Functional expression of TrkA receptors in hippocampal neurons

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.     

Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.     

Signal transduction in human hematopoietic cells by vascular endothelial growth factor related protein, a novel ligand for the FLT4 receptor

We have recently identified a novel ligand of the vascular endothelial growth factor (VEGF) family termed VEGF-related protein (VRP), which specifically binds to the FLT4 receptor. To characterize the signaling events after VRP engagement of its cognate receptor in hematopoietic cells, a population of human erythroleukemia (HEL) cells, termed HEL-JW, expressing high levels of FLT4 receptor was isolated. Stimulation of HEL-JW cells with VRP alone and in combination with the c-kit ligand/stem cell factor increased cell growth. VRP induced tyrosine phosphorylation of various proteins, including the FLT4 receptor. Further characterization of these tyrosine phosphorylated molecules revealed that Shc, Grb2, and SOS form a complex with the activated FLT4 receptor. HEL-JW cells also expressed RAFTK, a recently identified member of the focal adhesion kinase family. RAFTK was phosphorylated and activated upon VRP treatment, and there was an enhanced association of this kinase with the adaptor protein Grb2. Furthermore, the c-Jun NH2-terminal kinase (JNK), involved in growth activation and shown to mediate RAFTK signaling in other cell types, was activated by VRP stimulation. We also observed that VRP treatment of HEL-JW cells resulted in the phosphorylation of the cytoskeletal protein paxillin. This treatment resulted in an increased association of paxillin with RAFTK, which was mediated by the C-terminal region of RAFTK. These studies indicate that VRP stimulation induced the formation of a signaling complex at its activated receptor as well as activation of RAFTK. VRP-mediated activation of RAFTK may facilitate signal transduction to the cytoskeleton and downstream to the JNK pathway in FLT4-expressing blood cells.     

Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product

An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To elucidate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gain-of-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.     

Replacement of tyrosine 1251 in the carboxyl terminus of the insulin-like growth factor-I receptor disrupts the actin cytoskeleton and inhibits proliferation and anchorage-independent growth

Insulin-like growth factor (IGF)-I signaling through the IGF-I receptor modulates cellular adhesion and proliferation and the transforming ability of cells overexpressing the IGF-I receptor. Tyrosine phosphorylation of intracellular proteins is essential for this transduction of the IGF-I-induced mitogenic and tumorigenic signals. IGF-I induces specific cytoskeletal structure and the phosphorylation of proteins in the associated focal adhesion complexes. The determination of the exact pathways emanating from the IGF-I receptor that are involved in mediating these signals will contribute greatly to the understanding of IGF-I action. We have previously shown that replacement of tyrosine residues 1250 and 1251 in the carboxyl terminus of the IGF-I receptor abrogates IGF-I-induced cellular proliferation and tumor formation in nude mice. In this study, replacement of either tyrosine 1250 or 1251 similarly reduces the cells ability to grow in an anchorage-independent manner. The actin cytoskeleton and cellular localization of vinculin are disrupted by replacement of tyrosine 1251. Tyrosine residues 1250 and 1251 are not essential for tyrosine phosphorylation of two known substrates; insulin receptor substrate-1 and SHC, nor association of known downstream adaptor proteins to these substrates. In addition, these mutant IGF-I receptors do not affect IGF-I-stimulated p42/p44 mitogen-activated protein kinase activation or phosphatidylinositol (PI) 3'-kinase activity. Thus, it appears that in fibroblasts expressing tyrosine 1250 and 1251 mutant IGF-I receptors, the signal transduction pathways impacting on mitogenesis and tumorigenesis do not occur exclusively through the PI 3'-kinase or mitogen-activated protein kinase pathways.     

Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity

SLP-76 (SH2 domain leukocyte protein of 76 kDa) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression. In this report, we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region. Two tyrosines (Y113 and Y128) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation. Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function, mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation. A third tyrosine within the amino-terminal region (Y145) appears to be the most important for optimal SLP-76 function, as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity.     

Tyrosine 112 of latent membrane protein 2A is essential for protein tyrosine kinase loading and regulation of Epstein-Barr virus latency

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.     

Activation loop tyrosines contribute varying roles to TrkB autophosphorylation and signal transduction

The TrkB receptor tyrosine kinase (RTK) is a high affinity receptor for the neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Following exposure to BDNF or NT-4/5, TrkB is autophosphorylated on five cytoplasmic tyrosines: Y484, Y670, Y674, Y675, and Y785. Based on crystallographic analyses for others RTKs, TrkB tyrosines Y670, Y674, and Y675 are expected to lie within a putative kinase activation loop. Phosphorylation of these activation loop tyrosines is postulated to be a conserved event required for complete RTK activation. Here, we have assessed the importance these activation loop tyrosines play in regulating TrkB autophosphorylation, cytoplasmic signal transduction, and cell proliferation. We show that while tyrosine 670 is dispensable for BDNF-inducible TrkB autophosphorylation and the activation of certain signal transduction events, it is required for complete TrkB-mediated cellular proliferation. Combinatorial mutagenesis of tyrosines 674 and 675 only moderately affects TrkB autophosphorylation, but significantly impairs the BDNF-inducible stimulation of cytoplasmic signaling events and cellular proliferation. The combined mutation of all three activation loop tyrosines results in an inactive receptor, which is unable to autophosphorylate, stimulate signaling events, or induce mitogenesis. The data highlight the varying degrees of importance of the three activation loop tyrosines in TrkB mediated biological responses.     

Cellular uptake properties of a 2''-amino/2''-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA

PURPOSE: Rhabdomyosarcomas (RMS) are heterogeneous in their clinical presentation, histology, and cytogenetics. The growth of some RMS cells has been found to be regulated by the tyrosine kinase insulin-like growth factor (IGF) type I receptor. However, RMS cells exhibit variable sensitivity to inhibitors of tyrosine kinases and IGF receptors. Collectively, these heterogeneous features suggest that differences exist in the growth regulatory pathways of RMS. The objective of this study is to identify active tyrosine kinase signal transduction pathways in embryonal and alveolar RMS cells. METHODS: RMS tumor samples and cell lines representing both embryonal and alveolar histologic subtypes have been analyzed by immunoprecipitation and immunoblotting techniques to characterize phosphotyrosyl protein patterns and to identify tyrosine phosphorylated proteins. RESULTS: RMS cells can be characterized based on the patterns of phosphotyrosyl proteins, including the phosphorylation status of the catenin-like protein Cas1 and the signal adapter protein SHC, and the activation of IGF type I receptor signaling cascades including the formation of SHC-GRB2 signal protein complexes and MAP kinase activation. CONCLUSIONS: Rhabdomyosarcomas, especially the embryonal histologic subtype, are heterogeneous at the level of tyrosine kinase signal transduction. It will be important to characterize the growth regulatory pathways active in individual RMS tumors before targeting molecular therapies to this malignancy.     

Increased recruitment of hematopoietic progenitor cells underlies the ex vivo expansion potential of FLT3 ligand

The ligand for flt-3 (FLT3L) exhibits striking structural homology with stem cell factor (SCF) and monocyte colony-stimulating factor (M-CSF) and also acts in synergy with a range of other hematopoietic growth factors (HGF). In this study, we show that FLT3L responsive hematopoietic progenitor cells (HPC) are CD34+CD38-, rhodamine 123dull, and hydroperoxycyclophosphamide (4-HC) resistant. To investigate the basis for the capacity of FLT3L to augment the de novo generation of myeloid progenitors from CD34+CD38- cells, single bone marrow CD34+CD38- cells were sorted into Terasaki wells containing serum-free medium supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), SCF (4 HGF) +/- FLT3L. Under these conditions, FLT3L recruited approximately twofold more CD34+CD38- cells into division than 4 HGF alone. The enhanced proliferative response to FLT3L was evident by day 3 and was maintained at all subsequent time points examined. In accord with these findings, we also show that transduction of CD34+CD38- cells with the LAPSN retrovirus is enhanced by FLT3L. The results of these experiments therefore indicate that increased recruitment of primitive HPC into cell cycle underlies the ex vivo expansion potential of FLT3L and also its ability to improve retroviral transduction of HPC.     

Redundant and selective roles for erythropoietin receptor tyrosines in erythropoiesis in vivo

Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the pressence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development.     

The CBL-related protein CBLB participates in FLT3 and interleukin-7 receptor signal transduction in pro-B cells

The FLT3 receptor tyrosine kinase and its ligand, FL, play an important role in early hematopoietic development. We have found that CBLB, a recently characterized molecule closely related to the CBL protooncogene product, is phosphorylated on tyrosine(s) following FL treatment of JEA2 human pro-B cells and THP1 monocytic cells. Treatment of JEA2 cells with interleukin (IL)-7 induces CBLB phosphorylation as well. FL and IL-7, respectively, induce and increase association of tyrosine-phosphorylated SHC and the p85 subunit of phosphatidylinositol 3'-kinase with CBLB. In these cells, CBLB constitutively binds the GRB2 adaptor predominantly through its N-terminal SH3 domain, to form a complex that is distinct from the GRB2.CBL and GRB2.SOS1 complexes. Together with the fact that CBLB is consistently found in blast cells from acute leukemias and in peripheral blood mononuclear cells, this suggests that CBLB has a role in tyrosine kinase-regulated signaling pathways in many hematolymphoid cells.     

SH1 domain autophosphorylation of P210 BCR/ABL is required for transformation but not growth factor independence

P210 BCR/ABL is a chimeric oncogene implicated in the pathogenesis of chronic myelogenous leukemia. BCR sequences have been shown to be required for activation of the tyrosine kinase and transforming functions of BCR/ABL. In this work, we show that two other structural requirements for full transforming activity of P210 BCR/ABL include a functional tyrosine kinase and the presence of tyrosine 1294, a site of autophosphorylation within the tyrosine kinase domain. Replacement of tyrosine 1294 with phenylalanine (1294F) greatly diminishes the transforming activity of BCR/ABL without affecting the specific activity of the protein tyrosine kinase. Expression of an exogenous myc gene in fibroblasts partially complements the transforming capacity of mutant P210 BCR/ABL (1294F). Surprisingly, tyrosine 1294 is not required for efficient induction of growth factor-independence in hematopoietic cell lines by P210 BCR/ABL. These results suggest that autophosphorylation at tyrosine 1294 may be important for recognition and phosphorylation of cellular substrates in the pathway of transformation, but it is not critical for mediating the events which lead to growth factor independence.     

Tyrosine phosphorylation of the MUC1 breast cancer membrane proteins. Cytokine receptor-like molecules

Phosphorylation on tyrosine residues is a key step in signal transduction pathways mediated by membrane proteins. Although it is known that human breast cancer tissue expresses at least 2 MUC1 type 1 membrane proteins (a polymorphic high molecular weight MUC1 glycoprotein that contains a variable number of tandem 20 amino acid repeat units, and the MUC1/Y protein that is not polymorphic and is lacking this repeat array) their function in the development of human breast cancer has remained elusive. Here it is shown that these MUC1 proteins are extensively phosphorylated, that phosphorylation occurs primarily on tyrosine residues and that following phosphorylation the MUC1 proteins may potentially interact with SH2 domain-containing proteins and thereby initiate a signal transduction cascade. As with cytokine receptors, the MUC1 proteins do not harbor intrinsic tyrosine kinase activity yet are tyrosine phosphorylated and the MUC1/Y protein participates in a cell surface heteromeric complex whose formation is mediated by two cytoplasmically located MUC1 cysteine residues. Furthermore, the MUC1/Y protein demonstrates sequence similarity with sequences present in cytokine receptors that are known to be involved in ligand binding. Our results demonstrate that the two MUC1 isoforms are both likely to function in signal transduction pathways and to be intimately linked to the oncogenetic process and suggest that the MUC1/Y protein may act in a similar fashion to cytokine receptors.     

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.     

Specific binding of Fyn and phosphatidylinositol 3-kinase to the B cell surface glycoprotein CD19 through their src homology 2 domains

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.     

Regulation of cytotoxic T lymphocyte-associated molecule-4 by Src kinases

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is a cell surface receptor expressed on activated T cells that can inhibit T cell responses induced by activation of the TCR and CD28. Studies with phosphorylated peptides based on the CTLA-4 intracellular domain have suggested that tyrosine phosphorylation of CTLA-4 may regulate its interactions with cytoplasmic proteins that could determine its intracellular trafficking and/or signal transduction. However, the kinase(s) that phosphorylate CTLA-4 remain uncharacterized. In this report, we show that CTLA-4 can associate with the Src kinases Fyn and Lck and that transfection of Fyn or Lck, but not the unrelated kinase ZAP70, can induce tyrosine phosphorylation of CTLA-4 on residues Y201 and Y218. A similar pattern of tyrosine phosphorylation was found in pervanadate-treated Jurkat T cells stably expressing CTLA-4. Phosphorylation of CTLA-4 Y201 in Jurkat cells correlated with cell surface accumulation of CTLA-4. CTLA-4 phosphorylation induced the association of CTLA-4 with the tyrosine phosphatase SHP-2, but not with phosphatidylinositol 3-kinase. In contrast, Lck-induced phosphorylation of CD28 resulted in the recruitment of phosphatidylinositol 3-kinase, but not SHP-2. These findings suggest that phosphorylation of CD28 and CTLA-4 by Lck activates distinct intracellular signaling pathways. The association of CTLA-4 with Src kinases and with SHP-2 results in the formation of a CTLA-4 complex with the potential to regulate T cell activation.     

Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential

Autophosphorylation of type I receptor tyrosine kinases (RTKs) comprises one step in the signaling events mediated by erbB receptors such as p185neu and EGFR. Previous analysis of p185neu has indicated that there are at least five tyrosine autophosphorylation sites, Y882, Y1028, Y1143, Y1226/7 and Y1253, of which Y882 might be important because of its location in the kinase activity domain. We have specifically analysed the effect of a Y882F (phenylalanine substituted for tyrosine at position 882) mutation in the enzymatic active domain. We also deleted the carboxyl terminal 122 amino acids which contained three other autophosphorylation sites (TAPstop) and combined mutants of that deletion with Y882F (Y882F/APstop). Both in vitro and in vivo transformation assays showed that substitution of tyrosine882 by phenylalanine significantly decreased the transforming potential of activated, oncogenic p185neu, although no significant difference in the total phosphotyrosine levels of the mutant proteins were observed. To analyse mitogenic signaling in response to ligand, the intracellular domains of p185neu and Y882F were fused with the extracellular domain of the EGF receptor. The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were less responsive to EGF stimulation than those expressing EGFR/neu receptors. In vitro kinase assays demonstrated that abolishing the autophosphorylation site Y882 diminished the enzymatic tyrosine kinase activity of p185neu. These studies, taken together with the phenotypic inhibition observed with cells expressing Y882F, suggest that the tyrosine882 residue may be important for p185neu-mediated transformation by affecting the enzymatic kinase function of the p185neu receptor.