ArticlePDF Available

Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C

Authors:
  • AK Biotechnologies LLC

Abstract and Figures

The SH2 domain protein tyrosine phosphatases (PTPases) PTP1C and PTP1D were found associated with epidermal growth factor (EGF) receptor which was purified from A431 cell membranes by several steps of chromatography. Both PTPases also associated with the EGF receptor upon exposure of immunoprecipitated receptor to lysates of MCF7 mammary carcinoma cells. The associated PTPases had little activity toward the bound receptor when it was autophosphorylated in vitro. Receptor dephosphorylation could, however, be initiated by treatment of the receptor-PTPase complex with phosphatidic acid (PA). When autophosphorylated EGF receptor was exposed to lysates of PTP1C or PTP1D overexpressing 293 cells, the association of PTP1C but not of PTP1D was enhanced in the presence of PA. In intact A431 cells, an association of PTP1C and PTP1D with the EGF receptor was detectable by coimmunoprecipitation experiments. PA treatment reduced the phosphorylation state of ligand activated EGF receptors in A431 cells and in 293 cells overexpressing EGF receptors together with PTP1C but not in 293 cells overexpressing EGF receptors alone or together with PTP1D. We conclude that PTP1C but not PTP1D participates in dephosphorylation of activated EGF receptors. A possible role of PA for physiological modulation of EGF receptor signaling is discussed.
No caption available
… 
Content may be subject to copyright.
A preview of the PDF is not available
... However, in vitro studies described prooxidative effects. In mammalian cells, EGF enhances the production of intracellular reactive oxygen species by dual oxidase 1 [114] and these oxidative species, which are induced by EGF, modulate ADAMs (positive regulator of EGFR signaling) or protein tyrosine phosphatases (negative regulator of EGFR signaling) [115][116][117][118]. AKI is a multifactorial pathology characterized by renal tubular damage, inflammation, and, frequently, a transient decrease in renal function. ...
Article
Full-text available
Chronic kidney disease (CKD) is characterized by persistent inflammation and progressive fibrosis, ultimately leading to end-stage renal disease. Although many studies have investigated the factors involved in the progressive deterioration of renal function, current therapeutic strategies only delay disease progression, leaving an unmet need for effective therapeutic interventions that target the cause behind the inflammatory process and could slow down or reverse the development and progression of CKD. Epidermal growth factor receptor (EGFR) (ERBB1), a membrane tyrosine kinase receptor expressed in the kidney, is activated after renal damage, and preclinical studies have evidenced its potential as a therapeutic target in CKD therapy. To date, seven official EGFR ligands have been described, including epidermal growth factor (EGF) (canonical ligand), transforming growth factor- α , heparin-binding epidermal growth factor, amphiregulin, betacellulin, epiregulin, and epigen. Recently, the connective tissue growth factor (CTGF/CCN2) has been described as a novel EGFR ligand. The direct activation of EGFR by its ligands can exert different cellular responses, depending on the specific ligand, tissue, and pathological condition. Among all EGFR ligands, CTGF/CCN2 is of special relevance in CKD. This growth factor, by binding to EGFR and downstream signaling pathway activation, regulates renal inflammation, cell growth, and fibrosis. EGFR can also be “transactivated” by extracellular stimuli, including several key factors involved in renal disease, such as angiotensin II, transforming growth factor beta (TGFB), and other cytokines, including members of the tumor necrosis factor superfamily, showing another important mechanism involved in renal pathology. The aim of this review is to summarize the contribution of EGFR pathway activation in experimental kidney damage, with special attention to the regulation of the inflammatory response and the role of some EGFR ligands in this process. Better insights in EGFR signaling in renal disease could improve our current knowledge of renal pathology contributing to therapeutic strategies for CKD development and progression.
... The association of SHP1 with EGFR results in suppression of EGFR-mediated ERK activation. However, present knowledge has different interpretations of the action of SHP1 on the EGFR signaling such that SHP1 binding to the EGFR can cause an overall decrease in tyrosine phosphorylation status of the receptor and attenuation of the receptor signaling both in transient coexpression systems and in stably SHP1-transfected cells [111,115,116]. ...
Article
Post-translational protein modifications are critical regulators of protein functions as they expand the signaling potentials of the modified proteins, leading to diverse physiological consequences. Currently, increasing evidence suggests that protein methylation is as important as other post-translational modifications in the regulation of various biological processes. This drives us to ask whether methylation is involved in the EGFR (epidermal growth factor receptor) signaling, a biological process extensively regulated by multiple post-translational modifications including phosphorylation, glycosylation and ubiquitination. We found that EGFR R1175 is methylated by a protein arginine methyltransferase named PRMT5. During EGFR activation, PRMT5-mediated R1175 methylation specifically enhances EGF-induced EGFR autophosphorylation at Y1173 residue. This novel modification crosstalk increases SHP1 recruitment to EGFR and suppresses EGFR-mediated ERK activation, resulting in inhibition of cell proliferation, migration, and invasion of EGFR-expressing cells. Based on these findings, we provide the first link between arginine methylation and tyrosine phosphorylation and identify R1175 methylation as an inhibitory modification specifically against EGFR-mediated ERK activation.
... Cysteine S-nitrosylation of protein tyrosine phosphatase SHP-1 is associated with BK-mediated activation of the EGF-R Both SH-2 domains of the tyrosine phosphatase SHP-1, associates with phospho-Tyr1173 at the cytoplasmic domain of the EGF-R, this interaction negatively regulates receptor activity through dephosphorylation [4,34]. In addition, mild nitrosative stress conditions can inhibit EGF-R dephosphorylation through S-nitrosylation of a single conserved Cys residue at the catalytic domain of SHP-1 [35,36]. ...
Article
Objective Interleukin (IL)-12 has a pivotal profibrotic role in the development of idiopathic pulmonary fibrosis (IPF). Medical research trials based on IPF registry databases have actively recruited patients. Surfactant protein D (SP-D) is a useful biomarker in patients with IPF. SP-D binds to signal regulatory protein α (SIRPα), which acts as an inhibitory receptor, and this SP-D/SIRPα interaction may have an anti-inflammatory effect. Accordingly, the inhibitory effect of SP-D on IL-12p40 production by lipopolysaccharide (LPS)-stimulated macrophages was investigated. Materials and Methods Human granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated macrophages (day 9 of culture) was used to investigate IL-12p40 production after stimulation with SP-D. Results GM-CSF was found to upregulate SIRPα expression by macrophages. PD98059 (an extracellular signal-regulated kinase [ERK] inhibitor) blunted induction of SIRPα expression by GM-CSF. SP-D significantly attenuated IL-12p40 production by macrophages after stimulation with LPS. Silencing of SIRPα/β/γ significantly reversed this inhibitory effect of SP-D. In contrast, neither SB023580 (a p38α/β MAPK inhibitor) nor BIRB796 (a p38γ/δ MAPK inhibitor) attenuated the inhibitory effect of SP-D on LPS-stimulated production of IL-12p40. Silencing of SHP also had no influence on this effect of SP-D. Interestingly, a Rho-associated protein kinase (ROCK) inhibitor (Y-27632) abolished the inhibition of LPS-stimulated IL-12p40 production by SP-D, whereas silencing of ERK 2 significantly blunted this effect of Y-27632. Conclusions These findings suggest that SP-D inhibits LPS-stimulated production of IL-12p40 via the SIRPα/ROCK/ERK signaling pathway.
Article
Full-text available
Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.
Chapter
In this chapter, we will discuss the role of 14 non-transmembrane (NT) type protein-tyrosine phosphatases (PTPs, encoded by the genes PTPN3, PTPN4, PTPN5, PTPN6, PTPN7, PTPN9, PTPN13, PTPN14, PTPN18, PTPN20, PTPN21, PTPN22, PTPN23, and PTPRR) in cancer. The presentation of NTPTPs includes a brief description of general features of the individual molecules in structure and activity regulation, as well as key facts about their physiological functions. Genetic or epigenetic alterations of NTPTP genes in cancer cells and, if available, the molecular consequences for the specific phenotypic effects are subsequently discussed. A role for defects of PTPN6, PTPN13, or PTPN23 function in certain tumor types has been established best. For other NTPTPs, indications for putative roles in cancer rest on gene mutations in cancer tissues, phenotypes of cell lines with altered NTPTP status, and known physiological functions, but need confirmation in future studies. Clearly, NTPTP effects on cancer phenotypes are dependent on the specific cell context. Roles of NTPTPs in the cancer microenvironment and for therapeutic responses to anti-cancer drugs are emerging. Functions of NTPTPs in immune cells, notably of PTPN6, might also allow therapeutic exploitation in the future.
Chapter
The primary function of neutrophils is host defense but, paradoxically, unregulated activation of these phagocytes may result in tissue damage in conditions such as acute lung injury and the systemic inflammatory response syndrome (SIRS). Despite extensive research into the physiological and cellular basis of inflammatory injury and increasingly sophisticated intensive care, the morbidity and mortality of these syndromes remains distressingly high. The purpose of this chapter is to summarize recent developments in our understanding of the signaling pathways regulating leukocyte activation relevant to neutrophil-mediated tissue injury. Two main concepts will be explored: 1) that defective regulation of protein tyrosine phosphatases (PTPs) that function primarily in signal termination may predispose to uncontrolled neutrophil activation, and 2) that signals from adhesion receptors contribute to enhanced release of cytotoxic compounds by neutrophils during adhesion to endothelium, epithelium, or extracellular matrix proteins in the interstitium. This ‘adhesion-dependent activation’ of leukocytes can greatly potentiate the effects of soluble mediators on activation. Recent studies from our laboratory that illuminate these signahng pathways will be reviewed. It is hoped that the results of these studies will provide insights into the pathogenesis of lung injury and eventually lead to novel therapeutic strategies to prevent or ameliorate tissue injury.
Chapter
Dimerization of growth factor receptor tyrosine kinases (RTK) (Heldin 1995) leads in turn to a mutual phosphorylation of two receptor monomers at multiple tyrosine residues. This process, commonly designated as “autophosphorylation” is the initial and a crucial event of growth factor signaling, which is followed by initiation of a multitude of downstream signaling chains (Ullrich and Schlessinger 1990; Fantl et al. 1993). RTK autophosphorylation serves two purposes: (1) It regulates the activity of the RTK positively, albeit to a different extent for different receptor species. Determination of the three-dimensional structure of the catalytic center of the insulin receptor has recently shed some light on the structural basis for the pronounced activation of this RTK by autophosphorylation (Hubbard et al. 1994). Tyrosine 1162 in the unphosphorylated form sterically blocks access to the peptide substrate and the ATP binding sites. In the phosphorylated form it is expected to become disengaged from the active center and to allow access of the substrates. In terms of homology it is possible that such a mechanism might operate similarly in other RTK. (2) Autophosphorylation creates binding sites for intracellular proteins possessing phosphotyrosine binding domains such as SH2 or PTB domains (Pawson 1995).
Chapter
Members of the receptor tyrosine kinase family are frequently implicated in experimental models of neoplasia as well as in human cancer. One of the best studied receptor signaling systems from this family is the epidermal growth factor receptor (EGFR). The EGFR is widely expressed in mammals and has been implicated in various stages of embryonic development. Recently, EGFR knockout mice by targeted disruption of exon 1 (Sibilia and Wagner 1995) or exon 2 (Miettinen et al. 1995; Threadgill et al. 1995) of the mouse EGFR gene have been reported. The resulting phenotypes of EGFR-/- embryos or newborns were very similar. Growth retardation and epithelial immaturity were found, with the severity of defects being dependent on the mouse genetic background. These results indicate that the EGFR is of fundamental importance in the regulation of epithelial proliferation and differentiation. Experiments with transgenic mice suggest that an autocrine mechanism involving the EGFR could be expected to play a role in the initiation and/or progression of tumors. Transgenic mice expressing transforming growth factor-α (TGF-α) were reported to develop mammary and hepatocellular carcinoma as well as pancreatic hyperplasia (Jhappan et al. 1990; Sandgreen et al. 1990).
Chapter
As shown in figure 1, ethanol can be metabolized by oxidative and nonoxidative pathways (Lieber, 1995). In the oxidative pathway, ethanol is converted to acetal-dehyde through the action of alcohol dehydrogenases, the microsomal ethanol-oxidizing system, or catalase. Acetaldehyde is then subsequently metabolized to acetate through the action of aldehyde dehydrogenases. In one of the nonoxidative pathways of ethanol metabolism, ethanol undergoes esterification with fatty acids by the action of fatty acid ethyl ester synthase to form fatty acid ethyl esters. The nonoxidative ethanol pathway that is the focus of this review is the pathway leading to the synthesis of phosphatidylethanol. Figure 1 shows how ethanol can be inserted as the head group of a phospholipid to form phosphatidylethanol. The transformation occurs through the activation of phospholipase D, mainly on phosphatidylcholine in the presence of ethanol.
Article
Full-text available
The autophosphorylation, from [gamma-32P]ATP, of insulin and epidermal growth factor receptors in rat liver endosomes peaked at 2-5 min and declined thereafter. When autophosphorylation from either [gamma-32P]ATP or unlabeled ATP was stopped after 5 min by adding excess EDTA +/- ATP, the phosphotyrosine (PY) content of each receptor decreased at 37 degrees C with a t 1/2 of 1.6 min. This was equally so whether the PY content of 32P-labeled receptors was analyzed by autoradiography of KOH-treated gels or by Western blotting with PY antibodies of immunoprecipitated receptors. The dephosphorylation reaction was strictly dependent on the presence of sulfhydryl, was unaffected by the addition of rat liver cytosol, and was temperature-dependent. The phosphotyrosine phosphatase(s) (PTPase(s)) appeared to be tightly anchored to the endosomal membrane, since the dephosphorylation reaction was unaffected by sodium carbonate and 0.6 M KCl treatments. However, treatment with Triton X-100 abolished dephosphorylation, implying an intimate association between the PTPase(s) and its substrate in an intact membrane environment. The powerful insulinomimetic agent pervanadate was the most potent inhibitor (50% inhibition at 1 microM). Increasing the dose of injected ligand augmented the rate of insulin and decreased that of EGF receptor dephosphorylation, respectively. Immunoblotting with specific antibodies failed to identify PTPase 1B or T-cell PTPase in ENs, whereas positive signals were seen in plasma membrane. These studies indicate that the phosphorylation state of receptor tyrosine kinases is dynamically regulated, with dephosphorylation, by closely associated PTPase(s), playing an important role.
Article
Full-text available
Tyrosine phosphorylation is a mechanism of signal transduction shared by many growth factor receptors and oncogene products. Phosphotyrosine phosphatases (PTPases) potentially modulate or counter-regulate these signaling pathways. To test this hypothesis, the transmembrane PTPase CD45 (leukocyte common antigen) was expressed in the murine cell line C127. Hormone-dependent autophosphorylation of the platelet-derived growth factor (PDGF) and insulin-like growth factor-1 (IGF-1) receptors was markedly reduced in cells expressing the transmembrane PTPase. Tyrosine phosphorylation of other PDGF-dependent phosphoproteins (160, 140, and 55 kDa) and IGF-1-dependent phosphoproteins (145 kDa) was similarly decreased. Interestingly, the pattern of growth factor-independent tyrosine phosphorylations was comparable in cells expressing the PTPase and control cells. This suggests a selectivity or accessibility of the PTPase limited to a subset of cellular phosphotyrosyl proteins. The maximum mitogenic response to PDGF and IGF-1 in cells expressing the PTPase was decreased by 67 and 71%, respectively. These results demonstrate that a transmembrane PTPase can both affect the tyrosine phosphorylation state of growth factor receptors and modulate proximal and distal cellular responses to the growth factors.
Article
Full-text available
The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
Article
Mutations in the gene encoding the phosphotyrosine phosphatase PTP1C, a cytoplasmic protein containing a COOH-terminal catalytic and two NH2-terminal Src homology 2 (SH2) domains, have been identified in motheaten (me) and viable motheaten (mev) mice and are associated with severe hemopoietic dysregulation. The me mutation is predicted to result in termination of the PTP1C polypeptide within the first SH2 domain, whereas the mev mutation creates an insertion or deletion in the phosphatase domain. No PTP1C RNA or protein could be detected in the hemopoietic tissues of me mice, nor could PTP1C phosphotyrosine phosphatase activity be isolated from cells homozygous for the me mutation. In contrast, mice homozygous for the less severe mev mutation expressed levels of full-length PTP1C protein comparable to those detected in wild type mice and the SH2 domains of mev PTP1C bound normally to phosphotyrosine-containing ligands in vitro. Nevertheless, the mev mutation induced a marked reduction in PTP1C activity. These observations provide strong evidence that the motheaten phenotypic results from loss-of-function mutations in the PTP1C gene and imply a critical role for PTP1C in the regulation of hemopoietic differentiation and immune function.
Article
To clarify the role of protein tyrosine phosphatase containing Src homology 2 (SH2) regions on insulin signaling, we investigated the interactions among the insulin receptor, a pair of SH2 domains of SH-PTP2 coupled to glutathione-S-transferase (GST) and insulin receptor substrate-1 (IRS-1)-GST fusion proteins (amino-portion, IRS-1N; carboxyl portion, IRS-1C). GST-SH2 protein of SH-PTP2 bound to the wild type insulin receptor, but not to that with a carboxyl-terminal mutation (Y/F2). Furthermore, even though Y/F2 receptors were used, the SH2 protein was also co-immunoprecipitated with IRS-1C, but not with IRS-1N. These results indicate that SH2 domains of SH-PTP2 can directly associate with the Y1322TXM motif on the carboxyl terminus of insulin receptors and also may bind to the carboxyl portion of IRS-1, possibly via the V1172IDL motif in vitro.
Article
We have investigated the effect of vanadate on the phosphoserine- and phosphotyrosine-specific phosphoprotein phosphatase activities of A-431 cell membranes and have found that micromolar concentrations of vanadate strongly inhibit the membrane-dependent dephosphorylation of histones containing phosphotyrosine but that they do not inhibit the dephosphorylation of histones containing phosphoserine and phosphothreonine. In addition, the dephosphorylation of endogeneous membrane proteins of A-431 cells (which are known to be phosphorylated at tyrosine residues) was inhibited by vanadate. These results show that vanadate is a potent and selective inhibitor of phosphotyrosyl-protein phosphatase.
Article
Protein-tyrosine phosphatases (PTPases) play an essential role in the regulation of signal transduction mediated by reversible protein-tyrosine phosphorylation. In order to characterize individual rat hepatic PTPases that might have specificity for autophosphorylated receptor tyrosine kinases, we isolated cDNA segments encoding three PTPases (PTPase 1B, LAR and LRP) that are expressed in insulin-sensitive liver and skeletal muscle tissue, and evaluated their catalytic activity in vitro. The intrinsic PTPase activities of the full-length PTPase 1B protein and the cytoplasmic domains of LAR and LRP were studied by expression of recombinant cDNA constructs in the inducible bacterial vector pKK233-2 using extracts of a host strain of Escherichia coli that lacks endogenous PTPase activity. Each of the cloned cDNAs dephosphorylated a cognate phosphopeptide derived from the regulatory region of the insulin receptor. Despite having only 30-39% sequence identity in their catalytic domains, LAR and PTPase 1B had similar relative activities between the peptide substrate and intact insulin receptors, and also displayed similar initial rates of simultaneous dephosphorylation of insulin and epidermal growth factor (EGF) receptors. In contrast, LRP exhibited a higher rate of dephosphorylation of both intact receptors relative to the peptide substrate, and also dephosphorylated EGF receptors more rapidly than insulin receptors. These studies indicate that three PTPases with markedly divergent structures have the catalytic potential to dephosphorylate both insulin and EGF receptors in intact cells and that redundant PTPase activity may occur in vivo. For these PTPases to have specific physiological actions in intact cells, they must be influenced by steric effects of the additional protein segments of the native transmembrane enzymes, cellular compartmentalization and/or interactions with regulatory proteins.
Article
We describe the characterization of the Drosophila gene, corkscrew (csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the torso signal via tailless, to downstream terminal genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.