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 (EGFR) and the erbB-2 gene product, gp185erbB-2, exhibit distinct abilities to stimulate mitogenesis in different target cells. By using chimeric molecules between these two receptors, we have previously shown that their intracellular juxtamembrane regions are responsible for this specificity. Here we describe a genetically engineered EGFR mutant containing a threonine for arginine substitution at position 662 in the EGFR juxtamembrane domain, corresponding to threonine 694 in gp185erbB-2. This mutant, designated EGFRThr662, displayed affinity for EGF binding and catalytic properties that were indistinguishable from those of the wild type EGFR. However, EGFRThr662 behaved much as gp185erbB-2 in a number of bioassays which readily distinguish between the mitogenic effects of EGFR and gp185erbB-2. Moreover, significant differences were detected in the pattern of intracellular proteins phosphorylated on tyrosine in vivo by EGFR and EGFRThr662 in response to EGF. Thus, small differences in the primary sequence of two closely related receptors have dramatic effects on their ability to couple with mitogenic pathways.
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PMID:A single amino acid substitution is sufficient to modify the mitogenic properties of the epidermal growth factor receptor to resemble that of gp185erbB-2. 135 64

We inoculated rabbits with synthetic phosphopeptides, duplicating a major autophosphorylation site of the c-erbB-2 protooncogene product. The rabbits produced antisera that, after reverse immunoaffinity purification, selectively recognize the erbB-2 protein in its enzymatically active configuration. These anti-phosphopeptide antisera identify a subset of erbB-2-positive human cell lines wherein the protein is constitutively active as a tyrosine kinase. Synthetic phosphopeptides incorporating informative protein phosphorylation sites may prove useful for generating antibodies that indicate the activation state of additional tyrosine kinases and perhaps other proteins phosphorylated on serine and threonine residues.
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PMID:Synthetic phosphopeptide immunogens yield activation-specific antibodies to the c-erbB-2 receptor. 135 39

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.
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PMID:Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins. 137 Jun 7

Okadaic acid, a potent tumor promoter and inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types. The increases are limited to phosphoserine and phosphothreonine residues. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a distinct tumor promoter and protein kinase C activator, also induces serine/threonine phosphorylation of the EGF receptor and is known to modulate receptor functions. Comparison of okadaic acid and TPA influences on the EGF receptor show significant differences. Okadaic acid did not promote phosphorylation of Thr-654, a major site of TPA-induced phosphorylation. However, other sites of phosphorylation were similar for the two tumor promoters. In vitro experiments with purified protein phosphatase 2A demonstrate the insensitivity of Thr-654 phosphorylation, which regulates EGF receptor function, to dephosphorylation by this okadaic acid-sensitive protein phosphatase. In contrast to TPA, okadaic acid did not attenuate the tyrosine kinase activity or ligand binding capacity of the EGF receptor. However, okadaic acid did produce a decrease in EGF-stimulated inositol phosphate formation in a manner distinct from that of TPA.
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PMID:Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. Comparison with receptor phosphorylation and functions affected by another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. 165 56

P185 is a receptor-like protein encoded by the neu/erbB-2 proto-oncogene. A point mutation in the transmembrane domain renders this protein oncogenic. We report here that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates the phosphorylation of the normal neu protein (p185) and the oncogenic neu protein (p185*). The increased phosphorylation occurs mainly on serine and threonine residues. Phosphate labeling experiments showed that TPA causes a reduction of basal phosphotyrosine in p185 but not p185*. Immunoblotting with antiphosphotyrosine antibody yielded similar results. TPA also inhibited tyrosine phosphorylation of p185* in an in vitro immune complex kinase assay. These data suggest that protein kinase C, the receptor for TPA, regulates p185 function through serine or threonine phosphorylation.
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PMID:TPA inhibits the tyrosine kinase activity of the neu protein in vivo and in vitro. 167 82

Staurosporine is a potent microbial inhibitor of a number of protein kinases, including protein kinase C, cyclic AMP-dependent kinase, and the tyrosine kinase pp60src. We have used staurosporine to investigate the role of phosphorylation in the regulation of the epidermal growth factor (EGF) receptor in both human epidermal carcinoma A431 cells and mouse Swiss 3T3 fibroblasts. We report here that staurosporine treatment causes enhancement in high affinity EGF binding and a decrease in the phosphorylation state of the unstimulated receptor at a number of residues, including threonine 669. Staurosporine also antagonizes the inhibition of high affinity EGF binding and the increase in phosphorylation state of the unstimulated EGF receptor by phorbol esters and the calcium ionophore A23187. Staurosporine is an effective inhibitor of the EGF-stimulated receptor tyrosine kinase in vitro and thus does not enhance EGF stimulation of EGF receptor autophosphorylation in vivo. These results suggest that phosphorylation plays a major role in the regulation of the high affinity binding state of the EGF receptor in both unstimulated and mitogenically activated cells.
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PMID:Regulation of the epidermal growth factor receptor by growth-modulating agents: effects of staurosporine, a protein kinase inhibitor. 168 32

To investigate the functional significance of epidermal growth factor (EGF) receptor phosphorylation, experimental systems were explored in which receptor phosphorylation on tyrosine and serine/threonine could be differentially stimulated. Exposure of A431 cells to 20 nM EGF at 37 degrees C results in phosphorylation of serine, threonine, and tyrosine sites on the receptor. Monoclonal antibody (mAb) 225 binds to the EGF receptor with affinity comparable to EGF and competes with the binding of EGF. Exposure of A431 cells to 20 nM EGF in the presence of 300 nM anti-EGF receptor mAb 225 (15-fold excess) selectively activated serine and threonine phosphorylation of the receptor, but not tyrosine phosphorylation. This observation indicates that EGF-mediated receptor phosphorylation on tyrosine and on serine/threonine residues is dissociable. The intracellular fate of the EGF receptor was examined under conditions that produce different phosphorylation states of receptor amino acids. Exposure of A431 cells to EGF decreased the half-life (T1/2) of the receptor from 17.8 h to 5.6 h, with activation of tyrosine, serine, and threonine phosphorylation. Incubation with mAb 225 augmented the degradation rate (T1/2 = 8.5 h) without activation of receptor phosphorylation. Concurrent exposure to EGF (20 nM) and mAb 225 (300 nM) resulted in comparable enhanced degradation (T1/2 = 9.5 h), with increased phosphorylation only on serine and threonine residues. These results suggest that serine/threonine phosphorylation is irrelevant to the augmentation of receptor degradation. Methylamine, an inhibitor of lysosomal function that did not affect phosphorylation of the EGF receptor, completely protected EGF receptors from rapid degradation induced by EGF, but it only slightly altered the rate of EGF receptor degradation elicited by mAb 225 or by EGF plus 15-fold excess mAb 225. In contrast, mAb 455, which binds to the receptor but does not inhibit EGF binding and EGF-induced activation of phosphorylation on tyrosine, serine, and threonine residues, did not influence EGF-induced rapid, methylamine sensitive degradation of EGF receptor. The results suggest that when EGF receptors are internalized under conditions that do not activate the receptor tyrosine kinase, they are sorted into a nonlysosomal pathway that differs from the methylamine-sensitive lysosomal pathway traversed following activation by EGF. The data indicate the possibility of a function for tyrosine kinase activation and tyrosine autophosphorylation in determining the lysosomal intracellular pathway of EGF receptor processing and degradation.
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PMID:Modulation of tyrosine, serine, and threonine phosphorylation and intracellular processing of the epidermal growth factor receptor by antireceptor monoclonal antibody. 168 18

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.
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PMID:cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation. 169 23

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


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