Protein tyrosine phosphatase 1B interacts with and is tyrosine phosphorylated by the epidermal growth factor receptor (original) (raw)
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Tyrosine Phosphorylation Mapping of the Epidermal Growth Factor Receptor Signaling Pathway
Journal of Biological Chemistry, 2002
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.
Molecular and Cellular Biology, 2004
The platelet-derived growth factor (PDGF)  receptor mediates mitogenic and chemotactic signals. Like other tyrosine kinase receptors, the PDGF  receptor is negatively regulated by protein tyrosine phosphatases (PTPs). To explore whether T-cell PTP (TC-PTP) negatively regulates the PDGF  receptor, we compared PDGF  receptor tyrosine phosphorylation in wild-type and TC-PTP knockout (ko) mouse embryos. PDGF  receptors were hyperphosphorylated in TC-PTP ko embryos. Fivefold-higher ligand-induced receptor phosphorylation was observed in TC-PTP ko mouse embryo fibroblasts (MEFs) as well. Reexpression of TC-PTP partly abolished this difference. As determined with site-specific phosphotyrosine antibodies, the extent of hyperphosphorylation varied among different autophosphorylation sites. The phospholipase C␥1 binding site Y1021, previously implicated in chemotaxis, displayed the largest increase in phosphorylation. The increase in Y1021 phosphorylation was accompanied by increased phospholipase C␥1 activity and migratory hyperresponsiveness to PDGF. PDGF  receptor tyrosine phosphorylation in PTP-1B ko MEFs but not in PTP ko MEFs was also higher than that in control cells. This increase occurred with a site distribution different from that seen after TC-PTP depletion. PDGF-induced migration was not increased in PTP-1B ko cells. In summary, our findings identify TC-PTP as a previously unrecognized negative regulator of PDGF  receptor signaling and support the general notion that PTPs display site selectivity in their action on tyrosine kinase receptors.
Journal of Biological Chemistry, 2013
Background: Our understanding of the physiological relevance between receptor-like protein-tyrosine phosphatase (RPTP) and receptor protein-tyrosine kinase (RPTK) is limited. Results: Multiple RPTKs were identified as substrates for the R3 RPTP subfamily. Conclusion: Members of the R3 subfamily show a similar but distinct specificity toward RPTKs. Significance: This study sheds light on physiological roles of the R3 RPTP subfamily. Receptor-like protein-tyrosine phosphatases (RPTPs) are involved in various aspects of cellular functions, such as proliferation, differentiation, survival, migration, and metabolism. A small number of RPTPs have been reported to regulate activities of some cellular proteins including receptor protein-tyrosine kinases (RPTKs). However, our understanding about the roles of individual RPTPs in the regulation of RPTKs is still limited. The R3 RPTP subfamily reportedly plays pivotal roles in the development of several tissues including the vascular and nervous systems. Here, we examined enzyme-substrate relationships between the four R3 RPTP subfamily members and 21 RPTK members selected from 14 RPTK subfamilies by using a mammalian two-hybrid system with substrate-trapping RPTP mutants. Among the 84 RPTP-RPTK combinations conceivable, we detected 30 positive interactions: 25 of the enzyme-substrate relationships were novel. We randomly chose several RPTKs assumed to be substrates for R3 RPTPs, and validated the results of this screen by in vitro dephosphorylation assays, and by cell-based assays involving overexpression and knockdown experiments. Because their functional relationships were verified without exception, it is probable that the RPTKs identified as potential substrates are actually physiological substrates for the R3 RPTPs. Interestingly, some RPTKs were recognized as substrates by all R3 members, but others were recognized by only one or a few members. The enzyme-substrate relationships identified in the present study will shed light on physiological roles of the R3 RPTP subfamily. Cellular tyrosine phosphorylation levels are governed by the opposing activities of protein-tyrosine kinases (PTKs) 3 and * This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
Journal of Cell Science
Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity of a pathway, can be controlled by multiple tyrosine phosphatases, attesting to the intricate integration of these enzymes in cellular regulation. Lastly, we are starting to appreciate the roles of intracellular topology, tyrosine phosphorylation and oligomerization among the many mechanisms regulating tyrosine phosphatase activity.
Protein tyrosine phosphatase PTPRR isoforms : modulators of neuronal growth factor signalling
Cell Oncol, 2008
Community development and activity is critically dependent on communication between individuals. Likewise, proper growth and functioning of multicellular organisms requires communication between cells. In this process, extracellular stimuli that trigger the correct cellular responses through the activation of specific intracellular signalling pathways play a pivotal role. A key mechanism in cellular signal transduction is the reversible tyrosine phosphorylation of proteins, exerted by the concerted action of two classes of enzymes: protein tyrosine kinases (PTKs) that catalyse the addition of a phosphate group to a tyrosine residue, and protein tyrosine phosphatases (PTPs) that remove that phosphate group again. Although the number of genes in the human genome that encode PTPs roughly equals that of PTK-encoding genes, our knowledge on products of PTP genes is lagging behind that on PTK genes [1, 2]. Partly this can be explained by the fact that PTKs were discovered earlier than PTPs. In addition, immediately after their discovery, the role of PTKs was coupled to disease. Mutant forms of PTKs were found encoded by the genomes of tumour viruses and PTK signalling pathways appeared constitutively activated in malignant cells. In contrast, around the time of their first identification [3] PTPs were merely regarded as "housekeeping" proteins that served to terminate intracellular signalling initiated by PTKs. This view has changed considerably over the past decade. Nowadays, PTPs are known as downstream counteractors but also as upstream activators of PTKs. Furthermore, we now know that PTP activity is subjected to regulatory principles as well and, importantly, mutations in PTP genes are causative or directly involved in several developmental and acquired disorders, including several forms of cancer [4]. Thus, PTPs perform much more complex roles in intracellular signalling than initially anticipated. However, the physiological function(s) of most PTPs still remain(s) to be disclosed. This thesis provides an overview of our studies on PTPs that belong to the protein tyrosine phosphatase receptor-type R (PTPRR) isoform family. Using cell and animal models, information about the function of these proteins in • General introduction General introduction cellular signalling and control of locomotion was obtained. Before presenting this work, I will first introduce the reader to signalling mechanisms and components that are generally relevant for studies described in the upcoming chapters. Receptor tyrosine kinase signalling Many fundamental cellular processes like cell growth, differentiation, metabolism and survival are regulated by extracellular growth factors or cytokines. Since these factors are unable to pass the hydrophobic cell membrane, they exert their effects via binding to cell surface receptors. One such receptor family consists of the so-called receptor tyrosine kinases (RTKs) that have an intrinsic protein tyrosine kinase activity [5]. RTKs are single membrane spanning proteins consisting of an intracellular kinase domain and an extracellular ligand-binding domain that is usually glycosylated. This extracellular part often contains one or multiple copies of Ig-like, fibronectin type III-like, EGF-like or cysteine-rich domains [6]. In general, ligand binding to RTKs induces dimerisation of these receptors resulting in autophosphorylation of two different classes of tyrosine residues in their cytosolic domains. Autophosphorylation of tyrosine residues within the kinase domain increases the kinase activity and precedes phosphorylation of other tyrosine residues that are localized outside the kinase domain [7, 8]. These latter phosphotyrosines function as binding sites for SH2 (Src homology 2) or PTB (phosphotyrosine binding) domain-containing proteins. Binding provides a mechanism for the assembly and recruitment of downstream signalling complexes. Depending on the type of RTK that is activated, many different signalling pathways (e.g. the STAT, PLCγ, MAPK, PI3-K or Cdc42 cascade) can be switched on [5]. As an example of RTK signalling, the working mechanism of the TrkA signal transduction cascade will be described in detail.
Oncogene, 2002
We have used a proteomic approach using mass spectrometry to identify signaling molecules involved in receptor tyrosine kinase signaling pathways. Using affinity purification by anti-phosphotyrosine antibodies to enrich for tyrosine phosphorylated proteins, we have identified a novel signaling molecule in the epidermal growth factor receptor signaling pathway. This molecule, designated Odin, contains several ankyrin repeats, two sterile alpha motifs and a phosphotyrosine binding domain and is ubiquitously expressed. Using antibodies against endogenous Odin, we show that it undergoes tyrosine phosphorylation upon addition of growth factors such as EGF or PDGF but not by cytokines such as IL-3 or erythropoietin. Immunofluorescence experiments as well as Western blot analysis on subcellular fractions demonstrated that Odin is localized to the cytoplasm both before and after growth factor treatment. Deletion analysis showed that the phosphotyrosine binding domain of Odin is not required for its tyrosine phosphorylation. Overexpression of Odin, but not an unrelated adapter protein, Grb2, inhibited EGF-induced activation of c-Fos promoter. Microinjection of wild-type or a mutant version lacking the PTB domain into NIH3T3 fibroblasts inhibited PDGF-induced mitogenesis. Taken together, our results indicate that Odin may play a negative role in growth factor receptor signaling pathways.
European Journal of Biochemistry, 1992
The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor c(. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 6 1 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent V,,, value of 20 nmol min-' mg-' and K , values of 4.5 pM for ATP and 1.43 mM for angiotensin 11. This corresponds to a turnover number of 1.4 mol min-' mol-' .
Journal of Proteome Research, 2008
To identify phosphotyrosine (pY) sites in the epidermal growth factor receptor (EGFR) network, a tandem immunoprecipitation-mass spectrometry method (TIPY-MS) was applied wherein protease-digested EGFR immune complexes were extracted with anti-pY after Rush et al. (Nat. Biotech. 2005, 23, 94) and analyzed by LC-MS/MS. New pY sites in the pathway were found, including SOS1 Y1065, SOS2 Y1275, CBL-B Y889, and in the EGFR regulatory protein Mig-6 Y458. The novel human C19orf19 gene product was found EGFR-associated and phosphorylated at 5 tyrosines in response to EGFR activation and, therefore, represents a new component of the EGFR signaling network.