, HeLa, Jurkat, MCF7) subjected to diverse treatments (e.g. growth factors) to identify biomarkers for the actions of these agents. Reproducible results were obtained with as little as 25 μg of lysate protein, with a dynamic range of detection exceeding 6000-fold, and a median error range for duplicates measurements of ±12%. Typically 10-15% of the proteins tracked with these arrays demonstrated perturbations exceeding 50%. More than a third of the leads from our antibody microarrays were confirmed by immunoblotting studies. The major limitation associated with validation by Western blotting was the much lower sensitivity with immunoblotting compared with antibody microarrays. We also explored the specific interactions of heat shock proteins, adapter proteins, 14-3-3 and calcium-binding proteins with the antibody microarray captured lysate proteins from cancer cell lines. By combining these detection strategies, it was feasible to obtain over 7000 data points from use of a single antibody microarray slide with two lysate samples and duplicate measurements. The goal of our proteomics and bioinformatics studies is to use the experimental results from the application of these microarrays to map the architecture of signalling networks in a cell- or tissue-specific manner. Such multi-tiered microarray-based analyses permit target-directed identification of diverse regulatory protein changes in different experimental model systems with greater sensitivity, breadth, selectivity and economy when compared to any other competing proteomics methodologies. Citation Format: Steven Pelech, Lambert Yue, Jeffrey White, Ryan Hounjet, Dirk Winkler. Profiling signalling protein expression, modifications and interactions with multi-dimensional antibody microarrays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4552.

Production and Characterization of Polyclonal Generic Phosphotyrosine‐specific Antibodies

Reversible protein‐tyrosine phosphorylation plays critical roles in cell regulation under normal and pathological conditions, which has made generic phosphotyrosine (pY) antibodies invaluable tools for biomedical research. A recent study that compared the specificities of three widely‐used monoclonal pY antibodies has raised concerns about strong sequence motif selectivity and low overall coverage rates. These issues can potentially introduce significant bias in pY site identification and quantification. In this study we describe a novel strategy of generating a pool of polyclonal antibodies from a large set of physiological pY‐site sequences. Over 400 peptides spanning 7 to 20 amino acids in length, each featuring 1 to 7 phosphorylation sites and representing over 1000 phosphosites, were used to immunize over 100 rabbits. The sera from these rabbits were subjected to ammonium sulphate fractionation, pooled, and then affinity‐purified on pY‐agarose columns to produce the PYK antibody preparations. The specificity of the purified PYK antibody was assessed with phosphopeptide microarrays, including the Jerini Peptide Technologies Phosphatase Peptide Microarray, which features 6,099 peptides representing diverse human pY‐sites. The rabbit polyclonal PYK phosphotyrosine antibody proved to perform better than the well known 4G10, PY20 and PY100 mouse monoclonal antibodies. PYK was extremely stable to repeated freeze thaw, and found to be 4‐fold or more sensitive for detection of phosphopeptides on arrays and for proteins following Western blotting of EGF‐treated A431 cell lysates than the monoclonal antibodies. Like the other monoclonal pY antibodies, PYK reactivities with phosphothreonine and phosphoserine were over 200‐ and 300‐fold less, respectively, when compared to phosphotyrosine on phosphopeptide microarrays. PYK also detected a larger number of diverse pY‐containing peptides than 4G10, PY20 and PY100 on phosphopeptide microarrays, and unlike these other antibodies, acidic amino acid residues surrounding the pY‐sites were not negative determinants for antibody recognition. Since acidic amino acids are important positive determinants for substrate recognition for most protein‐tyrosine kinases and flank most protein‐tyrosine phosphosites, our findings indicate that the PYK antibody may be more useful for enrichment and analysis of physiological pY‐sites than the commonly used mouse monoclonal antibodies for such purposes. Further purification of the PYK antibody over an IgG‐agarose column also allowed development of preparation of a reporter antibody that was suitable for use in sandwich antibody microarrays to assess changes in the tyrosine phosphorylation status of over 500 signaling proteins simultaneously with as little as 50 μg of crude lysate protein from cells and tissues.Support or Funding InformationSupported by Kinexus Bioinformatics Corporation.

Homogeneous single-label tyrosine kinase activity assay for high throughput screening

Protein post-translational modifications (PTMs) are regulatory mechanisms carried out by different enzymes in a cell. Kinase catalyzed phosphorylation is one of the most important PTM affecting the protein activity and function. We have developed a single-label quenching resonance energy transfer (QRET) assay to monitor tyrosine phosphorylation in a homogeneous high throughput compatible format. Epidermal growth factor receptor (EGFR) induced phosphorylation was monitored using Eu3+-chelate labeled peptide and label-free phosphotyrosine specific antibody in presence of a soluble quencher molecule. In the QRET kinase assay, antibody binding to phosphorylated Eu3+-peptide protects the Eu3+-chelate from luminescence quenching, monitoring high time-resolved luminescence (TRL) signals. In the presence of specific kinase inhibitor, antibody recognition and Eu3+-chelate protection is prevented, allowing an efficient luminescence quenching. The assay functionality was demonstrated with a panel of EGFR inhibitors (AG-1478, compound 56, erlotinib, PD174265, and staurosporine). The monitored IC50 values ranged from 0.08 to 155.3 nM and were comparable to those found in the literature. EGFR activity and inhibition assays were performed using low nanomolar enzyme and antibody concentration in a 384-well plate format, demonstrating its compatibility for high throughput screening (HTS).

SRChing for the substrates of Src

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

S56 Unravelling VEGF165 Signalling in the Lung

IntroductionVascular endothelial growth factor (VEGF) is a potent mitogenic, angiogenic and permeability factor that has been implicated in the development of lung injury and repair in a number of respiratory...

Abstract LB-24: LPA-mediated oncogenic signaling in ovarian cancer involves p130Cas

Abstract Ovarian cancer is the most deadly gynecological cancer and the need for new therapies is evidenced by the fact no new drugs targeting ovarian cancer have been identified. Recent studies have shown that lysophosphatidic acid (LPA), a lipid growth factor, is implicated in the genesis and progression of ovarian cancer, yet the majority of the mechanisms through which LPA promotes cancer genesis and progression remain to be elucidated. Therefore, the underlying theme of our research is to identify novel LPA-activated signaling nodes that can be targeted for therapy in ovarian cancer. Subjecting the lysates from HeyA8 ovarian cancer cells to a high-throughput antibody array analysis, we identified that LPA potently stimulates the tyrosine-phosphorylation of p130Cas, a scaffolding protein, which, upon phosphorylation, recruits an array of signaling molecules that promotes tumor cell proliferation, survival and metastasis. The antibody-array data was validated via immunoprecipitation of p130Cas and immunoblotting with a phospho-tyrosine specific antibody in a panel of ovarian cancer cells consisting of HeyA8, OVCA429, OVCA432 and SKOV3, establishing cell-type independent activation of p130Cas by LPA. Analyzing further, we have identified that tyrosine-410 of p130Cas is phosphorylated in response to LPA. Several past studies, including ours, have shown oncogenic signaling by LPA involves the activation of LPA receptors that are coupled to the α-subunits of the heterotrimeric G proteins, Gi, Gq, G12, and/or G13. To define the specific α-subunit that mediates LPA-induced p130Cas-phosphorylation, we monitored Tyr-410 phosphorylation of p130Cas in response to 20 μM of LPA (20 min) in HeyA8 cells in which the expression of Gαi2, Gαq, Gα12, or Gα13 was silenced by specific shRNAs. Our results indicated that only the silencing of Gαi2 abrogated LPA-mediated Tyr-410 phosphorylation of p130Cas. Since Gαi2 has been shown to play a critical role in LPA-LPAR coupled cell migration, we hypothesized that the LPA-LPAR-Gαi2-p130Cas signaling axis is involved in the invasive migration of ovarian cancer cells. To test this hypothesis, we examined whether the knocking down of p130Cas, via p130Cas-specific siRNA, attenuated LPA-mediated migration of ovarian cancer cells. Our results indicated that the silencing of p130Cas significantly inhibited LPA-stimulated migration of ovarian cancer cells by 58%. Thus, our study is the first to establish LPA-LPAR-Gαi2-mediated stimulation of p130Cas is involved in invasive migration of ovarian cancer cells. Overall, the results presented in our current study point to p130Cas as a critical signaling node in pathological LPA signaling and that by targeting LPA receptors, LPA production and/or the downstream kinases that phosphorylate p130Cas, it may be possible to disable the oncogenic signaling cascades activated by p130Cas in ovarian cancer and enhance the survival of ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-24. doi:1538-7445.AM2012-LB-24

Covalent Tagging of Phosphorylated Peptides by Phosphate‐Specific Deoxyribozymes

Covalent Tagging of Phosphorylated Peptides by Phosphate‐Specific Deoxyribozymes

Abstract 2526: Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity

Abstract Topoisomerase (topo) II catalyzes topological changes in DNA. Although, both human isozymes, topo IIα and β, are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin we detected four +80-Da modified sites, tyrosine 656, serine 1395, threonine 1426 and serine 1545. Phosphorylation at serine 1395, threonine 1426 and serine 1545 was established based on neutral loss of H3PO4 (−98 Da) in the collision-induced dissociation (CID) spectra and on differences in 2D-phosphopeptide maps of 32P-labeled wild type (WT) and serine1395alanine or threonine1426alanine/serine1545alanine mutant topo IIβ. However, phosphorylation at tyrosine 656 could not be verified by 2D-phosphopeptide mapping of 32P-labeled WT and tyrosine656phenylalanine mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyrosine 656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions (characteristic of bromination) in CID spectra of two peptides containing tyrosine 656 and tyrosine 711, confirming bromination. Interestingly, mutation of only tyrosine 656, but not serine 1395, threonine 1426 or serine 1545, decreased topo IIβ activity suggesting a functional role for tyrosine 656. Homology modeling of the region of topo IIβ (amino acids 449-1108) containing these two reactive tyrosine residues revealed that tyrosine 711 is buried, whereas tyrosine 656 is highly exposed on the surface. This region is within, or very close to the dimerization interface, which suggests that these amino acids may be critical for topo IIβ dimer formation. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2526. doi:10.1158/1538-7445.AM2011-2526

Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.

Tyrosine Phosphorylation Enrichment and Subsequent Analysis by MALDI‐TOF/TOF MS/MS and LC‐ESI‐IT‐MS/MS

This unit describes methods to detect, identify, and characterize tyrosine phosphorylation in proteins by mass spectrometry, including sample preparation methods, enrichment strategies using phosphotyrosine-specific antibodies, and chromatographic separation methods.

Prothoracicotropic hormone induces tyrosine phosphorylation in prothoracic glands of the silkworm, Bombyx mori

In the present study, we investigated the tyrosine phosphorylation of Bombyx mori prothoracic glands using phosphotyrosine-specific antibodies and Western blot analysis. Results showed that prothoracicotropic hormone (PTTH) stimulates a rapid increase in tyrosine phosphorylation of at least 2 proteins in prothoracic glands, one of which was identified as extracellular signal-regulated kinase (ERK). The phosphorylation of another 120-kDa protein showed dose- and time-dependent stimulation by PTTH in vitro. In vitro activation of tyrosine phosphorylation was also verified by in vivo experiments: injection of PTTH into day-6 last-instar larvae greatly increased tyrosine phosphorylation. Treatment of prothoracic glands with the protein tyrosine phosphatase inhibitor, sodium orthovanadate, also resulted in tyrosine phosphorylation of several proteins and increased ecdysteroidogenesis. The PTTH-stimulated phosphorylation of the 120-kDa protein was markedly attenuated by genistein, a broad-spectrum tyrosine kinase inhibitor, but not by HNMPA-(AM)(3) , a specific inhibitor of insulin receptor tyrosine kinase. PP2, a more-selective inhibitor of the Src-family tyrosine kinases, partially inhibited PTTH-stimulated tyrosine phosphorylation, but not ecdysteroidogenesis. This result implies the possibility that in addition to ERK, the phosphorylation of the 120-kDa protein, which is not Src-family tyrosine kinase, is likely also involved in PTTH-stimulated ecdysteroidogenesis in B. mori.

Alteration of the PKCθ–Vav1 complex and phosphorylation of Vav1 in TCDD-induced apoptosis in the lymphoblastic T cell line, L-MAT

We have previously reported that protein kinase C (PKC) theta (θ) and protein tyrosine kinase are involved in 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced apoptosis of L-MAT, a human lymphoblastic T cell line. In the current report, we show that Vav1, a GDP/GTP exchange factor for Rho-like small GTPases, could be detected by Western blotting in the membrane fraction of L-MAT cells after TCDD treatment and was precipitated by incubating with an antibody against PKCθ. Furthermore, the degree of phosphorylation of Vav1, which can be detected using the phosphotyrosine-specific antibody PY-20 or 4G10, is significantly increased after treatment with TCDD. In addition, pretreatment of the cells with genistein, a protein tyrosine kinase inhibitor, abolished the phosphorylation of Vav1 and inhibited the apoptosis. These results suggest that TCDD treatment may activate an unidentified protein tyrosine kinase. Accordingly we hypothesize that this kinase phosphorylates Vav1, following which phosphorylated Vav1 may translocate to the membrane with PKCθ. Finally, PKCθ may mediate the transfer of the apoptotic signal to downstream components.

BCAR3 Regulates Src/p130Cas Association, Src Kinase Activity, and Breast Cancer Adhesion Signaling

The nonreceptor protein-tyrosine kinase c-Src is frequently overexpressed and/or activated in a variety of cancers, including those of the breast. Several heterologous binding partners of c-Src have been shown to regulate its catalytic activity by relieving intramolecular autoinhibitory interactions. One such protein, p130(Cas) (Cas), is expressed at high levels in both breast cancer cell lines and breast tumors, providing a potential mechanism for c-Src activation in breast cancers. The Cas-binding protein BCAR3 (breast cancer antiestrogen resistance-3) is expressed at high levels in invasive breast cancer cell lines, and this molecule has previously been shown to coordinate with Cas to increase c-Src activity in COS-1 cells. In this study, we show for the first time using gain- and loss-of-function approaches that BCAR3 regulates c-Src activity in the endogenous setting of breast cancer cells. We further show that BCAR3 regulates the interaction between Cas and c-Src, both qualitatively as well as quantitatively. Finally, we present evidence that the coordinated activity of these proteins contributes to breast cancer cell adhesion signaling and spreading. Based on these data, we propose that the c-Src/Cas/BCAR3 signaling axis is a prominent regulator of c-Src activity, which in turn controls cell behaviors that lead to aggressive and invasive breast tumor phenotypes.

Autoantibody-mediated regulation of B cell responses by functional anti-CD22 autoantibodies in patients with systemic sclerosis.

Studies have demonstrated that B cells play important roles in systemic sclerosis (SSc), especially through the CD19/CD22 autoimmune loop. CD22 is a B cell-specific inhibitory receptor that dampens B cell antigen receptor (BCR) signalling via tyrosine phosphorylation-dependent mechanism. In this study, we examined the presence and functional property of circulating autoantibodies reacting with CD22 in systemic sclerosis. Serum samples from 10 tight skin (TSK/+) mice and 50 SSc patients were assessed for anti-CD22 autoantibodies by enzyme-linked immunosorbent assays using recombinant mouse or human CD22. The association between anti-CD22 antibodies and clinical features was also investigated in SSc patients. Furthermore, the influence of SSc serum including anti-CD22 autoantibodies for CD22 tyrosine phosphorylation was examined by Western blotting using phosphotyrosine-specific antibodies reacting with four major tyrosine motifs of CD22 cytoplasmic domain. Anti-CD22 autoantibodies were positive in 80% of TSK/+ mice and in 22% of SSc patients. Patients positive for anti-CD22 antibodies showed significantly higher modified Rodnan skin thickness score compared with patients negative for anti-CD22 antibodies. Furthermore, anti-CD22 antibodies from patients' sera were capable of reducing phosphorylation of all four CD22 tyrosine motifs, while sera negative for anti-CD22 antibodies did not affect CD22 phosphorylation. Thus, a subset of SSc patients possessed autoantibodies reacting with a major inhibitory B cell response regulator, CD22. Because these antibodies can interfere CD22-mediated suppression onto B cell activation in vitro, SSc B cells produce functional autoantibodies that can enhance their own activation. This unique regulation may contribute to the autoimmune aspect of SSc.

C-Abl tyrosine kinase interacts with MAVS and regulates innate immune response

The tyrosine kinase, c-Abl, plays important roles in many aspects of cellular function. Previous reports showed that c-Abl is involved in NF-κB signaling. However, the functions of c-Abl in innate immunity are still unknown. Here we demonstrate that the mitochondrial antiviral signaling (MAVS) protein can be physically associated with c-Abl in vivo and in vitro. MAVS interacted with c-Abl through its Card and TM domain. A phosphotyrosine-specific antibody indicated that MAVS was phosphorylated by c-Abl. Functional impairment of c-Abl attenuated MAVS or VSV induced type-I IFN production. Importantly, c-Abl knockdown in MCF7 cells displayed impaired MAVS-mediated NF-κB and IRF3 activation. Taken together, our results suggest that c-Abl modulates innate immune response through MAVS. Structured summary MINT- 7297498, MINT- 7297511, MINT- 7297557, MINT- 7297574: MAVS (uniprotkb: Q7Z434) physically interacts (MI: 0915) with c-Abl (uniprotkb: P00519) by anti tag coimmunoprecipitation (MI: 0007)MINT- 7297542: c-Abl (uniprotkb: P00519) physically interacts (MI: 0915) with MAVS (uniprotkb: Q7Z434) by anti bait coimmunoprecipitation (MI: 0006)MINT- 7297526: c-Abl (uniprotkb: P00519) physically interacts (MI: 0915) with MAVS (uniprotkb: Q7Z434) by far western blotting (MI: 0047)

Differential protein kinase activity in ER-positive and ER-negative breast cancer

e22142 Background: Specific protein tyrosine kinases (PTKs) promote cancer progression and are potential drug targets. The presence of the estrogen receptor (ER) is an important criterion in deciding on treatment of patients with breast cancer. Since PTKs can affect the tumors' receptor function, we investigated in our first proof-of-principle study whether it is possible to discriminate ER-positive (ER+) from ER-negative (ER-) tumors based on PTK activity using microarrays containing many PTK peptide substrates. Methods: Cryosections of 12 ER+ and 12 ER- breast tumors were lysed in the presence of appropriate protease and phosphatase inhibitors. The protein kinase activity in the lysates was monitored in vitro using PamChip peptide microarrays, which comprise of 253 PTK peptide substrates derived from known human phosphorylation sites. Peptide phosphorylation through active kinases can be monitored in samples in real time, using a fluorescently labeled phospho-tyrosine specific antibody. Results: Phosphorylation activity profiles were determined in quadruplicate using multiple independent sample preparations. Using ANOVA analysis, out of 253 peptides, several peptides with a different phosphorylation signal in ER+ and ER- samples were found: 22 with p < 0.02 including VEGFR1, 2 and 3 and ADAM9. Multivariate unsupervised analysis using Principle Component Analysis showed a clustering of ER+ samples vs. ER- samples. Predictive analysis was performed using Partial-Least Squares Discriminant Analysis without explicit feature selection. The prediction error obtained from within experiment cross validation was typically in the range 10–20%. The error rate for between experiment prediction was 20%. An average predictive profile was constructed in which peptides with a relatively large weight were included, e.g. ADAM9 and BCAR1. Conclusions: Using PamChip peptide microarrays we have shown that differences in protein kinase activity exist between ER+ and ER- breast tumors. Our in vitro assay is a promising tool to investigate the interplay between kinase and nuclear receptor mediated signaling, with potential relevance to patient selection for targeted therapies. No significant financial relationships to disclose.