Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P04626 (erbB-2)
5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.
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PMID:Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34cdc2. 132 Apr 11

The epidermal growth factor (EGF) receptor is both an activator and a target of growth factor-stimulated kinases involved in cellular signaling. Threonine-669 (T669) of the EGF receptor is phosphorylated in response to a wide variety of growth-modulating agents. MAP kinase is similarly phosphorylated as well as stimulated by growth activators, including EGF. To determine whether a MAP-type kinase is responsible for T669 kinase activity in EGF-stimulated 3T3-L1 cells, we partially purified and characterized the T669 peptide kinase. The results indicate that a MAP kinase phosphorylates the T669 peptide and raise the possibility that this enzyme may participate in a feedback loop, being activated by the EGF receptor and in turn phosphorylating the receptor.
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PMID:Epidermal growth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells is an EGF-stimulated "MAP" kinase. 184 6

Although signaling by the epidermal growth factor (EGF) receptor is thought to be dependent on receptor tyrosine kinase activity, it is clear that mitogen-activated protein (MAP) kinase can be activated by receptors lacking kinase activity. Since analysis of the signaling pathways used by kinase-defective receptors could reveal otherwise masked capabilities, we examined in detail the tyrosine phosphorylations and enzymes of the MAP kinase pathway induced by kinase-defective EGF receptors. Following EGF stimulation of B82L cells expressing a kinase-defective EGF receptor mutant (K721M), we found that ERK2 and ERK1 MAP kinases, as well as MEK1 and MEK2 were all activated, and SHC became prominently tyrosine-phosphorylated. By contrast, kinase-defective receptors failed to induce detectable phosphorylations of GAP (GTPase-activating protein), p62, JAK1, or p91STAT1, all of which were robustly phosphorylated by wild-type receptors. These data demonstrate that kinase-defective receptors induce several protein tyrosine phosphorylations, but that these represent only a subset of those seen with wild-type receptors. This suggests that kinase-defective receptors activate a heterologous tyrosine kinase with a specificity different from the EGF receptor. We found that kinase-defective receptors induced ErbB2/c-Neu enzymatic activation and ErbB2/c-Neu binding to SHC at a level even greater than that induced by wild-type receptors. Thus, heterodimerization with and activation of endogenous ErbB2/c-Neu is a possible mechanism by which kinase-defective receptors stimulate the MAP kinase pathway.
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PMID:An incomplete program of cellular tyrosine phosphorylations induced by kinase-defective epidermal growth factor receptors. 753 32

A single point mutation, Glu627--> Val, equivalent to the activating mutation in the Neu oncogene, was inserted in the transmembrane domain of the human epidermal growth factor (EGF) receptor. Unlike the wild type, Glu627-EGF receptor, transfected in NIH3T3 cells, gave rise to focal transformation and growth in agar even in the absence EGF. Constitutive activity of mutant EGF receptor amounted to 20% of that of wild type receptor stimulated by EGF. In addition, the mutant receptor was more sensitive to EGF, reaching maximum transforming activity at 5 ng/ml EGF. NIH3T3 cells expressing Glu627-EGF receptor showed a transformed phenotype and were not arrested in G0 upon serum deprivation. The mutant receptor was constitutively autophosphorylated, and several other cellular proteins were phosphorylated on tyrosine in absence of the ligand. Among these, the SHC adaptor protein was phosphorylated in absence of EGF, the other adaptor, GRB-2 was constitutively associated with the Glu627-EGF receptor in vivo and in vitro, and mitogen-activated protein kinase was constitutively phosphorylated. In contrast, other EGF receptor substrates, like phospholipase C gamma, were not phosphorylated in absence of EGF. The mutant receptor showed a higher sensitivity to cleavage by calpain both in absence and presence of EGF, appeared as a 170- and 150-kDa doublet in cell extracts, and a specific calpain inhibitor blocked the appearance of the 150-kDa form. Since the calpain cleavage site is located in the receptor cytoplasmic tail, this finding suggests that the Glu627 mutation induces a slightly different conformation in the EGF receptor intracellular domain. In conclusion, our data show that a point mutation in the EGF receptor transmembrane domain was able to constitutively activate the receptor and to induce transformation via constitutive activation of the Ras pathway.
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PMID:SHC and GRB-2 are constitutively by an epidermal growth factor receptor with a point mutation in the transmembrane domain. 764 41

A putative mitogen-activated protein kinase (MAPK) has recently been identified, which potentially phosphorylates the human epidermal growth factor (EGF) receptor at a physiological site (Thr-669) and is distinguished from other MAPKs/extracellular signal-regulated protein kinases (ERKs) on the basis of chromatographic, immunological, and kinetic data. Here we report that this newly discovered MAPK is physically associated with the EGF receptor in A431 cells and with the related receptor/tyrosine kinase HER2 (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. This human EGF receptor-associated kinase is characterized as a 40-kDa Thr-669 kinase that exists in a high molecular mass complex with the respective growth factor receptor. EGF treatment of A431 cells stimulates the tyrosine phosphorylation of p40 and increases Thr-669 kinase activity in p40-containing fractions. The 40-kDa kinase is recognized by affinity-purified polyclonal antibodies directed against the sea star p44mpk and a Pan-ERK antibody directed against the conserved subdomain VIII of MAPKs/ERKs, but is not recognized by antibodies selective for the rat p44erk1 and/or the p42mapk/erk2 isoforms, thus identifying the EGF receptor-associated kinase as a novel MAPK that may regulate receptor function in vivo.
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PMID:Identification of a human epidermal growth factor receptor-associated protein kinase as a new member of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase family. 768 42

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.
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PMID:Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF. 781 43

Insulin-stimulated glucose transport in adipocytes is mediated by the insulin receptor. To ascertain whether a related receptor could also trigger this response, the epidermal growth factor (EGF) receptor (EGFR) was introduced into adipocytes. 3T3-L1 fibroblasts were infected by a retroviral construct encoding either the full-length (WT) or a carboxy-terminal truncated (c'973) human EGFR; truncation of the amino acids distal to 973 removes all autophosphorylation motifs. After selection and conversion to adipocytes, the level of EGFR expression was retained in infectant adipocytes (150,000 and 250,000/cell, respectively), but not in the parental 3T3-L1 adipocytes (< 5000/cell). WT and c'973 EGFR exhibited ligand-dependent tyrosine kinase activity and stimulated mitogen-activated protein kinase activity equivalently; neither phosphorylated insulin receptor substrate-1. WT EGFR, but not c'973 EGFR, underwent ligand-induced autophosphorylation. EGF did not stimulate tyrosine phosphorylation of the insulin receptor or insulin receptor substrate-1. EGF had a minimal effect on glucose transport by parental 3T3-L1 adipocytes. Glucose transport in the WT EGFR adipocytes was stimulated equivalently by insulin and EGF; exposure to insulin and EGF in combination did not result in augmented transport. Glucose transport in the c'973 EGFR adipocytes was stimulated by insulin, but not by EGF. GLUT4 was translocated to the plasma membrane to a similar extent in response to insulin or EGF in the WT EGFR adipocytes; only insulin caused a significant GLUT4 translocation in the parental or c'973 EGFR adipocytes. These data suggest that the insulin and EGF signaling pathways that lead to glucose transport converge in these adipocytes down-stream of the insulin receptor, and that activation of this pathway requires signaling motifs in the carboxy-terminus of the EGFR. This model system represents a novel approach with which to dissect signal transduction pathways in terminally differentiated adipocytes.
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PMID:Epidermal growth factor (EGF) receptor carboxy-terminal domains are required for EGF-induced glucose transport in transgenic 3T3-L1 adipocytes. 783 73

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.
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PMID:Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669. 794 18

The c-erbB-2 proto-oncogene encodes a receptor tyrosine kinase (RTK) closely related to the epidermal growth factor receptor (EGFR). Overexpression of erbB-2 occurs in approximately 20% of human breast tumours, where increased expression correlates with poor patient prognosis. The EGFR is coupled to the Ras signalling pathway by interaction with the adaptor protein Grb2, and Sos, a Ras GDP-GTP exchange factor. In this study, activation of the erbB-2 receptor and its association with Grb2 and Sos was investigated in breast cancer cell lines which overexpress erbB-2. The receptor was found to be tyrosine phosphorylated in all cell lines in which it is overexpressed. Western blotting of Grb2 and Sos immuneprecipitates from such cells revealed co-precipitation of erbB-2, demonstrating association of the Grb2/Sos complex with erbB-2 in vivo. Furthermore, a fusion protein containing only the SH2 domain of Grb2 bound to erbB-2 immobilized on nitrocellulose, indicating that association with Grb2 is direct and mediated by the SH2 domain of Grb2. The degree of association between the erbB-2 receptor and Grb2 in vivo was related to erbB-2 overexpression, and MAP kinase, which functions downstream from Ras, displayed markedly increased activity in cell lines overexpressing erbB-2. These results demonstrate that erbB-2 is coupled to Ras signalling via the Grb2/Sos complex, and that overexpression of this receptor in breast cancer cells leads to amplification of the Ras signalling pathway.
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PMID:Activation of the Ras signalling pathway in human breast cancer cells overexpressing erbB-2. 797 Jul 20

The importance of epidermal growth factor (EGF) receptor expression level and autophosphorylation sites in src homology and collagen protein (SHC) tyrosine phosphorylation has been studied. In contrast to EGF-induced tyrosine phosphorylation of the GTPase-activating protein for ras (rasGAP) and phospholipase C-gamma 1 (PLC-gamma 1), SHC tyrosine phosphorylation occurs at a very low receptor density in parental NIH3T3 mouse fibroblasts expressing less than 1 x 10(4) EGF receptors per cell. In transfected NIH3T3 cells expressing human EGF receptors (approximately 4 x 10(5) receptors per cell), maximal levels of SHC and PLC-gamma 1 tyrosine phosphorylation occur when approximately 4 x 10(4) receptors or more are occupied by ligand. At lower levels of receptor occupancy only SHC phosphorylation was significant. Also, EGF treatment of mouse keratinocytes, which represent a physiological target of EGF, express a low number of EGF receptors (approximately 2 x 10(4) receptors per cell), and stringently require EGF to grow, results in intense SHC tyrosine phosphorylation, compared to rasGAP or PLC-gamma 1. SHC is also efficiently tyrosine phosphorylated by an EGF receptor deletion mutant (Dc214) that is devoid of autophosphorylation sites, but which remains mitogenically responsive to EGF. The EGF receptor mutant Dc214 is able to activate the ras guanine nucleotide exchanger and phosphorylate mitogen-activated protein kinase (MAPK), presumable as a result of complex formation between tyrosine phosphorylated SHC and GRB2. These results indicate that potent EGF-induced SHC tyrosine phosphorylation can be triggered in cells having relatively few receptors. Also, our data show that EGF receptors are able to phosphorylate SHC, activate the exchange of guanine nucleotide on ras and phosphorylate MAPK by a mechanism that does not require receptor autophosphorylation sites and, therefore, the src homology 2 (SH2):phosphotyrosine-dependent interaction of SHC or GRB2 with the EGF receptor.
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PMID:Potent SHC tyrosine phosphorylation by epidermal growth factor at low receptor density or in the absence of receptor autophosphorylation sites. 803 6


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