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Biomedical subjects

W T Wong

Publications and source records attributed to W T Wong.

At least 37 records · Page 2Linked to original sources
PubMed1997

The SH3 domain of Eps8 exists as a novel intertwined dimer.

SH3 domains are structurally well-characterized as monomeric modular units of protein structure that mediate protein-protein recognition in numerous signal transduction proteins. The X-ray crystallographic structure of the Eps8 SH3 domain reveals a novel variation of the canonical SH3 fold: the SH3 domain from Eps8 is a dimer formed by strand interchange. In addition, co-immunoprecipitation experiments show that intact Eps8 is multimeric in vivo. Hence, the SH3 domain of Eps8 may represent a dimerization motif.

Adaptor Proteins, Signal Transducing↗
PubMed1996

RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain.

Eps8 is a recently identified SH3-containing substrate for tyrosine kinase receptors. To understand the role of eps8 in receptor-mediated signaling, we cloned cDNAs encoding proteins that bind to its SH3 domain. One of these cDNAs predicts the synthesis of an 828 amino acid protein with homology to the N-terminal region of the tre oncogene. We designated this protein RN-tre for Related to the N-terminus of tre. RN-tre is ubiquitously expressed and maps to 10p13, a region known to be involved in translocations in various leukemias. In addition, a 10p13 monosomy syndrome, characterized by developmental alterations, has been reported. The regional homology between RN-tre and tre, which is limited to their N-terminal portion, prompted us to investigate the origin of the tre oncogene transcriptional unit. We were able to show that tre is the fusion product of a 5' genetic element, homologous to RN-tre and a 3' element, encoding a de-ubiquinating enzyme. Moreover, we identified, within the N-terminus of RN-tre and tre, a domain (named TrH, for Tre Homology), which is conserved within several proteins from yeast to mammals and has protein-binding properties in vitro.

3T3 Cells↗
PubMed1996

RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation.

We isolated a cDNA encoding a protein, RN-tre, which shows homology to the N-terminus of the tre oncogene product and has SH3-binding ability as well as an evolutionarily conserved domain, termed TrH, with protein-binding ability in vitro. In the present study, we identify the product of the RN-tre gene as a 97-100 kDa protein. We demonstrate stable association in vivo and in vitro between RN-tre and eps8, mediated by the SH3 domain of the latter. In vitro, RN-tre displayed remarkable preference for binding to the eps8-SH3, as compared to eight other SH3s. The Kd for the in vitro interaction between RN-tre and eps8-SH3 was between 10(-8) and 10(-7) M. A role for RN-tre in cell proliferation was suggested by the finding that a C-terminal truncated mutant was able to confer proliferative advantage and reduced serum-requirement to NIH3T3 fibroblasts. Finally, comparison of the structure and biological activities of RN-tre and of the tre oncogene product, provided insight into the mechanism of oncogenic activation of tre.

3T3 Cells↗
PubMed1995

A protein-binding domain, EH, identified in the receptor tyrosine kinase substrate Eps15 and conserved in evolution.

In this report we structurally and functionally define a binding domain that is involved in protein association and that we have designated EH (for Eps15 homology domain). This domain was identified in the tyrosine kinase substrate Eps15 on the basis of regional conservation with several heterogeneous proteins of yeast and nematode. The EH domain spans about 70 amino acids and shows approximately 60% overall amino acid conservation. We demonstrated the ability of the EH domain to specifically bind cytosolic proteins in normal and malignant cells of mesenchymal, epithelial, and hematopoietic origin. These observations prompted our search for additional EH-containing proteins in mammalian cells. Using an EH domain-specific probe derived from the eps15 cDNA, we cloned and characterized a cDNA encoding an EH-containing protein with overall similarity to Eps15; we designated this protein Eps15r (for Eps15-related). Structural comparison of Eps15 and Eps15r defines a family of signal transducers possessing extensive networking abilities including EH-mediated binding and association with Src homology 3-containing proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.

Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.

3T3 Cells↗
PubMed1995

Expression of the receptor tyrosine kinase substrate genes eps8 and eps15 during mouse development.

Receptor tyrosine kinases (RTKs) control proliferation and differentiation through their ability to bind and/or phosphorylate intracellular substrates. The repertoire of substrates recruited by different RTK is largely overlapping. It is not clear, therefore, how a cell distinguishes among signals originating from different RTKs. One possibility is that selective availability of substrates participates in the regulation of this process. To gain insight into this issue, we studied the expression pattern, during mouse embryogenesis, of the eps8 and eps15 genes, which encode two recently identified RTK substrates. Both genes are expressed from E 10 in a restricted fashion. eps8 is first expressed in frontonasal neural crest-derived cells, in the mesenchyme of branchial arches and in the liver primordium. At E 12.5-E 14, eps8 is additionally expressed in the central nervous system (CNS) in a regional restricted pattern at the met-mesencephalic transition area and in the developing submandibular salivary glands. eps15 is expressed at E 10 in the liver primordium, in the spinal ganglia and in the encephalic ganglia derived from the hindbrain neural crest. In addition, at E 12.5-E 14, eps15 is expressed, along all the CNS, in the ventricular zone where undifferentiated neuroblasts are located. The regional pattern of developmental expression of these two substrates sharply contrasts with their ubiquitous expression in adults, raising the possibility that their expression during embryogenesis is linked to selective proliferative and/or differentiative responses of specific neuroectodermal regions and body organs.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent.

Several signal transducers bind through their SH2 domains to phosphotyrosine-containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor-transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates.

3T3 Cells↗
PubMed1995

Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation.

eps8, a recently identified tyrosine kinase substrate, has been shown to augment epidermal growth factor (EGF) responsiveness, implicating it in EGF receptor (EGFR)-mediated mitogenic signaling. We investigated the status of eps8 phosphorylation in normal and transformed cells and the role of eps8 in transformation. In NIH 3T3 cells overexpressing EGFR (NIH-EGFR), eps8 becomes rapidly phosphorylated upon EGF stimulation. At receptor-saturating doses of EGF, approximately 30% of the eps8 pool is tyrosine phosphorylated. Under physiological conditions of activation (i.e., at low receptor occupancy), corresponding to the 50% effective dose of EGF for mitogenesis, approximately 3 to 4% of the eps8 contains phosphotyrosine. In human tumor cell lines, we detected constitutive tyrosine phosphorylation of eps8, with a stoichiometry (approximately 5%) similar to that associated with potent mitogenic response in NIH-EGFR cells. Overexpression of eps8 was able to transform NIH 3T3 cells under limiting conditions of activation of the EGFR pathway. Concomitant tyrosine phosphorylation of eps8 and shc, but not of rasGAP, phospholipase C-gamma, and eps15, was frequently detected in tumor cells. This suggested that eps8 and shc might be part of a pathway which is preferentially selected in some tumors. Cooperation between these two transducers was further indicated by the finding of their in vivo association. This association was, at least in part, dependent on recognition of shc by the SH3 domain of eps8. Our results indicate that eps8 is physiologically part of the EGFR-activated signaling and that its alterations can contribute to the malignant phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Tracheal laceration from endotracheal intubation in a cat.

Iatrogenic trauma to the upper airway as a result of the clinical use of endotracheal tubes has rarely been reported in domestic animals. This report describes a case of tracheal laceration in a cat after endotracheal intubation for a dental procedure under general anaesthesia. The diagnosis and successful management of the condition are discussed, and other complications of the use of cuffed endotracheal and tracheostomy tubes are considered.

Animals↗
PubMed1994

Symphyseal distraction-osteotomy using an ulnar autograft for the treatment of pelvic canal stenosis in three cats.

Three adult cats with obstipation due to stenosis of the pelvic canal were treated by symphyseal distraction-osteotomy using an ulnar autograft. The duration of clinical signs before the surgery in the three cats was one, three and 19 months; the first two cats had no recurrence of signs after surgery, and the third had less severe and less frequent bouts of constipation.

Animals↗
PubMed1994

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway.

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

3T3 Cells↗
PubMed1994

The high transforming potency of erbB-2 and ret is associated with phosphorylation of paxillin and a 23 kDa protein.

Two-dimensional gel maps of proteins phosphorylated by the epidermal growth factor receptor (EGFR) and erbB-2 kinases were obtained, to investigate the molecular basis of the different biological properties of these two molecules. Several proteins were phosphorylated by EGFR or erbB-2 with different stoichiometry. Differences were either quantitative or qualitative. In NIH3T3 cells, erbB-2 is 100-fold more transforming than EGFR. In the same cell line several proteins were preferentially phosphorylated by erbB-2, as compared to EGFR. To identify which of these substrates might be directly involved in mitogenic signaling, we obtained two-dimensional maps of proteins phosphorylated on tyrosine by EGFR/ret and an EGFR/erbB-2TK chimeric receptors. Both these chimerae behaved indistinguishably from erbB-2 in a number of bioassays and potently transformed NIH3T3 cells. Paxillin and a 23 kDa substrate were invariably phosphorylated to higher stoichiometry whenever potent mitogenic and transforming signals were activated. We propose that paxillin and the 23 kDa substrate are important elements in the erbB-2 and ret-activated mitogenic and transforming signaling.

3T3 Cells↗
PubMed1994

Evolutionary conservation of the EPS8 gene and its mapping to human chromosome 12q23-q24.

We have previously isolated the coding sequence for a novel substrate for tyrosine kinases, eps8, from NIH3T3 fibroblasts. Eps8 was phosphorylated in vivo by several receptor tyrosine kinases (RTKs) and, upon overexpression, was able to enhance EGFR-mediated mitogenic signaling in NIH3T3 cells. To gain understanding of eps8 function as well as its role in normal and neoplastic proliferation, we cloned the human eps8 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. In addition to a previously identified SH3 domain, the predicted amino acid sequence of human eps8 revealed a non-random distribution of prolines, clustered in a way to suggest SH3-binding sites and a putative PH domain. Eps8 was expressed in all epithelial and fibroblastic lines examined and in some, but not all, hematopoietic cells. An essential function of eps8 in cell growth regulation was underscored by its conservation during evolution, where eps8-related sequences were detected as early as in Saccharomyces cerevisiae. Finally, the human EPS8 locus was mapped to chromosome 12q23-q24.

3T3 Cells↗
PubMed1994

The human eps15 gene, encoding a tyrosine kinase substrate, is conserved in evolution and maps to 1p31-p32.

Employing an expression cloning approach for tyrosine kinase substrates, we have previously isolated the coding sequence for a novel putative EGFR substrate, eps15, from NIH3T3 fibroblasts. Eps15 displayed a receptor-specific pattern of tyrosine phosphorylation in vivo and was able to transform NIH3T3 cells upon overexpression. To gain understanding of eps15 function as well as its role in normal and neoplastic proliferation, we cloned the human eps15 coding sequence and studied expression of the human RNA and protein, evolutionary conservation, and chromosomal location. The close structural similarity of human eps15 with the murine homologue is indicated by 89% and 90% identity of nucleotide and predicted amino acid sequences, respectively. Using the human eps15 coding sequence as probe, we demonstrate that eps15 is member of a gene family that is highly conserved during evolution. An essential function of eps15 in cell growth regulation is underscored by our observation of ubiquitous expression at the transcript and the protein level in normal and malignant human cells. The human EPS15 locus maps to chromosome 1p31-p32, a region involved in deletion in neuroblastoma, translocations in acute lymphoblastic leukemia, and exhibiting a fragile site.

3T3 Cells↗
PubMed1993

Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals.

A method which allows direct cloning of intracellular substrates for receptor tyrosine kinases (RTKs) was developed. By applying this technique to the study of the epidermal growth factor receptor (EGFR) signaling pathway, we have isolated a cDNA, designated eps8, which predicts a approximately 92 kDa protein containing an SH3 domain. Eps8 also contains a putative nuclear targeting sequence. Antibodies specific to the eps8 gene product recognize a protein of M(r) 97 kDa and a minor 68 kDa component, which are closely related, as demonstrated by V8 proteolytic mapping. The product of the eps8 gene is tyrosine-phosphorylated in vivo following EGF stimulation of intact cells and associates with the EGFR, despite the lack of a functional SH2 domain. Several other RTKs are also able to phosphorylate p97eps8. Thus, the eps8 gene product represents a novel substrate for RTKs. Adoptive expression of the eps8 cDNA in fibroblastic or hematopoietic target cells expressing the EGFR resulted in increased mitogenic response to EGF, implicating the eps8 gene product in the control of mitogenic signals.

3T3 Cells↗
PubMed1993

Surgical management of the post-phlebitic leg syndrome.

Twenty-five patients (32 limbs) with severe retrograde ejection venous syndrome (post-phlebitic leg syndrome) were surgically treated from 1971 to 1986 using a "very complete fasciotomy" with or without other individualized procedures. Clinical data, venographic findings, operative choices, and outcome were analyzed retrospectively. The theoretic benefit of adopting this new and conceptually accurate diagnostic label and the principle of "no incompetent perforators, no ulcers" is discussed. The patients generally had been managed for 10 to 20 years with conservative neglect or operative inadequacy. With correct conceptual understanding, anatomic stratification, defined indications for surgery, and individualization of procedures, an excellent result has been obtained with a low rate of serious complications. The very complete fasciotomy should be suitable for general use in treating this difficult and controversial syndrome.

Adult↗
PubMed1993

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

3T3 Cells↗