Epidermal Growth Factor Receptor Protein Tyrosine Kinase Research Articles

MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis.

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.

Upregulation of programmed death ligand-1 by radiation through the pathways downstream of epidermal growth factor receptor in glioma

Objective To investigate the role of epidermal growth factor receptor (EGFR) signaling pathway in the upregulation of programmed death ligand-1 (PD-L1) caused by radiation (RT) and explore new strategies for the treatment of glioma. Methods Tissue microarrays (TMA) which contained glioma cancer specimens from 64 patients was used to examine the correlation between PD-L1 and EGFR levels. Furthermore, we performed experiments in vitro to investigate the role of EGFR pathway in RT-induced PD-L1 expression using human glioma cell lines U87 and U251. Results High PD-L1 expression was identified in 28 (43.8%) of 64 glioma specimens, and high EGFR expression was evident in 31 (48.4%) of 64 glioma specimens. There was a significant correlation between PD-L1 and EGFR protein levels in glioma specimens (χ2=5.000, P=0.025). High PD-L1 expression was significantly associated with patients’ age (χ2=11.460, P=0.001) and grade (χ2=7.110, P=0.008). The protein and mRNA levels of PD-L1 were both significantly upregulated by RT (t=10.292, P=0.000; t=52.171, P=0.000). Only the phosphorylated EGFR and janus protein tyrosine kinase (JAK) 2 were induced by RT (t=10.485, P=0.001; t=8.911, P=0.001). Besides, inhibition of EGFR pathway could abrogate the upregulation of PD-L1 caused by RT (t=0.758, P=0.490). The combination of RT with EGFR inhibitor had the same effect on enhancing antitumor immune response compared with the combination of RT with PD-L1 neutralizing antibody (Ab). However, RT combined with PD-L1 Ab plus AG490 did not further enhance the effect. Conclusion RT could upregulate the PD-L1 expression through the pathways downstream of EGFR in glioma. RT combined with EGFR inhibitor may become a new strategy for glioma. Key words: Programmed death ligand-1; Epidermal growth factor receptor; Radiotherapy; Combination therapy; Glioma

Role of Epidermal Growth Factor Receptor in Ovine Fetal Pulmonary Vascular Remodeling Following Exposure to High Altitude Long-Term Hypoxia

High altitude long-term hypoxia (LTH) in the fetus may result in pulmonary vascular smooth muscle cell (PVSMC) proliferation and pulmonary vascular remodeling. Our objective was to determine if epidermal growth factor receptor (EGFR) is involved in hypoxia induced PVSMC proliferation or in pulmonary vascular remodeling in ovine fetuses exposed to high altitude LTH. Fetuses of pregnant ewes that were held at 3820-m altitude from *30 to 140 days (LTH) gestation and sea level control pregnant ewes were delivered near term. Morphometric analyses and immunohistochemistry were done on fetal lung sections. Pulmonary arteries of LTH fetuses exhibited medial wall thickening and distal muscularization. Western blot analyses done on protein isolated from pulmonary arteries demonstrated an upregulation of EGFR. This upregulation was attributed in part to PVSMC in the medial wall by immunohistochemistry.Proliferation of fetal ovine PVSMC after 24 h of hypoxia (2% O2) was attenuated by inhibition of EGFR with 250 nmol tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), a specific EGFR protein tyrosine kinase inhibitor, when measured by [3H]-thymidine incorporation. Our data indicate that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term fetal hypoxia and that inhibition of EGFR signaling may ameliorate hypoxia-induced pulmonary vascular remodeling.

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

Next generation of adeno-associated virus 2 vectors: Point mutations in tyrosines lead to high-efficiency transduction at lower doses

Recombinant adeno-associated virus 2 (AAV2) vectors are in use in several Phase I/II clinical trials, but relatively large vector doses are needed to achieve therapeutic benefits. Large vector doses also trigger an immune response as a significant fraction of the vectors fails to traffic efficiently to the nucleus and is targeted for degradation by the host cell proteasome machinery. We have reported that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects transduction by AAV2 vectors by impairing nuclear transport of the vectors. We have also observed that EGFR-PTK can phosphorylate AAV2 capsids at tyrosine residues. Tyrosine-phosphorylated AAV2 vectors enter cells efficiently but fail to transduce effectively, in part because of ubiquitination of AAV capsids followed by proteasome-mediated degradation. We reasoned that mutations of the surface-exposed tyrosine residues might allow the vectors to evade phosphorylation and subsequent ubiquitination and, thus, prevent proteasome-mediated degradation. Here, we document that site-directed mutagenesis of surface-exposed tyrosine residues leads to production of vectors that transduce HeLa cells approximately 10-fold more efficiently in vitro and murine hepatocytes nearly 30-fold more efficiently in vivo at a log lower vector dose. Therapeutic levels of human Factor IX (F.IX) are also produced at an approximately 10-fold reduced vector dose. The increased transduction efficiency of tyrosine-mutant vectors is due to lack of capsid ubiquitination and improved intracellular trafficking to the nucleus. These studies have led to the development of AAV vectors that are capable of high-efficiency transduction at lower doses, which has important implications in their use in human gene therapy.

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.

A Dual Role of EGFR Protein Tyrosine Kinase Signaling in Ubiquitination of AAV2 Capsids and Viral Second-strand DNA Synthesis

A 52 kd cellular protein, FK506-binding protein (FKBP52), phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), inhibits adeno-associated virus 2 (AAV2) second-strand DNA synthesis and transgene expression. FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and TC-PTP over-expression leads to improved viral second-strand DNA synthesis and improved transgene expression. In these studies, we observed that perturbation of EGFR-PTK signaling by a specific inhibitor, Tyrphostin 23 (Tyr23), augmented the transduction efficiency of the single-stranded AAV (ssAAV) vector as well as the self-complementary AAV (scAAV) vector. Similarly, tyrosine-dephosphorylation of FKBP52 by TC-PTP resulted in increased transduction by both vectors. These data suggested that EGFR-PTK signaling also affects aspects of AAV transduction other than viral second-strand DNA synthesis. We document that inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsids which, in turn, facilitates nuclear transport by limiting proteasome-mediated degradation of AAV vectors. We also document that Tyr23-mediated increase in AAV2 transduction efficiency is not further enhanced by a specific proteasome inhibitor, MG132. Thus, EGFR-PTK signaling modulates ubiquitin (Ub)/proteasome pathway-mediated intracellular trafficking as well as FKBP52-mediated second-strand DNA synthesis of AAV2 vectors. This has implications in the optimal use of AAV vectors in gene therapy.

8. Adeno-Associated Virus 2-Mediated Gene Transfer: A Complex Interaction between Epidermal Growth Factor Receptor Protein Tyrosine Kinase Signaling and Ubiquitin/Proteasome Pathway in Transgene Expression

The transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein, FKBP52, inhibits the viral second-strand DNA synthesis, and consequently, transgene expression (J. Virol., 75: 9818, 2001). FKBP52 is phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), which inhibits AAV second-strand DNA synthesis by greater than 90% (J. Virol. 72: 9835, 1998; J. Virol., 75: 9818, 2001). Tyrosine-phosphorylated FKBP52 is dephosphorylated by T-cell protein tyrosine phosphatase (TC-PTP), which negatively regulates EGFR-PTK signaling, and leads to a significant increase in AAV transduction (J. Virol. 77: 2741, 2003). We have documented high-efficiency transduction of hepatocytes in TC-PTP-transgenic mice (Gene Ther., 11: 1165, 2004). We have also documented that intracellular trafficking of AAV from cytosol to nucleus is improved in hematopoietic cells from TC-PTP transgenic mice (Hum. Gene Ther., 15: 1207, 2004). In the present studies, we studied the role of EGFR-PTK signaling in AAV-mediated transgene expression. Consistent with our previous studies, less than 5% of HeLa cells were transduced by single-stranded (ss) AAV-EGFP vectors, and pretreatment of cells with Tyrphostin 23 (Tyr23), a specific inhibitor of EGFR-PTK, or stable expression of TC-PTP resulted in respectively |[sim]|12-fold and |[sim]|15-fold increase in AAV transduction. Although transduction by self-complementary (sc) AAV2-EGFP vectors was |[sim]|8-fold higher compared with their ss counterparts, pretreatment with Tyr23 or stable expression of TC-PTP resulted in a further |[sim]|10-fold increase in transduction. These data suggested that Tyr23 and TC-PTP affected other aspects of EGFR-PTK signaling involved in regulating vector transduction. We next examined whether Tyr23 or TC-PTP modulated the ubiquitin/proteasome pathway, both because proteasomes have been implicated in the regulation of EGFR endocytosis, and proteasome inhibitors have been shown to augment AAV transduction. Treatment of HeLa cells with MG132, a specific inhibitor of proteasome, resulted in respectively |[sim]|6-fold and |[sim]|8-fold increase in transduction efficiency of ss and scAAV vectors, However, neither a synergistic nor an additive effect was observed following pretreatment with MG132+Tyr23 or MG132+TC-PTP with either ss or scAAV vectors in HeLa cells. Similar results were obtained with murine fibroblast cell line NIH3T3, adult mouse hepatocyte cell line H2.35, and fetal mouse hepatocyte cell line FL83B. Taken together, these data indicate that complex interactions between EGFR-PTK signaling and ubiquitin/ proteasome pathway affect various aspects of intracellular trafficking and second-strand DNA synthesis of AAV vectors. A better understanding of these interactions is likely to be important in yielding new insights in the optimal use of recombinant AAV vectors in human gene therapy.

Inhibitory Activities of Epidermal Growth Factor Receptor Tyrosine Kinase-Targeted Dihydroxyisoflavone and Trihydroxydeoxybenzoin Derivatives on Sarcocystis neurona , Neospora caninum , and Cryptosporidium parvum Development

Several gene sequences of parasitic protozoa belonging to protein kinase gene families and epidermal growth factor (EGF)-like peptides, which act via binding to receptor tyrosine kinases of the EGF receptor (EGFR) family, appear to mediate host-protozoan interactions. As a clue to EGFR protein tyrosine kinase (PTK) mediation and a novel approach for identifying anticoccidial agents, activities against Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum grown in BM and HCT-8 cell cultures of 52 EGFR PTK inhibitor isoflavone analogs (dihydroxyisoflavone and trihydroxydeoxybenzoine derivatives) were investigated. Their cytotoxicities against host cells were either absent, mild, or moderate by a nitroblue tetrazolium test. At concentrations ranging from 5 to 10 microg/ml, 20 and 5 analogs, including RM-6427 and RM-6428, exhibited an in vitro inhibitory effect of > or = 95% against at least one parasite or against all three, respectively. In immunosuppressed Cryptosporidium parvum-infected Mongolian gerbils orally treated with either 200 or 400 mg of agent RM-6427/kg of body weight/day for 8 days, fecal microscopic oocyst shedding was abolished in 6/10 animals (P of <0.001 versus untreated controls) and mean shedding was reduced by 90.5% (P of <0.0001) and 92.0% (P of <0.0001), respectively, higher levels of inhibition than after nitazoxanide (200 mg/kg/day for 8 days) or paromomycin (100 mg/kg/day for 8 days) treatment (55.0%, P of <0.001, and 17.5%, P of >0.05, respectively). After RM-6427 therapy (200 mg/kg/day for 8 days), the reduction in the ratio of animals with intracellular parasites was nearly significant in ileum (P = 0.067) and more marked in the biliary tract (P < 0.0013) than after nitazoxanide or paromomycin treatment (0.05 < P < 0.004). RM-6428 treatment at a regimen of 400 mg/kg/day for 12 days inhibited oocyst shedding, measured using flow cytometry from day 4 (P < 0.05) to day 12 (P < 0.02) of therapy, when 2/15 animals had no shedding (P < 0.0001) and 11/15 were free of gut and/or biliary tract parasites (P < 0.01). No mucosal alteration was microscopically observed for treated or untreated infected gerbils. To our knowledge, this report is the first to suggest that the isoflavone class of agents has the potential for anticoccidial therapy.

The epidermal growth factor-pathway is not involved in down-regulation of Ca 2+-induced Cl − secretion in rat distal colon

Ca 2+-dependent secretagogues such as carbachol induce a transient Cl − secretion followed by long-lasting inhibition (run-down) of secretion. In the colonic tumour cell line, T84, epidermal growth factor (EGF) inhibits Ca 2+-dependent secretion, whereas antagonists of the EGF-signalling pathway slow down its run-down. The aim of the present study was to investigate whether a similar mechanism underlies the down-regulation of carbachol-induced Cl − secretion measured as change in short-circuit current (I sc) in a native intestinal epithelium, i.e. rat distal colon. In contrast to the colonic tumour cell line, EGF (1–100 μg/l) induced a transient secretory I sc and did not interfere with a subsequent administration of carbachol. Pretreatment with inhibitors of enzymes involved in the signalling cascade induced by EGF, i.e. tyrphostin AG1478, an inhibitor of the EGF receptor protein tyrosine kinase, PD 98059, an inhibitor of MAP kinase, and wortmannin, a blocker of the phosphatidylinositol-3-kinase, did also not affect the action of carbachol on transepithelial I sc. In order to investigate potential effects of these inhibitors on apical Cl − channels, the basolateral membrane was depolarized and a Cl − current across the apical membrane was driven by a Cl − gradient. Under these conditions, carbachol evoked a transient increase in I sc, caused by the stimulation of Ca 2+-dependent Cl − channels, followed by a long-lasting down-regulation of apical Cl − conductance leading to a decrease in I sc. All blockers of the EGF-signalling pathway tested did not interfere with the action of carbachol at the apical membrane. Consequently, the EGF-pathway seems not to be involved in the down-regulation of Ca 2+-dependent Cl − secretion across rat colon.

Involvement of epidermal growth factor receptor-protein tyrosine kinase transactivation in endothelin-1-induced vascular contraction.

Endothelin-1 (ET-1) is one of the major inducers of vasospasm following subarachnoid hemorrhage (SAH). It is generally accepted that extracellular signal-regulated kinase 1 and 2 (ERK1/2) are involved in ET-1-induced vascular contraction. In addition, ET-1 transactivates epidermal growth factor receptor (EGFR) protein tyrosine kinase (PTK), which leads to ERK1/2 stimulation. Therefore, the authors examined whether EGFR-PTK transactivation contributes to ET-1-induced vascular contraction in this study. Mitogen-activated protein kinase inhibitor, PD98059, inhibited ET-1-induced ERK1/2 stimulation in rabbit basilar artery (BA) vascular smooth-muscle cells (VSMCs). Moreover, PD98059 inhibited ET-1-induced contraction of rabbit BA rings. A specific inhibitor of EGFR PTK, AG1478, inhibited ET-1-induced EGFR-PTK transactivation, ERK1/2 stimulation, and contraction of BA rings in a concentration-dependent manner. The concentration of AG1478 required for 50% inhibition of the ET-1-induced contraction of BA rings was similar to that for ET-1-induced EGFR-PTK transactivation. Furthermore, AG1478 also inhibited ET-1-induced BA vasospasm in vivo. The results indicate that EGFR-PTK transactivation pathway plays an important role in ET-1-induced vascular contraction.

Meet the Guest Editor

Gerard Rosse grew up in Chatillon, a small town located in the French speaking region of Switzerland. He received his B.S. and Ph.D. in Chemistry from the University of Basle, Switzerland. His undergraduate studies, completed in 1991, encompassed a Diploma work on natural products total synthesis under the supervision of Prof. Christoph Tamm. Dr. Rosses doctoral thesis on the design, synthesis and biological characterization of multisubstrate-complex analogues as inhibitors of the EGF-Receptor protein tyrosine kinase was completed in 1995 with Prof. Urs Sequin and in collaboration with Dr. Heinz Fretz and Dr. Peter Traxler from Novartis (formerly Ciba-Geigy), Basle, Switzerland. He then studied as a post-doctoral fellow at Stanford University under Prof. John Griffin and working on combinatorial libraries of vancomycin derivatives. In 1997, Dr. Rosse moved back to Basle, where he was hired as a research chemist by F. Hoffmann-La Roche Inc. While at Roche, he worked within the combinatorial chemistry group of Dr. Lutz Weber on the synthesis of libraries small molecules. He also was involved in a variety of other projects including the development of antiinfective agents, high throughput NMR analysis and the investigation and application of bead-based methods for deorphaning genomically derived proteases. Since 1999, Dr. Rossé is working with Aventis, at the Aventis Combinatorial Technologies Center (ACTC), Tucson, Arizona. He is a Chemistry Group Leader and is leading the ACTC purification group as well. The research interests of Dr. Rossé include many aspects of drug discovery, with special emphasis on understanding the chemical biology of GPCRs and Kinases by designing and synthesizing focused libraries. He is also involved in the optimization of hits to leads, in the development of information technologies and instrumentations for parallel synthesis and purification. He has published many articles and patents. Dr. Rosse currently lives with his wife, Frederique, and two daughters, Alyssa and Noanne, in Oro Valley, AZ.

The Protein-tyrosine Phosphatase TCPTP Regulates Epidermal Growth Factor Receptor-mediated and Phosphatidylinositol 3-Kinase-dependent Signaling

In this study we have investigated the down-regulation of epidermal growth factor (EGF) receptor signaling by protein-tyrosine phosphatases (PTPs) in COS1 cells. The 45-kDa variant of the PTP TCPTP (TC45) exits the nucleus upon EGF receptor activation and recognizes the EGF receptor as a cellular substrate. We report that TC45 inhibits the EGF-dependent activation of the c-Jun N-terminal kinase, but does not alter the activation of extracellular signal-regulated kinase 2. These data demonstrate that TC45 can regulate selectively mitogen-activated protein kinase signaling pathways emanating from the EGF receptor. In EGF receptor-mediated signaling, the protein kinase PKB/Akt and the mitogen-activated protein kinase c-Jun N-terminal kinase, but not extracellular signal-regulated kinase 2, function downstream of phosphatidylinositol 3-kinase (PI 3-kinase). We have found that TC45 and the TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, inhibit almost completely the EGF-dependent activation of PI 3-kinase and PKB/Akt. TC45 and TC45-D182A act upstream of PI 3-kinase, most likely by inhibiting the recruitment of the p85 regulatory subunit of PI 3-kinase by the EGF receptor. Recent studies have indicated that the EGF receptor can be activated in the absence of EGF following integrin ligation. We find that the integrin-mediated activation of PKB/Akt in COS1 cells is abrogated by the specific EGF receptor protein-tyrosine kinase inhibitor tyrphostin AG1478, and that TC45 and TC45-D182A can inhibit activation of PKB/Akt following the attachment of COS1 cells to fibronectin. Thus, TC45 may serve as a negative regulator of growth factor or integrin-induced, EGF receptor-mediated PI 3-kinase signaling.

D-Cbl, the Drosophila homologue of the c-Cbl proto-oncogene, interacts with the Drosophila EGF receptor in vivo, despite lacking C-terminal adaptor binding sites.

The c-Cbl proto-oncogene encodes a multidomain phosphoprotein that has been demonstrated to interact with a wide range of signalling proteins. The biochemical function of c-Cbl in these complexes is, however, unclear. Recent studies with the C. elegans Cbl homologue, sli-1, have suggested that Cbl proteins may act as negative regulators of EGF receptor (EGFR) signalling. As the EGFR and other protein tyrosine kinase receptor signalling pathways are highly conserved between insects and vertebrates, we sought a Drosophila homologue of c-Cbl for a detailed genetic analysis. We report here that Drosophila melanogaster has a single gene, D-cbl, that is homologous to c-cbl. We find that D-cbl encodes a 52 kDa protein that has a high degree of similarity to c-Cbl and SLI-1 across novel phosphotyrosine-binding (PTB) and RING finger domains. Surprisingly, however, D-Cbl is C-terminally truncated relative to c-Cbl and SLI-1 and consequently is unable to bind SH3-domain containing adaptor proteins, including the Drosophila Grb2 homologue, Drk. Although the D-Cbl protein lacks Drk binding sites it can nevertheless associate with a tyrosine phosphorylated protein, or is itself tyrosine phosphorylated in an DER dependent manner and associates with activated Drosophila EGF receptors (DER) in vivo. Consistent with a role for D-Cbl in DER dependent patterning in the embryo and adult, D-Cbl is expressed at a high level in early embryos and throughout the imaginal discs in third instar larvae. This study forms the basis for future genetic analysis of D-Cbl, aimed at gaining insights into the role of Cbl proteins in signal transduction.

Differential inhibition of epidermal growth factor signaling pathways in rat hepatocytes by long-term ethanol treatment

BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF- induced PLC gamma tyrosine phosphorylation and inositol 1,4,5- trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism. (Gastroenterology 1997 Jun;112(6):2073-88)

A Defective EGF-Receptor inWaved-2Mice Attenuates Intestinal Adaptation

While the pathophysiology of intestinal adaptation following small bowel resection (SBR) is not well understood, there is evidence to suggest an important role for epidermal growth factor (EGF) in this process. Inwaved-2mice, a naturally occurring mutation results in reduced EGF receptor protein tyrosine kinase activity. We tested the hypothesis that an intact EGF receptor is essential for adaptation by subjecting this strain of mice to SBR. A 50% proximal SBR or sham operation (bowel transection with reanastomosis only) was performed inwaved-2,heterozygous, and wild-type mice. After 3 days, adaptation was characterized in the remnant ileum as changes in DNA and protein content per unit length. Villus height and crypt depth were measured, and crypt cell proliferation rates were determined by the percentage of crypt cells taking up 5-bromodeoxyuridine. Following sham surgery, all mice regained their preoperative weight by the third postoperative day. After SBR, all mice gained weight while thewaved-2mice did not. Ileal DNA and protein significantly increased after SBR in wild-type and heterozygous mice while these parameters were unchanged in thewaved-2mice. Villus height and crypt cell proliferation increased in response to SBR in all groups; however, the changes were less pronounced in thewaved-2mice. Adaptation after SBR is impaired inwaved-2mice. Signal transduction by the EGF receptor is a critical component of this response. These data endorse a crucial role for EGF and its receptor in the pathogenesis of intestinal adaptation.

Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.

The Juxtamembrane, Cytosolic Region of the Epidermal Growth Factor Receptor Is Involved in Association with α-Subunit of Gs

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha. However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha. However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.

In vivo pharmacology and anti-tumour evaluation of the tyrphostin tyrosine kinase inhibitor RG13022.

Amplification and increased expression of many growth factor receptors, including the epidermal growth factor receptor (EGFR), has been observed in human tumours. One therapeutic strategy for overcoming EGF autocrine control of tumour growth is inhibition of EGFR protein tyrosine kinase (PTK). A series of low molecular weight molecules have been identified which inhibit the EGFR PTK in vitro and demonstrate antiproliferative activity against human cancer cell lines with high expression of EGFR. A significant growth delay in squamous cancer xenografts has been reported for one of these compounds, the tyrphostin RG13022. Based on these encouraging results, we sought to confirm the activity of RG13022 in vivo and relate the effects to the in vivo plasma disposition. RG13022 and three additional peaks were detected by HPLC following intraperitoneal administration of 20 mg kg-1 RG13022 in MF1 nu/nu mice. RG13022 demonstrated rapid biexponential elimination from plasma with a terminal half-life of 50.4 min. RG13022 plasma concentrations were less than 1 microM by 20 min post injection. A primary product was identified as the geometrical isomer (E)-RG13022. Both RG13022 and its geometrical isomer inhibited DNA synthesis in HN5 cells after a 24 h in vitro incubation (IC50 = 11 microM and 38 microM respectively). Neither RG13022 nor its geometrical isomer displayed significant cytotoxicity. RG13022 had no influence on the growth of HN5 tumours when administered chronically, starting either on the day of tumour inoculation or after establishment of tumour xenografts. The rapid in vivo elimination of RG13022 has potential significance to the development of this and other related tyrphostin tyrosine kinase inhibitors, as plasma concentrations fell below that required for in vitro activity by 20 min post injection. The lack of in vivo tumour growth delay suggests that a more optimal administration schedule for RG13022 would include more frequent injections or continuous administration. An improved formulation for RG13022 is therefore required before further development of this or other similar protein tyrosine kinase inhibitors can be made. Alternative strategies should also be sought which display longer lasting in vivo exposures.

Activation of Gsα by the Epidermal Growth Factor Receptor Involves Phosphorylation

Previous studies from our laboratory have shown that epidermal growth factor (EGF) stimulates cAMP accumulation in the heart via a process involving Gsalpha and the EGF receptor (EGFR) protein tyrosine kinase activity (Nair, B. G., Parikh, B., Milligan, G., and Patel, T. B. (1990) J. Biol. Chem. 265, 21317-21322; Nair, B. G., and Patel, T. B. (1993) Biochem. Pharmacol. 46, 1239-1245). Therefore, studies were performed to investigate the hypothesis that the EGFR protein tyrosine kinase phosphorylates Gsalpha and activates this protein. Employing purified EGFR and Gsalpha, we have demonstrated that the EGFR kinase phosphorylates Gsalpha in a time-dependent manner with a stoichiometry of 2 mol of phosphate incorporated/mol of Gsalpha. As determined by phosphoamino acid analysis, the phosphorylation of Gsalpha by the EGFR kinase was exclusively on tyrosine residues. Interestingly, GDP and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) inhibited the phosphorylation of Gsalpha without altering EGFR autophosphorylation. However, G protein betagamma subunits protected against GDP- and GTPgammaS-mediated inhibition of phosphorylation of Gsalpha. In functional studies, phospho-Gsalpha demonstrated a greater GTPase activity and also a greater capacity to bind GTPgammaS as compared to the nonphosphorylated Gsalpha. Moreover, the phospho-Gsalpha augmented adenylyl cyclase activity in S49 cyc- cell membranes to a greater extent than its nonphosphorylated counterpart. Therefore, we conclude that phosphorylation of Gsalpha on tyrosine residues by the EGFR kinase activates this G protein and increases its ability to stimulate adenylyl cyclase.