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1.
Evidence for epidermal growth factor (EGF)-induced intermolecular autophosphorylation of the EGF receptors in living cells. 总被引:19,自引:7,他引:12
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A M Honegger A Schmidt A Ullrich J Schlessinger 《Molecular and cellular biology》1990,10(8):4035-4044
In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries. 相似文献
2.
Pesesse X Dewaste V De Smedt F Laffargue M Giuriato S Moreau C Payrastre B Erneux C 《The Journal of biological chemistry》2001,276(30):28348-28355
The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged full-length enzyme was expressed in COS-7 cells and stained with anti-His6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc. 相似文献
3.
Reconstitution of human epidermal growth factor receptors and its deletion mutants in cultured hamster cells 总被引:40,自引:0,他引:40
E Livneh R Prywes O Kashles N Reiss I Sasson Y Mory A Ullrich J Schlessinger 《The Journal of biological chemistry》1986,261(27):12490-12497
DNA sequences encoding the human epidermal growth factor (EGF) receptor and various EGF-receptor deletion mutants were transfected into chinese hamster ovary (CHO) cells devoid of endogenous EGF receptors. A functional human EGF-receptor is expressed on the surface of heterologous CHO cells with the following properties: it exhibits typical high affinity (10%; Kd = 3 X 10(-10) M) and low affinity (90%; Kd = 3 X 10(-9) M) binding sites for 125I-EGF; it is expressed as a polypeptide of 170,000 molecular weight with intrinsic protein tyrosine kinase activity. EGF stimulates the kinase activity leading to self-phosphorylation and to phosphorylation of exogenous substrate; 125I-EGF is rapidly internalized into the CHO cells by receptor mediated endocytosis and; EGF stimulates DNA synthesis in the cells expressing the human EGF-receptor. Deletion of 63 amino acids from the C-terminal end of EGF-receptor, which removes two autophosphorylation sites, abolishes the high affinity state of the receptor. Nevertheless, this receptor mutant is able to undergo endocytosis and to respond mitogenically to EGF to a similar extent as the "wild type" receptor. Further deletions from the cytoplasmic domain give rise to low affinity endocytosis-defective receptor mutants. Finally, deletion of the transmembrane domain of the human receptor yields an EGF-receptor ligand binding domain which is secreted from the cells. 相似文献
4.
T Fujioka J H Kim H Adachi K Saito M Tsujimoto S Yokoyama M Ui 《European journal of biochemistry》2001,268(15):4158-4168
In accordance with our recent results obtained with cultured rat hepatocytes [Fujioka, T. & Ui, M. (2001) Eur. J. Biochem. 268, 25-34], epidermal growth factor (EGF) gave rise to transient tyrosine phosphorylation of insulin receptor substrates (IRS-1 and IRS-2), thereby activating the bound phosphatidylinositol 3-kinase in human epidermoid carcinoma A431 cells normally abundant in EGF receptors (EGFR) and Chinese hamster ovary (CHO) cells transfected with full-length EGFR. These actions of EGF, although much smaller in magnitude than those of insulin or IGF-I in the same cells, were accompanied by tyrosine phosphorylation of EGFR rather than insulin or IGF-I receptors, never observed in wild-type CHO cells expressing no EGFR, and totally inhibited by an inhibitor of EGFR kinase, AG1478, that was without effect on insulin or IGF-I actions. Recombinant IRS-1 was phosphorylated on tyrosines upon incubation with purified EGFR from A431 cells and 32P-labeled ATP. When CHO cells were transfected with C-terminal truncated EGFR lacking three NPXY motifs responsible for direct binding to phosphotyrosine-binding domains of IRSs, no effect of EGF could be observed. We suggest that tyrosine phosphorylation of IRS-1 or IRS-2 could mediate EGFR-induced activation of phosphatidylinositol 3-kinase in mammalian cells. 相似文献
5.
Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line. 相似文献
6.
Mutations in the cytoplasmic domain of EGF receptor affect EGF binding and receptor internalization. 总被引:39,自引:6,他引:33
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Binding of epidermal growth factor (EGF) to its receptor results in a cascade of events that culminate in cell division. The receptor is present on the cell surface in two forms of high and low affinity binding for EGF. EGF binding activates the receptor's intracellular tyrosine kinase activity and subsequently causes the receptor to be rapidly internalized into the cell via clathrin-coated pits. We have cloned the EGF receptor cDNA into a retroviral expression vector and made mutations in vitro to investigate the function of different receptor domains. Deletion of cytoplasmic sequences abolishes high but not low affinity sites as well as impairing the ability of the protein to internalize into cells. Thus, cytoplasmic sequences must be involved in the regulation of high affinity sites and are required for EGF-induced receptor internalization. A four amino acid insertion mutation at residue 708 abolishes the protein-tyrosine kinase activity of the immunoprecipitated receptor. However, this receptor mutant exhibits both the high and low affinity states, internalizes efficiently and is able to cause cells to undergo DNA synthesis in response to EGF. Another four amino acid insertion mutation (residue 888) abolishes protein-tyrosine kinase activity, high affinity binding, internalization and mitogenic responsiveness. Finally, a chimaeric receptor composed of the extracellular EGF binding domain and the cytoplasmic v-abl kinase region transforms Rat-I cells. This chimaeric receptor possesses intrinsic protein tyrosine kinase activity which cannot be regulated by EGF. Moreover, EGF fails to induce the internalization of the chimaeric receptor. 相似文献
7.
Expression of the human EGF receptor with ligand-stimulatable kinase activity in insect cells using a baculovirus vector. 总被引:19,自引:2,他引:17
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The mechanism by which the binding of epidermal growth factor (EGF) to specific cell surface receptors induces a range of biological responses remains poorly understood. An important part of the study of signal transduction in this system involves the production of sufficient native and mutant EGF receptor species for X-ray crystallographic and spectroscopic analysis. Baculovirus vectors containing the cDNA encoding the human EGF receptor protein have here been utilized to infect insect cells. This results in expression of a 155-kb transmembrane protein which is recognized by four antibodies against different regions of the human EGF receptor. Studies with tunicamycin, monensen and endoglycosidase H show the difference in size between the recombinant and the native receptor is due to alterations in glycocsylation. Studies of [125I] EGF binding shows a Kd of 2 X 10(-9) M in intact infected insect cells which falls to 2 X 10(-7) M upon detergent solubilization. The recombinant protein exhibits an EGF-stimulated tyrosine protein kinase activity and an analysis of tryptic peptides shows that the phosphate acceptor sites are similar to those of the EGF receptor isolated from A431 cells. These observations indicate that functional EGF receptor can be expressed in insect cells, and furthermore, this system can be used for large-scale production. 相似文献
8.
A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis. 总被引:25,自引:11,他引:25
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A M Honegger D Szapary A Schmidt R Lyall E Van Obberghen T J Dull A Ullrich J Schlessinger 《Molecular and cellular biology》1987,7(12):4568-4571
Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction. 相似文献
9.
It has been shown that the transduction of the proliferative signal induced by EGF in ciliata Tetrahymena pyriformis cells is not connected with autophosphorylation of the receptor tyrosine kinases. The results obtained indicate that EGF in ciliata cells initiates the mitogenic pathway including the membrane proteins of the tyrosine kinases-like type (without tyrosine phosphorylation sites), adenylate cyclase, tyrosine- and Ca2+ -dependent ERK-like kinases. 相似文献
10.
The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma. 总被引:60,自引:13,他引:47
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B Margolis N Li A Koch M Mohammadi D R Hurwitz A Zilberstein A Ullrich T Pawson J Schlessinger 《The EMBO journal》1990,9(13):4375-4380
Phospholipase C-gamma (PLC-gamma) and GTPase activating protein (GAP) are substrates of EGF, PDGF and other growth factor receptors. Since either PLC-gamma or GAP also bind to the activated receptors it was suggested that their SH2 domains are mediating this association. We attempted to delineate the specific region of the EGF receptor that is responsible for the binding, utilizing EGF receptor mutants, PLC-gamma, and a bacterially expressed TRP E fusion protein containing the SH2 domains of GAP. As previously shown, tyrosine autophosphorylation of the wild-type receptor wsa crucial in mediating the association and in agreement, a kinase negative EGF receptor could bind PLC-gamma or TRP E GAP SH2, but only when cross tyrosine phosphorylated by an active EGF receptor kinase. The importance of autophosphorylation for association was confirmed by demonstrating that a carboxy-terminal deletion of the EGFR missing four autophosphorylation sites bound these proteins poorly. To study the role of EGF receptor autophosphorylation further, a 203 amino acid EGF receptor fragment was generated with cyanogen bromide that contained all known tyrosine autophosphorylation sites. This fragment bound both TRP E GAP SH2 and PLC-gamma but only when tyrosine phosphorylated. This data localizes a major binding site for SH2 domain containing proteins to the carboxy-terminus of the EGF receptor and points to the importance of tyrosine phosphorylation in mediating this association. 相似文献
11.
A population of stable NIH 3T3 transfectants with two molecular weight classes of membrane-bound EGF receptors encoded by a human EGF receptor cDNA has been identified and characterized. In addition to intact EGF receptors, these cells also express a molecule with an extensive cytosolic deletion. This deletion includes the ligand-activated intrinsic protein tyrosine kinase catalytic domain. Treatment with EGF caused dimerization of intact and truncated receptors, allowing us to assess protein tyrosine kinase activity in the heterodimer isolated from living cells. In contrast to homodimeric complexes with intact EGF receptor only, heterodimers were deficient in protein tyrosine kinase activity. Moreover, physical association between intact and truncated molecules suppressed receptor auto-phosphorylation by EGF receptor protein tyrosine kinase activated by antibody binding in vitro. Evidence presented here supports the idea that protein tyrosine kinase activation is facilitated by interaction between adjacent receptor molecules with intact catalytic domains. Furthermore, molecules with cytoplasmic deletions that are physically associated with kinase-active EGF receptors appear to behave as dominant negative mutations. The HerC cl cells used in this study were selected with methotrexate to amplify the EGF receptor cDNA, and in that sense may resemble certain tumor-derived cells characterized by overexpressed and rearranged EGF receptor genes. 相似文献
12.
Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation of proteins was examined in cells expressing wild-type (WT-EGFR) EGF receptors or EGF receptors truncated at residue 973 (973-EGFR). A much broader spectrum of tyrosine phosphorylated proteins was found following EGF treatment of 973-EGFR expressing cells compared with cells expressing wild-type receptors. Several additional ras GTPase activating protein-associated tyrosine phosphorylated proteins were found in EGF-treated 973-EGFR cells relative to WT-EGFR cells. Additional tyrosine-phosphorylated proteins were also found to co-immunoprecipitate with phospholipase C gamma 1 (PLC gamma 1) following EGF treatment of cells expressing 973-EGFR relative to cells expressing WT-EGFR. EGF-stimulated tyrosine phosphorylation of PLC gamma 1 was found in cells expressing WT-EGFR, but not in cells expressing 973-EGFR. WT-EGF receptor from EGF-treated cells bound well to bacterially expressed src homology (SH) regions of PLC gamma 1 and to a lesser extent to bacterially expressed GTPase activating protein SH regions. No binding of 973-EGF receptor to SH regions of either protein could be detected. EGF treatment greatly reduced the half-life of WT-EGFR, but had relatively little effect on the half-life of 973-EGFR. EGF induced internalization of 973-EGFR at a slower rate than WT-EGFR and caused the appearance of discrete receptor degradation products for both cell types. The data indicate that truncation of the EGF receptor at residue 973 alters receptor substrate specificity, decreases the rate of receptor internalization, and has an inhibitory effect on receptor degradation. 相似文献
13.
Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor. 总被引:16,自引:8,他引:8
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H App R Hazan A Zilberstein A Ullrich J Schlessinger U Rapp 《Molecular and cellular biology》1991,11(2):913-919
Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction. 相似文献
14.
Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor 总被引:16,自引:5,他引:11
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Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function. 相似文献
15.
Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha. 总被引:30,自引:6,他引:24
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I Lax A Johnson R Howk J Sap F Bellot M Winkler A Ullrich B Vennstrom J Schlessinger D Givol 《Molecular and cellular biology》1988,8(5):1970-1978
The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors. 相似文献
16.
17.
Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts 总被引:1,自引:0,他引:1
E Sturani R Zippel L Morello R Brambilla P M Comoglio L Alberghina 《Experimental cell research》1990,191(2):323-327
Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment. 相似文献
18.
Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing 总被引:100,自引:0,他引:100
A M Honegger T J Dull S Felder E Van Obberghen F Bellot D Szapary A Schmidt A Ullrich J Schlessinger 《Cell》1987,51(2):199-209
Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface. 相似文献
19.
Metabolic effects induced by epidermal growth factor (EGF) in cells expressing EGF receptor mutants 总被引:2,自引:0,他引:2
J C Scimeca R Ballotti F Alengrin A M Honegger A Ullrich J Schlessinger E V Obberghen 《The Journal of biological chemistry》1989,264(12):6831-6835
The epidermal growth factor (EGF) receptor tyrosine kinase activity is required for both the earliest EGF-stimulated post-binding events (enhancement of inositol phosphate formation and Ca2+ influx, activation of Na+/H+ exchange), and the ultimate EGF-induced mitogenic response. To assess the role of EGF receptor kinase in EGF-induced metabolic effects (2-deoxyglucose and 2-aminoisobutyric acid uptake), we used NIH3T3 cells (clone 2.2), which do not possess endogenous EGF receptors and which were transfected with cDNA constructs encoding either wild type or kinase-deficient human EGF receptor (HER). In addition, we tested the importance of three HER autophosphorylation sites (Tyr-1068, Tyr-1148, and Tyr-1173) in transduction of EGF-stimulated 2-deoxyglucose uptake. Taking our data together, we conclude the following: (i) HER tyrosine kinase activity is required to elicit EGF stimulation of both 2-deoxyglucose and 2-aminoisobutyric acid uptake; (ii) mutations on individual HER autophosphorylation sites, Tyr-1068, Tyr-1148, and Tyr-1173 do not impair EGF-stimulated 2-deoxyglucose uptake. 相似文献
20.
Boeri Erba E Matthiesen R Bunkenborg J Schulze WX Di Stefano P Cabodi S Tarone G Defilippi P Jensen ON 《Journal of proteome research》2007,6(7):2768-2785
Using stable isotope labeling and mass spectrometry, we performed a sensitive, quantitative analysis of multiple phosphorylation sites of the epidermal growth factor (EGF) receptor. Phosphopeptide detection efficiency was significantly improved by using the tyrosine phosphatase inhibitor sodium pervanadate to boost the abundance of phosphorylation of the EGF receptor. Nine phosphorylation sites (pT669, pS967, pS1002, pY845, pY974, pY1045, pY1086, pY1148, and pY1173) of EGF receptor were quantified from EGF-stimulated cells in suspension and adherent conditions. Our data sets revealed that EGF stimulation of adherent cells induced higher levels of tyrosine phosphorylation relative to EGF stimulation of suspended cells. In contrast, EGF stimulation of adherent cells induced lower levels of serine and threonine phosphorylation relative to EGF stimulation of suspended cells. These findings are consistent with the hypothesis that cellular adhesion modulates phosphorylation of plasma membrane receptor tyrosine kinases relevant for EGF-induced signal transduction processes. Furthermore, our results suggest that strong phosphatase inhibitors should be used to generate reference datasets in comparative phosphoproteomics experiments. 相似文献