Temperature dependence of the epidermal growth factor receptor signaling network can be accounted for by a kinetic model.

Stimulation of isolated hepatocytes with epidermal growth factor (EGF) causes rapid tyrosine phosphorylation of the EGF receptor (EGFR) and adapter/target proteins, which was monitored with 1 and 2 s resolution at 37, 20, and 4 degrees C. The temporal responses detected for multiple signaling proteins involve both transient and sustained phosphorylation patterns, which change dramatically at low temperatures. To account quantitatively for complex responses, we employed a mechanistic kinetic model of the EGFR pathway, formulated in molecular terms as cascades of protein interactions and phosphorylation and dephosphorylation reactions. Assuming differential temperature dependencies for different reaction groups, such as SH2 and PTB domain-mediated interactions, the EGFR kinase, and the phosphatases, good quantitative agreement was obtained between computer-simulated and measured responses. The kinetic model demonstrates that, for each protein-protein interaction, the dissociation rate constant, k(off), strongly decreases at low temperatures, whereas this decline may or may not be accompanied by a large decrease in the k(on) value. Temperature-induced changes in the maximal activities of the reactions catalyzed by the EGFR kinase were moderate, compared to such changes in the V(max) of the phosphatases. However, strong changes in both the V(max) and K(m) for phosphatases resulted in moderate changes in the V(max)/K(m) ratio, comparable to the corresponding changes in EGFR kinase activity, with a single exception for the receptor phosphatase at 4 degrees C. The model suggests a significant decrease in the rates of the EGF receptor dimerization and its dephosphorylation at 4 degrees C, which can be related to the phase transition in the membrane lipids. A combination of high-resolution experimental monitoring and molecular level kinetic modeling made it possible to quantitatively account for the temperature dependence of the integrative signaling responses.     

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.     

Enhanced erythropoietin heterogeneity in a CHO culture is caused by proteolytic degradation and can be eliminated by a high glutamine level

The molecular heterogeneity of recombinant humanerythropoietin (EPO) increased during the course of abatch culture of transfected Chinese hamster ovary(CHO) cells grown in serum-free medium. This wasshown by both an increased molecular weight and pIrange of the isolated EPO at the end of the culture. However, analysis of the N-glycan structures of themolecule by fluorophore-assisted carbohydrateelectrophoresis (FACE) and HPLC anion exchangechromatography indicated a consistent pattern ofglycosylation. Seven glycoforms were identified, thepredominant structure being a fully sialylatedtetra-antennary glycan. The degree of sialylationwas maintained throughout the culture. Analysis ofthe secreted EPO indicated a time-dependent increasein the molecular weight band width of the peptideconsistent with proteolytic degradation. A highglutamine concentration (16–20 mM) in the culturedecreased the apparent degradation of the EPO.     

The epidermal growth factor receptor from prostate cells is dephosphorylated by a prostate-specific phosphotyrosyl phosphatase.

Human prostatic acid phosphatase (PAcP) has been found to have phosphotyrosyl-protein phosphatase activity (H. C. Li, J. Chernoff, L. B. Chen, and A. Kirschonbaun, Eur. J. Biochem. 138:45-51, 1984; M.-F. Lin and G. M. Clinton, Biochem. J. 235:351-357, 1986) and has been suggested to negatively regulate phosphotyrosine levels, at least in part, by inhibition of tyrosine protein kinase activity (M.-F. Lin and G. M. Clinton, Adv. Protein Phosphatases 4:199-228, 1987; M.-F. Lin, C. L. Lee, and G. M. Clinton, Mol. Cell. Biol. 6:4753-4757, 1986). We investigated the molecular interaction of PAcP with a specific tyrosine kinase, the epidermal growth factor (EGF) receptor, from prostate carcinoma cells. Of several proteins phosphorylated in membrane vesicles from prostate carcinoma cells, PAcP selectively dephosphorylated the EGF receptor. The prostate EGF receptor was more efficiently dephosphorylated by PAcP than by another phosphotyrosyl phosphatase, potato acid phosphatase. Further characterization of the interaction of PAcP with the EGF receptor revealed that the optimal rate of dephosphorylation occurred at neutral rather than at acid pH. Thus, the enzyme that we formerly referred to as PAcP we now call prostatic phosphotyrosyl-protein phosphatase. Hydrolysis of phosphate from tyrosine residues in the immunoprecipitated EGF receptor catalyzed by purified prostatic phosphotyrosyl-protein phosphatase caused a 40 to 50% decrease in the receptor tyrosine kinase activity with angiotensin as the substrate. In contrast, autophosphorylation of the receptor was associated with an increase in tyrosine kinase activity.     

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7 × 10⁵ receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-α). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.     

Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. Evidence for a common mechanism

Previously, we and others have shown that epidermal growth factor (EGF) stimulates the synthesis of its own receptor and the accumulation of EGF receptor mRNA. Here, we demonstrate that the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), like EGF, also stimulates receptor synthesis in the human breast carcinoma cell line, MDA468 cells. The receptor synthesis rate increased 5-fold with a peak at 8 h after exposure to TPA with half-maximal stimulation at a dose of 5 ng/ml TPA. This stimulation of receptor synthesis occurred despite a 30% decrease in general cellular protein synthesis. The increased receptor synthesis rate resulted in the accumulation of 60% more receptor protein as determined by quantitative immunoblotting using a newly developed monoclonal antibody, H9B4. Although TPA treatment resulted in an immediate loss of high affinity EGF-binding sites, the long-term effect was an increase in both the low and high affinity binding sites. The effects of EGF and TPA on receptor synthesis were not additive. Furthermore, down-regulation of protein kinase C (the Ca2+/phospholipid-dependent enzyme) by long-term TPA treatment resulted in cells unable to respond to the stimulatory effects of both TPA and EGF on receptor synthesis. Nevertheless, the TPA-pretreated cells were still growth-inhibited by EGF. These results suggest that the stimulatory effect of EGF on receptor synthesis requires protein kinase C, whereas the inhibitory effect of EGF on the proliferation of these cells does not. Although we confirmed that EGF stimulated the incorporation of phosphate into phosphatidylinositol in A431 cells, it failed to do so in the MDA468 cells. Thus, in MDA468 cells, EGF may require protein kinase C for part of its action, but we could not demonstrate an associated activation of phosphatidylinositol turnover by EGF. The exact mechanism of involvement of protein kinase C in EGF action is still not clear.     

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.     

Cell movement elicited by epidermal growth factor receptor requires kinase and autophosphorylation but is separable from mitogenesis

The EGF receptor (EGFR) upon activation signals increased cell movement. However, the domains within the receptor, and the pathway which trigger movement are undefined. We expressed EGFR mutants at physiologic levels in receptor-devoid NR6 cells to investigate this biologic response. The receptors possessed kinase activity and underwent autophosphorylation as predicted by primary amino acid sequence. EGF-induced cell motility was assessed in vitro by excess migration into an acellular area and colony scatter in the presence of saturating concentrations of EGF. Wild-type (WT)-EGFR signaled increased motility. However, replacing the conserved lysine721 with methionine resulted in a kinase-inactive receptor which did not elicit movement. Removal of the entire terminus by truncation (c'973) also abrogated ligand-induced motility. Thus, we concentrated on the carboxy- terminal domains. EGF-induced movement was seen with a less-truncated mutant (c'1000) that contained a single autophosphorylated tyrosine (tyrosine992). Other mutants, c'991 and c'1000F992, in which this tyrosine was removed did not signal motility. Fusion mutants which presented other autophosphorylated tyrosine domains also exhibited EGF- induced movement. These findings suggested that the presence of both an autophosphorylated tyrosine signaling domain and the kinase activity are necessary for this biologic response. All kinase-positive mutants signaled cell proliferation but only those that contained autophosphorylatable tyrosines induced movement. The motility responses mediated by these EGFR were identical in the presence or absence of mitomycin-C, at a dose (0.5 micrograms/ml) which completely inhibited cell proliferation. On the other side, D-actinomycin (50 ng/ml) blocked EGF-induced motility but did not affect thymidine incorporation. Thus, EGF-induced mitogenesis and cell motility are mediated through different pathways.     

Modulation of drug-induced cytotoxicity by a bispecific monoclonal antibody that recognizes the epidermal growth factor receptor and doxorubicin

A hybrid hybridoma secreting a bispecific hybrid mAb (bsmAb), which recognizes both the epidermal growth factor receptor (EGF-R) and the drug doxorubicin, was produced by somatic hybridization of two hybridomas. The bsmAb obtained was able to retarget doxorubicin cytotoxicity in vitro specifically on EGF-R-positive cells exerting at the same time an antidotal effect on cells that did not overexpress the EGF-R. Distribution studies in mice indicate that the bsmAb selectively delivers the drug to tumour cells and modifies doxorubicin biodistribution with a statistically significant decrease of drug concentration in the intestine, which is the main target of early anthracycline toxicity. In keeping with this finding is the remarkable antidotal activity exerted by bsmAb in mice treated with doxorubiein, which is proved by retardation in loss of body weight and mortality. The effectiveness on tumour growth of the mAb followed by the administration of doxorubicin appears to be equal to that of the drug alone; however, the bsmAb exerts a remarkable antidotal activity.     

Evidence that the hepatic asialoglycoprotein receptor is internalized during endocytosis and that receptor recycling can be uncoupled from endocytosis at low temperature

Rat hepatocytes in the continuous presence of [³H]asialo-orosomucoid quickly establish a steady state number of free and occupied surface receptors and rate of endocytosis. These values do not change even though many times more glycoprotein is internalized than there are surface receptors per cell. However, when cells endocytose only one round of surface bound [³H]asialo-orosomucoid at 37°C the internalization of glycoprotein is about 5 times faster than the increase of functional receptors on the cell surface. At 18°C new surface receptors appear at only 6% of the rate of internalization of pre-bound asialoglycoprotein. The results suggest that reutilization of asialoglycoprotein receptors is preferentially inhibited at low temperature and that receptor-ligand complexes enter the cell.     

A chromosomal rearrangement hotspot can be identified from population genetic variation and is coincident with a hotspot for allelic recombination

Insights into the origins of structural variation and the mutational mechanisms underlying genomic disorders would be greatly improved by a genomewide map of hotspots of nonallelic homologous recombination (NAHR). Moreover, our understanding of sequence variation within the duplicated sequences that are substrates for NAHR lags far behind that of sequence variation within the single-copy portion of the genome. Perhaps the best-characterized NAHR hotspot lies within the 24-kb-long Charcot-Marie-Tooth disease type 1A (CMT1A)-repeats (REPs) that sponsor deletions and duplications that cause peripheral neuropathies. We investigated structural and sequence diversity within the CMT1A-REPs, both within and between species. We discovered a high frequency of retroelement insertions, accelerated sequence evolution after duplication, extensive paralogous gene conversion, and a greater than twofold enrichment of SNPs in humans relative to the genome average. We identified an allelic recombination hotspot underlying the known NAHR hotspot, which suggests that the two processes are intimately related. Finally, we used our data to develop a novel method for inferring the location of an NAHR hotspot from sequence variation within segmental duplications and applied it to identify a putative NAHR hotspot within the LCR22 repeats that sponsor velocardiofacial syndrome deletions. We propose that a large-scale project to map sequence variation within segmental duplications would reveal a wealth of novel chromosomal-rearrangement hotspots.     

Mapping and sequencing of a gene from myxoma virus that is related to those encoding epidermal growth factor and transforming growth factor alpha.

Myxoma virus, a Leporipoxvirus and agent of myxomatosis, was shown to possess a gene with the potential to encode an epidermal growth factorlike factor. Its relationship to other members of this family, including the poxvirus growth factors from Shope fibroma virus and vaccinia virus, was analyzed. Alignment of DNA sequences and related open reading frames of myxoma virus and Shope fibroma virus indicated colinearity of genes between these poxviruses.     

Placental overgrowth and fertility defects in mice with a hypermorphic allele of epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) is a member of the ERBB family of receptor tyrosine kinases that has been shown to play an important developmental and physiologic role in many aspects of pregnancy. We have previously shown in mice that Egfr ^tm1Mag nullizygous placentas have fewer proliferative trophoblasts than wild-type and exhibit strain-specific defects in the spongiotrophoblast and labyrinth layers. In this study we used mice with the hypermorphic Egfr ^Dsk5 allele to study the effects of increased levels of EGFR signaling on placental development. On three genetic backgrounds, heterozygosity for Egfr ^Dsk5 resulted in larger placental size with a more prominent spongiotrophoblast layer and increased expression of glycogen cell-specific genes. The C3HeB/FeJ strain showed additional placental enlargement of Egfr ^Dsk5 homozygotes with a significant number of homozygous embryos dying prior to 15.5 days post-coitus (dpc). We also observed strain-specific subfertility in Egfr ^Dsk5 heterozygous females and pregnancy loss was dependent on maternal factors rather than embryo genotype. Higher levels of phospho-EGFR were detected in the uterus of Egfr ^Dsk5 heterozygotes but the structure of Egfr ^Dsk5 heterozygous nonpregnant uteri appeared similar to wild-type. Collectively, our results demonstrate that mice with increased levels of EGFR signaling exhibit an extensive level of genetic background-dependent phenotypic variability. In addition, EGFR promotes growth of the placental spongiotrophoblast layer in mice, and EGFR expressed in the uterine stroma may play an underappreciated role in preparation of the uterus for embryo implantation.     

Activation of Rho is required for ligand-independent oncogenic signaling by a mutant epidermal growth factor receptor

Mutations in the epidermal growth factor receptor have been identified in several human tumor types, including gliomas. These receptor mutants have deletions in their extracellular ligand-binding domains and are, therefore, no longer regulated by ligand, resulting in constitutive activation of the receptor kinase. These mutants have been proposed to transduce oncogenic signals via ligand-independent signaling pathways. Avian viral homologues of these oncogenic epidermal growth factor receptors exhibit structurally homologous deletions and form tumors in chickens. One such mutant, S3v-ErbB, transforms fibroblasts in vitro, and transformation has been correlated with the formation of a novel tyrosine phosphoprotein complex. V-ErbB-mediated complex formation and transformation have been shown to occur independently of Ras activation. The major aims of this study are to further characterize this ligand-independent v-ErbB oncogenic signaling pathway. Here we show that both v-ErbB-mediated phosphoprotein complex formation and transformation are inhibited by a dominant negative mutant of Rho. This inhibition is specific for dominant negative Rho; dominant negative mutants of Rac and Cdc42 have no effect on transformation or on tyrosine phosphorylation of the phosphoprotein complex. Based on these observations, we propose that S3v-ErbB stimulates a Rho-dependent tyrosine kinase, resulting in complex formation and ultimately oncogenic transformation.     

Generation and validation of mice carrying a conditional allele of the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is important for normal homeostasis in a variety of tissues and, when abnormally expressed or mutated, contributes to the development of many diseases. However, in vivo functional studies are hindered by the lack of adult mice lacking EGFR because of the pre‐ and postnatal lethality of EGFR deficient mice. We generated a conditional allele of Egfr (Egfr^tm1Dwt) by flanking exon 3 with loxP sites in order to investigate tissue‐specific functions of this widely expressed receptor tyrosine kinase. The activity of the Egfr^tm1Dwt allele is indistinguishable from wildtype Egfr. Conversely, the Egfr^Δ allele, generated by Cre‐mediated deletion of exon 3 using the germline EIIa‐Cre transgenic line, functions as a null allele. Egfr^Δ/Δ embryos that have complete ablation of EGFR activity and die at mid‐gestation with placental defects identical to those reported for mice homozygous for the Egfr^tm1Mag null allele. We also inactivated the Egfr^tm1Dwt allele tissue‐specifically in the skin epithelium using the K14‐Cre transgenic line. These mice were viable but exhibited wavy coat hair remarkably similar to mice homozygous for the Egfr^wa2 hypomorphic allele or heterozygous for the Egfr^Wa5 antimorphic allele. These results suggest that the hairless phenotype of Egfr nullizygous mice is not solely due to absence of EGFR in the epithelium, but that EGFR activity is required also in skin stromal cells for normal hair morphogenesis. This new mouse model should have wide utility to inactivate Egfr conditionally for functional analysis of EGFR in adult tissues and disease states. genesis 47:85–92, 2009. © 2008 Wiley‐Liss, Inc.     

Cell cycle- and apoptosis-regulatory protein-1 is involved in apoptosis signaling by epidermal growth factor receptor

CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a pan-ErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results in elevated CARP-1 levels, whereas antisense-dependent depletion of CARP-1 causes inhibition of apoptosis by ERRP. CARP-1 is a tyrosine-phosphorylated protein, and ERRP treatments cause elevated tyrosine phosphorylation of CARP-1. CARP-1 contains multiple, nonoverlapping apoptosis-inducing subdomains; one such subdomain is present within amino acids 1-198. Wild-type or CARP-1-(1-198) proteins that have substitution of tyrosine 192 to phenylalanine abrogate apoptosis by ERRP. In addition, apoptosis mediated by wild type or CARP-1-(1-198), and not CARP-1-(1-198(Y192F)), results in activation of caspase-9 and increased phosphorylation of p38 MAPK. However, the expression of dominant-negative forms of p38 MAPK activators MKK3 or MKK6 proteins inhibits apoptosis induced by both the full-length and truncated (amino acids 1-198) proteins. Together, data demonstrate that tyrosine 192 of CARP-1 is a target of apoptosis signaling, and CARP-1, in turn, promotes apoptosis by activating p38 MAPK and caspase-9.     

Insulin-like growth factor I-mediated degradation of insulin receptor substrate-1 is inhibited by epidermal growth factor in prostate epithelial cells

We have sought to determine whether insulin-like growth factor I (IGF-I) regulates the levels of insulin receptor substrate-1 (IRS-1) in prostate epithelial cells. Exposure of prostate epithelial cells to IGF-I in the absence of other growth factors leads to a reduction in IRS-1 levels. Ubiquitin content of IRS-1 is increased in the presence of IGF-I, and inhibitors of the proteasome prevented the reduction of IRS-1 levels seen following IGF-I exposure. These results imply that IRS-1 is targeted to the proteasome upon exposure to IGF-I. The addition of epidermal growth factor (EGF) maintained IRS-1 levels even in the presence of IGF-I and inhibits IGF-I-dependent ubiquitination of IRS-1. Thus, these two growth factors, IGF-I and EGF, had antagonistic effects on IRS-1 protein levels in prostate epithelial cells. This regulation of IRS-1 reveals a novel level of cross-talk between the IGF-I and EGF signal pathways, which may have implications in tumors that harbor activating mutations in the EGF receptor.     

Separation and characterization of a phosphatidylinositol kinase activity that co-purifies with the epidermal growth factor receptor

Two retroviral protein-tyrosine kinases, v-src and v-ros, have been reported to possess phosphatidylinositol (PtdIns) kinase activity. Because the epidermal growth factor (EGF) receptor is a protein-tyrosine kinase with structural homology to p60v-src and because EGF stimulates PtdIns turnover in A431 cells, the EGF receptor has been examined for PtdIns kinase activity. Preparations of the EGF receptor, isolated from A431 cells and purified by two different methods of affinity chromatography, possessed an associated PtdIns kinase activity. This activity which co-purified with the EGF receptor represented only about 2% of the total PtdIns kinase activity of A431 membranes, and there was no correlation between the number of EGF receptors and the amount of PtdIns kinase activity in membranes from various cell types. A peptide substrate, angiotensin II, and PtdIns did not compete with each other as substrates for the protein-tyrosine and PtdIns kinase activities of the EGF receptor. When self-phosphorylated EGF receptor was fractionated by Sephacryl S-300 gel permeation chromatography, the peak of PtdIns kinase activity was separated from the comigrating peak of protein-tyrosine kinase activity and the self-phosphorylated EGF receptor. These results indicate that the protein-tyrosine kinase and PtdIns kinase activities which co-purify with the EGF receptor reside on different molecules. Angiotensin II and PtdIns did not compete as substrates for p60v-src isolated by immunoabsorption with a monoclonal antibody, suggesting that PtdIns kinase activity may also not be intrinsic to p60v-src.     

Identification of an N-cadherin motif that can interact with the fibroblast growth factor receptor and is required for axonal growth

In this study, we show that the neurite outgrowth response stimulated by N-cadherin is inhibited by a recently developed and highly specific fibroblast growth factor receptor (FGFR) antagonist. To test whether the N-cadherin response also requires FGF function, we developed peptide mimetics of the receptor binding sites on FGFs. Most mimetics inhibit the neurite outgrowth response stimulated by FGF in the absence of any effect on the N-cadherin response. The exceptions to this result were two mimetics of a short FGF1 sequence, which has been shown to interact with the region of the FGFR containing the histidine-alanine-valine motif. These peptides inhibited FGF and N-cadherin responses with similar efficacy. The histidine-alanine-valine region of the FGFR has previously been implicated in the N-cadherin response, and a candidate interaction site has been identified in extracellular domain 4 of N-cadherin. We now show that antibodies directed to this site on N-cadherin inhibit the neurite outgrowth response stimulated by N-cadherin, and peptide mimetics of the site inhibit N-cadherin and FGF responses. Thus, we can conclude that N-cadherin contains a novel motility motif in extracellular domain 4, and that peptide mimetics of this motif can interact with the FGFR.     

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.