Tyrosine Kinase Receptor Type Research Articles

Expression of receptor-type tyrosine kinase, Axl, and its ligand, Gas6, in pediatric thyroid carcinomas around chernobyl.

Protein-tyrosine kinases (PTKs) play a crucial role in regulating thyroid cell growth and differentiation. It is known that PTKs are activated by ionizing radiation, but the critical pathways through ionizing radiation-induced malignant transformation are not well defined. The Axl protein is a member of a new family of receptor tyrosine kinases, which the ligand, Gas6, a protein S-related molecule, has recently been identified. We previously clarified the expression of Axl and mitogenic action of Gas6 in thyroid carcinoma cells. To investigate the involvement of Gas6-Axl signaling in pediatric thyroid carcinoma further, we examined tissues obtained from 17 patients with thyroid papillary carcinomas from Gomel, Belarus, by immunohistochemistry and in situ hybridization. Axl and Gas6 were overexpressed in 76.5% and 70.6 % of cases, respectively. Solid component and invasive front tended to overexppress Gas6 and Axl in papillary carcinomas. Eighty-five percent of Axl positive cases coexpressed Gas6. In situ hybridization also confirmed the presence of axl mRNA in thyroid carcinoma tissues. These findings suggest that Axl and Gas6 expression might be involved in childhood thyroid tumorigenesis around Chernobyl.

Expression of the Palmitoylation-deficient CD151 Weakens the Association of α3β1 Integrin with the Tetraspanin-enriched Microdomains and Affects Integrin-dependent Signaling

Transmembrane proteins of the tetraspanin superfamily are assembled in multimeric complexes on the cell surface. Spatial orientation of tetraspanins within these complexes may affect signaling functions of the associated transmembrane receptors (e.g. integrins, receptor-type tyrosine kinases). The structural determinants that control assembly of the tetraspanin complexes are unknown. We have found that various tetraspanins and the alpha(3) integrin subunit are palmitoylated. The stability and molecular composition of the palmitoylated alpha(3)beta(1)-tetraspanin complexes are not affected by adhesion. To assess the significance of palmitoylation in the function of the alpha(3)beta(1)-tetraspanin complexes we mapped the sites of palmitoylation for CD151. Mutation of six cysteines, Cys(11), Cys(15), Cys(79), Cys(80), Cys(242), and Cys(243) was necessary to completely abolish palmitoylation of CD151. The association of the palmitoylation-deficient mutant of CD151 (CD151Cys8) with CD81 and CD63 was markedly decreased, but the interaction of the alpha(3)beta(1)-CD151Cys8 complex with phosphatidylinositol 4-kinase was not affected. Ectopic expression of CD151Cys8 in Rat-1 cells impaired the interactions of the endogenous CD63 and CD81 with the alpha(3)beta(1) integrin. Although the expression of the palmitoylation-deficient CD151 does not change cell spreading on the extracellular matrix, the number of focal adhesions increased. Adhesion-induced phosphorylation of PKB/c-Akt is markedly increased in cells expressing a palmitoylation-deficient mutant, thereby providing direct evidence for the role of the tetraspanin microdomains in regulation of the integrin-dependent phosphatidylinositol 3-kinase signaling pathway. In contrast, activation of FAK and ERK1/2 were not affected by the expression of CD151Cys8. Our results demonstrate that palmitoylation of tetraspanins is critical not only for the organization of the integrin-tetraspanin microdomains but also has a specific role in modulation of adhesion-dependent signaling.

Essential role of growth factor receptor-mediated signal transduction through the mitogen-activated protein kinase pathway in early embryogenesis of the echinoderm.

In this study it was shown that growth factor receptors (GFR) play a crucial role in early embryogenesis of the echinoderms Hemicentrotus pulcherrimus and Clypeaster japonicus by transmitting signals to the mitogen-activated protein kinase (MAPK) pathway. The phosphorylation ratio of extracellular signal-regulated kinase 1 (ERK1) changed dynamically during early embryogenesis and showed a peak at the swimming blastula (sBl) stage. Suramin, an inhibitor of GFR, when applied during the sBl stage perturbed morphogenesis, including primary mesenchyme cell (PMC) migration, cell proliferation, archenteron elongation, spiculogenesis, pigment cell differentiation and phosphorylation of myosin light chains (MLC). Genistein, a receptor-type protein tyrosine kinase inhibitor, severely inhibited PMC migration, gastrulation and the phosphorylation of MLC. Manumycin A, a Ras inhibitor, inhibited spiculogenesis and invagination. PD98059, a MAPK/ERK kinase inhibitor, perturbed early PMC migration and pigment cell differentiation, but not spiculogenesis and gastrulation (although these two events were significantly delayed). PMC ingression was not perturbed by genistein, suramin, manumycin A or PD98059. All of the inhibitors perturbed the phosphorylation of ERK1, which was completely restored by exogenous platelet-derived growth factor (PDGF)-AB. PDGF-AB also partially restored elongation of the archenteron by restoring cell proliferation that had been perturbed by suramin.

Tyrosine kinase-type receptor ErbB4 in chondrocytes: interaction with connective tissue growth factor and distribution in cartilage

In order to identify receptor molecules that participate in the growth and differentiation of chondrocytes, we cloned a number of cDNA fragments from HCS-2/8 chondrocytic cells, by using tyrosine kinase-specific primers for amplification. The mRNA expression of one such receptor, ErbB4, was increased by connective tissue growth factor/hypertrophic chondrocyte-specific gene product (CTGF/Hcs24), which promotes all stages of the endochondral ossification in vitro. ErbB4 expression was observed through all stages of chondrocytic differentiation in vitro, corresponding to the wide distribution of CTGF/Hcs24 target cells. Furthermore, positive signals for erbB4 mRNA were detectable throughout most populations of chondrocytes, in growth and articular cartilage in vivo. These results demonstrate for the first time that ErbB4 is expressed in chondrocytes and may play some roles in chondrocytic growth and differentiation along with CTGF/Hcs24.

Activin responsive PAI-1 promoter activity in granulosa cells is dependent on attachment to extracellular matrix

Objective: We have previously shown in a rodent organ culture model that preantral follicle development may be regulated by an interaction between extracellular matrix (ECM) and activin-a (1). The objectives of this study were 1) to characterize the activin receptors in a spontaneously immortalized rodent granulosa cell line (SIGC), 2) to show that these receptors in SIGC are functional, 3) to demonstrate that activin responsiveness is dependent on the presence of ECM. Design: An in vitro study of SIGC. SIGC carry the characteristics of preantral/undifferentiated granulosa cells (2) and express integrins that bind collagen (C), and laminin (L)(3). Materials/Methods: Expression of activin receptor type IB, IIA, and IIB were determined by Western blotting. To determine whether these receptors are functional, SIGC were transfected by electroporation with 40 mg of p3TP-lux and 10 mg of GFP plasmid in a serum-free medium. The p3TP-lux reporter gene contains the Activin-responsive PAI-1 promoter. As determined by the percentage of GFP positive cells the transfection efficiency was approximately 20–30%. After serum starvation SIGC were plated on laminin (L), collagen (C), or polylysine (P, negative control) coated coverslips and treated with 0–20 ng/mL of activin-a for 48 hours. The promoter activity was measured by a luminometer and corrected for protein concentration. Results: SIGC expressed activin type IB, IIA, and IIB receptors. The results of transfection experiments are summarized in the figure. The p3TP-lux promoter activity was increased with activin-a treatment in a dose dependent fashion on ECM but not on polylysine. There was no significant difference in promoter activity between cells plated on L and C. The promoter activity was negligible in cells plated on P. Conclusions: 1) SIGC express functional activin receptors and thus are suitable for studying the effects of activin treatment on granulosa cells, 2) In the SIGC model, promoter response to activin-a is dependent on the presence of ECM. Given the known interaction between tyrosine kinase-type receptor signaling (growth factor signaling) and ECM-induced integrin signaling on cell growth and differentiation (4), a similar interaction may exist between the serine/threonine kinase type receptor (i.e activin) signaling and integrin signaling pathways. 1. Oktay et al. Biol Reprod. 2000;63:457–61. 2. Stein et al. Cancer Res. 1991;51:696–706. 3. Oktay et al. J Soc Gynecol Investig. 2002;9:338A. 4. Oktay et al. J Cell Biol. 1999;145:1461–9. Supported by: ASRM-Serono Research grant to K.O., and by the Center for Reproductive Medicine & Infertility.

A Ligand-inducible Epidermal Growth Factor Receptor/Anaplastic Lymphoma Kinase Chimera Promotes Mitogenesis and Transforming Properties in 3T3 Cells

Oncogenic rearrangements of the anaplastic lymphoma kinase (ALK) gene, encoding a receptor type tyrosine kinase, are frequently associated with anaplastic large cell lymphomas. Such rearrangements juxtapose the intracellular domain of ALK to 5'-end sequences belonging to different genes and create transforming fusion proteins. To understand how the oncogenic versions of ALK contribute to lymphomagenesis, it is important to analyze the biological effects and the biochemical properties of this receptor under controlled conditions of activation. To this aim, we constructed chimeric receptor molecules in which the extracellular domain of the ALK kinase is replaced by the extracellular, ligand-binding domain of the epidermal growth factor receptor (EGFR). Upon transfection in NIH 3T3 fibroblasts, the EGFR/ALK chimera was correctly synthesized and transported to the cell surface, where it was fully functional in forming high versus low affinity EGF-binding sites and transducing an EGF-dependent signal intracellularly. Overexpression of the EGFR/ALK chimera in NIH 3T3 was sufficient to induce the malignant phenotype; the appearance of the transformed phenotype was, however, conditionally dependent on the administration of EGF. Moreover, the EGFR/ALK chimera was significantly more active in inducing transformation and DNA synthesis than the wild type EGFR when either was expressed at similar levels in NIH 3T3 cells. Comparative analysis of the biochemical pathways implicated in the transduction of mitogenic signals did not show any increased ability of the EGFR/ALK to phosphorylate PLC-gamma and MAPK compared with the EGFR. On the contrary, EGFR/ALK showed to have a consistently greater effect on phosphatidylinositol 3-kinase activity compared with the EGFR, indicating that this enzyme plays a major role in mediating the mitogenic effects of ALK in NIH 3T3 cells.

Distinctive gene expression of receptor-type tyrosine kinase families during rat hepatocarcinogenesis

Growth factor receptor tyrosine kinase signaling plays key roles in regulating growth of normal hepatocytes, however, which receptor-type tyrosine kinase (RTK) is involved in hepatocarcinogenesis remains undetermined. The aim of this study was to characterize the expression of these receptors in different stages of rat liver carcinogenesis. We compared the expression profile of RTK genes in rat normal liver and diethylnitrosamine-induced hepatoma tissues using a homology cloning method with degenerated primers. In situ hybridization, immunohistochemical staining, and RT-PCR were performed to analyze the cell type-specific expression of target RTKs during the chemically-induced hepatocarcinogenesis. Sequence analysis of 459 clones identified 23 different RTK genes. The Tie-2, c-Met, and Flk-1 genes were the most abundant RTK genes cloned in rat hepatoma compared to normal liver. In situ hybridization and immunohistochemical studies showed overexpression of c-Met and Flk-1 in GST-P positive preneoplastic lesions as well as neoplastic lesions. Tie-2 was expressed not only in endothelial cells but also in so-called oval cells, which are thought to be liver stem cells. Tie-2 ligand, angiopointin-1, mRNA was detected in both normal livers and hepatoma cells/tissues. In contrast, angiopoietin-2 mRNA was detected only in hepatoma tissues. These results indicate that c-Met, Tie-2 and Flk-1 signals play important roles in different stages of chemically-induced hepatocarcinogenesis. Distinctive gene expression of RTK may contribute to epigenic implication of hepatoma formation.

Evidence of Both Extra- and Intracellular Cysteine Targets of Protein Modification for Activation of RET Kinase

By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase.

We Found It, You Can Relax Now

The hormone relaxin has long been recognized as important to mammalian reproductive processes (see the Perspective by Ivell), and the development of therapeutics to treat preterm labor disorders and delivery problems has been stifled by the elusiveness of its cognate receptor. Hsu et al. have identified a G protein-coupled receptor that mediates the effects of relaxin. This is somewhat surprising because a structurally similar hormone, insulin, signals through a receptor-type tyrosine kinase. S. Y. Hsu, K. Nakabayashi, S. Nishi, J. Kumagai, M. Kudo, O. D. Sherwood, A. J. W. Hsueh, Activation of orphan receptors by the hormone Relaxin. Science 295 , 671-674 (2002). [Abstract] [Full Text] R. Ivell, This hormone has been relaxin' too long! Science 295 , 637-638 (2002). [Full Text]

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.

Dendritic Spine Formation

STKE Changes in the morphology of neuronal dendritic spines are correlated with changes in synaptic plasticity. The cell surface proteoglycan syndecan-2 is clustered at the surface of mature hippocampal neurons and is thought to regulate structural changes of the spines. Now Ethell et al. have demonstrated that syndecan-2 is phosphorylated on at least two tyrosine residues by the receptor-type tyrosine kinase EphB2 in vitro and in vivo. The two transmembrane proteins colocalized in dendrites of hippocampal neurons and were isolated in a complex from mouse brain neurons. Phosphorylation was required for their interaction, for syndecan-2 to cluster, and for normal dendritic spine formation in transfected neurons. Phosphorylation could trigger clustering of syndecan-2, and the EphB2-syndecan-2 complex may subsequently initiate the recruitment of downstream signaling molecules that control dendritic spine formation.— LC Neuron 31 , 1001 (2001).

Dendritic Spine Formation

Changes in the morphology of neuronal dendritic spines are correlated with changes in synaptic plasticity. The cell surface proteoglycan syndecan-2 is clustered at the surface of mature hippocampal neurons and is thought to regulate structural changes of the spines. Ethell et al. demonstrate that syndecan-2 is phosphorylated on at least two tyrosine residues by the receptor-type tyrosine kinase EphB2 in vitro and in vivo. The two transmembrane proteins colocalized in dendrites of hippocampal neurons and were isolated in a complex from mouse brain neurons. Phosphorylation was required for their interaction, for syndecan-2 to cluster, and for normal dendritic spine formation in transfected neurons. The authors propose that phosphorylation triggers clustering of syndecan-2, and that the EphB2-syndecan-2 complex subsequently initiates the recruitment of downstream signaling molecules that control dendritic spine formation. I.M. Ethell, F. Irie, M.S. Kalo, J.R. Couchman, E.B. Pasquale, Y. Yamaguchi, EphB/Syndecan-2 signaling in dendritic spine morphogenesis. Neuron 31 , 1001-1013 (2001). [Full Text]

Destructive Rampage of Cbl

Receptor-type tyrosine kinases are not the only kinase targets for Cbl-mediated ubiquitination and degradation. Yokouchi et al. report that this E3 ligase also ubiquitinates the cytoplasmic tyrosine kinase Src, thus explaining its negative regulatory effect on Src. The ubiquitination process required Src kinase activity, and because Cbl is a known Src substrate, the authors propose that its phosphorylation on tyrosine by Src somehow activates ubiquitin ligase action. But Cbl also appears to be self destructive, ubiquitinating itself in the process of acting on Src. Cbl is constitutively associated with other components of the ubiqutin system as well as with inactive Src. Upon Src activation, the trimolecular complex dissociated, a release event that may facilitate the transfer of ubiquitin to two different substrates. M. Yokouchi, T. Kondo, A. Sanjay, A. Houghton, A. Yoshimura, S. Komiya, H. Zhang, R. Baron, Src-catalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins. J. Biol. Chem. 276 , 35185-35193 (2001). [Abstract] [Full Text]

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis.

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G(-/-) homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.

Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Suppresses TKR-Growth Factor Signaling Independent of Metalloproteinase Inhibition

The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. Here we demonstrate a direct role for TIMP-2 in regulating tyrosine kinase-type growth factor receptor activation. We show that TIMP-2 suppresses the mitogenic response to tyrosine kinase-type receptor growth factors in a fashion that is independent of MMP inhibition. The TIMP-2 suppression of mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and implicates cAMP as the second messenger in these effects. TIMP-2 neither altered the release of transforming growth factor alpha from the cell surface, nor epidermal growth factor (EGF) binding to the cognate receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (K(d) = 147 pm), that is not competed by the synthetic MMP inhibitor BB-94 and is independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation of EGFR and a concomitant reduction in Grb-2 association. TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation from immunoprecipitable EGFR complex and a selective increase in total SHP-1 activity. These studies represent a new functional paradigm for TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by short-circuiting EGFR activation.

Constitutive activation of FLT3 stimulates multiple intracellular signal transducers and results in transformation.

Aberrant expression of FLT3 has been found in most cases of B-lineage ALL and AML, and subsets of T cell ALL, CML in blast crisis and CLL. In 20% of patients with AML the receptor has small internal tandem duplications of the juxtamembrane region which appear to contitutively activate the receptor. To investigate whether FLT3 activation could play a role in leukemia, we generated a constitutively activated FLT3 by fusing its cytoplasmic domain to the helix-loop-helix domain of TEL in analogy to the fusion that occurs with TEL-PDGFR in CMML. In vitro translation assays demonstrated oligomerization and intrinsic tyrosine kinase activity of the TEL-FLT3 chimeric receptor. Constitutively activated TEL-FLT3 conferred IL-3 independence and long-term proliferation to transfected Ba/F3 cells. Immunoblot analyses showed that JAK 2, STAT 3, STAT 5a, STAT 5b and CBL were tyrosine-phosphorylated in TEL-FLT3 expressing Ba/F3 cells in the absence of IL-3. These data suggest a possible role for the JAK/STAT pathway in FLT3 signaling. Transplantation of TEL-FLT3 expressing Ba/F3 cells into syngeneic mice caused mortality in all mice by 3 weeks after injection. Histopathologic analysis demonstrated a massive infiltration of mononuclear cells in the liver, spleen and bone marrow. The mimicking of naturally occurring TEL fusions provides an approach to assess aspects of the biology of activated FLT3, or other receptor-type tyrosine kinases (RTKs) in leukemic transformation.

Expression of the c-kit protein is associated with certain subtypes of salivary gland carcinoma

The c-Kit protein, a receptor type tyrosine kinase that plays an important role in the development of hematopoietic cells, melanocytes, and germ cells, is expressed in mastocytosis, gastrointestinal stromal cell tumors (GISTs), germ cell tumors, and several other tumors. Gain-of-function mutations in exon 11 and exon 17 have been shown as a mechanism of c-kit activation in some tumors. To study the role of c-kit in salivary gland carcinomas, we analyzed the c-kit protein expression in 79 carcinomas of major and minor salivary glands by immunohistochemistry. Although varying in intensity of staining, c-kit expression was identified very often in adenoid cystic carcinomas (20/25), lymphoepithelioma-like carcinomas (6/6) and myoepithelial carcinomas (2/2), but not in other types of salivary gland carcinoma (0/46), P<0.00001. By DNA sequencing, genetic alteration of c-kit juxtamembrane domain (exon 11) and tyrosine kinase domain (exon 17) was not found in all the three types of salivary carcinoma that had c-kit protein expression. In conclusion, c-kit protein overexpression is involved in the pathogenesis of certain types of salivary gland carcinoma, but mutation of the gene is not the mechanism of c-kit activation.

C-met tyrosine kinase receptor expression is associated with abnormal beta-catenin expression and favourable prognostic factors in invasive breast carcinoma.

Receptor-type tyrosine kinases are important in cell signal transduction and proliferation. Abnormal expression of tyrosine kinases often leads to malignant transformation. c-met is a tyrosine kinase receptor and its ligand is hepatocyte growth factor (HGF). In this study, we have evaluated c-met expression in 69 invasive breast carcinomas and statistically analysed this expression with known clinicopathological prognostic parameters and patients' survival. We also studied for the first time c-met expression in association with E-cadherin and beta-catenin expression. Immunohistochemistry (ABC-HRP method) was peformed for the detection of c-met, E-cadherin and beta-catenin. c-met immunoreactivity was observed in 58% of cases and was associated with the lobular type of breast carcinomas (P = 0.012), low histological grade ductal carcinomas (P = 0.05), favourable prognostic and predictive factors such as oestrogen and progesterone receptor immunohistochemical expression and negative c-erbB-2 expression (P = 0.05, P = 0.014 and P = 0.03, respectively). c-met immunoreactivity did not correlate with lymph node status, tumour size and stage of the disease. Cox's proportional hazard regression model demonstrated that tumours with positive c-met immunoreactivity correlated significantly with favourable patients' survival (P = 0.028). When c-met staining was compared with E-cadherin and beta-catenin expression, a statistical significant correlation was established between c-met immunoreactivity and abnormal beta-catenin expression (P = 0.025) suggesting possible involvement of c-met in the downregulation of the E-cadherin-catenin complex, possibly through tyrosine phosphorylation of beta-catenin. c-met immunohistochemical expression seems to be associated with abnormal beta-catenin expression, good prognostic and predictive factors and favourable outcome in breast cancer patients.

Chat, a Cas/HEF1-associated Adaptor Protein That Integrates Multiple Signaling Pathways

Cas (Crk-associated substrate) and HEF1 (human enhancer of filamentation) are related adaptor proteins that function in integrin-mediated cell adhesion and antigen receptor signaling pathways. We report here a molecular cloning of Chat (Cas/HEF1-associated signal transducer) that associates with Cas and HEF1. Chat is a 78-kDa signaling molecule with an N-terminal SH2 domain and is expressed in a wide range of tissues. In hematopoietic cells, a 115-kDa isoform of Chat (Chat-H) was specifically expressed. Chat is associated with Cas in brain, and Chat-H is associated with HEF1 in splenocytes. Deletion analyses revealed that Chat and Cas are associated with each other by their C-terminal domains. Treatment of PC12 cells with epidermal growth factor or nerve growth factor increased the phosphorylation level of Chat. This increase was suppressed by an inhibitor of mitogen-activated protein (MAP) kinase kinase, PD98059, suggesting the phosphorylation of Chat by MAP kinase. In Chat-overexpressed COS7 cells, the activity of c-Jun N-terminal kinase was up-regulated. After the epidermal growth factor stimulation, Chat and Cas were colocalized with actin filaments at ruffling membranes. These findings suggest that Chat transduces signals of tyrosine kinases and MAP kinase to Cas signaling pathway.

UV Irradiation and Superactivation

UV irradiation damages DNA and can promote oncogenesis. Kato et al. now show that UV light can further affect already oncogenic, contitutively active tyrosine kinases at the cell surface by superactivating them. The c-Ret proto-oncogene is a receptor-type tyrosine kinase, and constitutively activated forms have been associated with certain cancers. The study shows that when cells expressing c-Ret or the activated receptor forms are exposed to UV light, there is an increase in receptor dimerization and activation, and an increase in overall cell phosphorylation. A cysteine residue in one of the intracellular kinase domains was critical for disulfide bond formation and dimerization. Expression of a superoxide dismutase, which scavenges free radicals, counteracted the UV effect. Thus, UV light can activate and superactivate Ret by changing cellular redox levels to oxidative, thereby promoting disulfide bond-mediated dimerization of Ret. Kato, M., Iwashita, T., Takeda, K., Akhand, A.A., Liu, W., Yoshihara, M., Asai, N., Suzuki, H., Takahashi, M., and Nakashima, I. (2000) Ultraviolet light induces redox teaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases. Mol. Biol. Cell 11: 93-101. [Abstract] [Full Text]