The efficacy of local anaesthesia for percutaneous epididymal sperm aspiration and testicular sperm aspiration

The actin filament-disrupting agent cytochalasin D strikingly increased tyrosine phosphorylation of a 75 kDa protein (p75) in rabbit aortic vascular smooth muscle cells. The microtubule-disrupting agent, colchicine had no effect on p75 tyrosine phosphorylation. Cytochalasin D also stimulated p75-directed kinase activity as determined by kinase assays of anti-Tyr(P) immunoprecipitates. Cytochalasin D stimulated tyrosine phosphorylation of the F-actin-binding protein, p80/85 cortactin, but p75 was not immunologically related either to cortactin, the phosphatidylinositol 3'-kinase p85 alpha subunit, or the 80 kDa isoform of caldesmon. These results suggest that p75 may represent a cytochalasin D-inducible kinase or kinase-associated component and provide evidence for the existence of a potentially novel kinase pathway regulated by disruption of the actin cytoskeleton.     

Identification of a novel Drosophila SMAD on the X chromosome

The mechanism of human platelet activation by thrombopoietin (TPO) was investigated in vitro. We found that rHuTPO stimulated thromboxane A2 formation and serotonin secretion, despite the absence of shape change and aggregation. Blockade of the arachidonic acid pathway did not inhibit rHuTPO-induced platelet secretion. rHuTPO stimulated the tyrosine phosphorylation of 64, 80/85, 95, 130 and 140 kDa proteins, but phosphoproteins of 100-105 and 125 kDa obtained when platelets aggregated in the presence of thrombin were absent. rHuTPO stimulated and potentiated the thrombin-induced tyrosine phosphorylation of a 80 kDa protein identified as the cortical actin-associated protein, p80/85 cortactin. When platelets were aggregated in the presence of rHuTPO and fibrinogen, cortactin phosphorylation was enhanced as compared to rHuTPO alone. Treatment with RGDS or cytochalasin D respectively reduced or abolished cortactin tyrosine phosphorylation. This confirms the existence of fibrinogen binding-dependent and independent pools of phosphorylated cortactin, both requiring intact actin polymerization. Cytoskeleton-binding proteins may be implicated in in vitro platelet activation by rHuTPO.     

Physical and functional association of cortactin with Syk in human leukemic cell line K562

Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80-kDa protein at an early stage of megakaryocytic differentiation and that this 80-kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment.     

Tyrosine phosphorylation events during different stages of collagen-platelet activation

Three groups of phosphoproteins have been distinguished, basing on the velocity and extent of phosphorylation in platelets stimulated with collagen. pp60c-src constituted the first group; the increase in its phosphorylation was the highest and most rapid (maximal in 30 s after the addition of collagen). pp80/85 and non-identified protein of 65 kDa formed the second group; the increase in their phosphorylation was twice smaller than that of pp60c-src, and reached its maximum 60 s after the addition of collagen. pp120, pp72syk, and two non-identified phosphoproteins of 90 and 75 kDa constituted the third group; the increase in their phosphorylation was 4-10-fold lower than that of pp60c-src and reached its maximum after 180 s. We conclude that the phosphorylation of pp60c-src is important for the change of shape of platelets, the phosphorylation of pp80/85 and pp65 for the initiation of the formation of aggregates and the phosphorylation of the third group of phosphoproteins for the formation of massive aggregates. This conclusion was supported by using a monoclonal anti-GPIb antibody, which did not inhibit the shape change of platelets and did not inhibit pp60c-src phosphorylation. This antibody inhibited aggregate formation as well as tyrosine phosphorylation of proteins belonging to the second and the third group of phosphoproteins.     

Depolarization-induced tyrosine phosphorylation in PC12h cells

Depolarization induced by KCl was found to induce tyrosine phosphorylation of cellular proteins in PC12h cells. By Western blotting with anti-phosphotyrosine antibody, we detected tyrosine phosphorylation of proteins with molecular weights of 120, 110, 105, 95, 75, 70, 66, 44, and 42 kDa in response to KCl. The immunoprecipitates from KCl-treated cells with the antibody contained large amounts of tyrosine-phosphorylated proteins and increased activity of tyrosine kinase. Incubation of the immunoprecipitates with [gamma-32P]ATP resulted in tyrosine phosphorylation of two proteins with the molecular weights of 120 and 140 kDa. These effects were completely abolished by the addition of EGTA before KCl treatment, suggesting that the depolarization-induced tyrosine phosphorylation may require calcium entry into the cells from the medium. Increased activity of tyrosine kinase phosphorylating the 120 and 140 kDa proteins was also recovered from cells stimulated with nerve growth factor, basic fibroblast growth factor, epidermal growth factor, and vasoactive intestinal peptide. Among them, depolarization by KCl elicited the strongest effect. These results indicate that a protein tyrosine kinase that phosphorylate the 120 and 140 kDa proteins is phosphorylated or activated in response to calcium ion, cAMP, and growth factors acting through tyrosine kinase receptors.     

Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells

Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-alpha inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-alpha. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-alpha treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-alpha inhibits this Src-dependent signaling pathway.     

Src family tyrosine kinase Lyn binds several proteins including paxillin in rat basophilic leukemia cells

Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.     

Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins

The consequences of Helicobacter pylori attachment to human gastric cells were examined by transmission electron microscopy and immunofluorescence microscopy. H. pylori attachment resulted in (i) effacement of microvilli at the site of attachment, (ii) cytoskeletal rearrangement directly beneath the bacterium, and (iii) cup/pedestal formation at the site of attachment. Double-immunofluorescence studies revealed that the cytoskeletal components actin, alpha-actinin, and talin are involved in the process. Immunoblot analysis showed that binding of H. pylori to AGS cells induced tyrosine phosphorylation of two host cell proteins of 145 and 105 kDa. These results indicate that attachment of H. pylori to gastric epithelial cells resembles that of enteropathogenic Escherichia coli. Coccoid H. pylori, which are thought to be terminally differentiated bacterial forms, are capable of binding and inducing cellular changes of the same sort as spiral H. pylori, including tyrosine phosphorylation of host proteins.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

T cell activation-dependent association between the p85 subunit of the phosphatidylinositol 3-kinase and Grb2/phospholipase C-gamma 1-binding phosphotyrosyl protein pp36/38

Tyrosine phosphorylation of cellular proteins is an early and an essential step in T cell receptor-mediated lymphocyte activation. Tyrosine phosphorylation of transmembrane receptor chains (such as zeta and CD3 chains) and membrane-associated proteins provides docking sites for SH2 domains of adaptor proteins and signaling enzymes, resulting in their recruitment in the vicinity of activated receptors. pp36/38 is a prominent substrate of early tyrosine phosphorylation upon stimulation through the T cell receptor. The tyrosine-phosphorylated form of pp36/38 is membrane-associated and directly interacts with phospholipase C-gamma 1 and Grb2, providing one mechanism to recruit downstream effectors to the cell membrane. Here, we demonstrate that in Jurkat T cells, pp36/38 associates with the p85 subunit of phosphatidylinositol 3-kinase (PI-3-K p85) in an activation-dependent manner. Association of pp36/38 with PI-3-K p85 was confirmed by transfection of a hemagglutinin-tagged p85 alpha cDNA into Jurkat cells followed by anti-hemagglutinin immunoprecipitation. In vitro binding experiments with glutathione S-transferase fusion proteins of PI-3-K p85 demonstrated that the SH2 domains, but not the SH3 domain, mediated binding to pp36/38. This binding was selectively abrogated by phosphopeptides that bind to p85 SH2 domains with high affinity. Filter binding assays demonstrated that association between pp36/38 and PI-3-K p85 SH2 domains was due to direct binding. These results strongly suggest the role of pp36/38 in recruiting PI-3-K to the cell membrane and further support the idea that pp36/38 is a multifunctional docking protein for SH2 domain-containing signaling proteins in T cells.     

Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells. Implications for carbachol-stimulated chloride secretion

We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 microM) stimulated a time-dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T84 cells. The tyrosine kinase inhibitor, genistein (5 microM), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase. Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 microM). Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nM), but was not altered by the PKC inhibitor GF 109203X (1 microM). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml). Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co-immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr. An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 microM), reversed CCh-stimulated phosphorylation of both EGFr and ERK. Tyrphostin AG1478 also potentiated chloride secretory responses to CCh. We conclude that CCh activates ERK in T84 cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated.     

p75, a member of the heat shock protein family, undergoes tyrosine phosphorylation in response to oxidative stress

The combination of H2O2 and vanadate generates aqueous peroxovanadium (pV) species, which are effective cell-permeable oxidants, and potent inhibitors of protein-tyrosine phosphatases. As a result, treatment of intact cells with pV compounds significantly enhances protein Tyr phosphorylation. Here we demonstrate that treatment of intact rat hepatoma Fao cells with pV markedly enhances Tyr phosphorylation of a 75-kDa protein, termed pp75. Amino-terminal sequencing of pp75 revealed that this protein is a member of the 70-75-kDa heat shock protein family, which includes PBP-74, glucose-related protein (GRP)-75, and mortalin. Tyr phosphorylation of pp75 is selective, because other proteins that belong to the heat shock protein 70 family, such as GRP-72, Bip (GRP-78), and HSC-70 fail to undergo Tyr phosphorylation when cells are treated with pV. Our findings suggest that heat shock proteins such as pp75 may undergo tyrosine phosphorylation when intact cells are subjected to oxidative stress induced by pV compounds.     

p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells

Flt3/Flk2 belongs to class III receptor tyrosine kinases. Like other members of type III receptor tyrosine kinases, murine Flt3 induces tyrosine phosphorylation of p85 and subsequently activation of PI3 kinase upon FL binding. While p85 binds murine Flt3 at Y958 in the carboxyl terminus of the receptor, human Flt3 does not have a potential p85-binding site in the carboxyl terminus. In this study, we examined whether p85 binds to human Flt3 in Baf3/Flt3 and THP-1 cells. In contrast to murine Flt3, p85 is not tyrosine phosphorylated after FL stimulation, nor does it bind Flt3 in both cell lines. Instead p85 associates inducibly with tyrosine phosphorylated SHP-2 and constitutively with SHIP and two tyrosine phosphorylated proteins with molecular weights about 100-kDa (p100) and 120-kDa (p120) in Baf3/Flt3 cells. The p100 associates with both p85 and SHP-2. In THP-1 cells, p85 associates inducibly with tyrosine phosphorylated SHIP, p100 and p120. These results indicate that p85 does not bind human Flt3, but forms a complex with SHP-2, SHIP, p100 and p120 in hematopoietic cells.     

Induction of host signal transduction pathways by Helicobacter pylori

Adherence of Helicobacter pylori to cultured gastric epithelial cells is associated with several cellular events, including the tyrosine phosphorylation of a 145-kDa host protein; the reorganization of the host cell actin and associated cellular proteins, like vasodilator-stimulated phosphoprotein, adjacent to the attached bacterial cell; and the subsequent release of the cytokine, interleukin 8 (IL-8). H. pylori isolated from patients with ulcer disease and gastric cancer contain a DNA insertion, the cag pathogenicity island (PAI), that is not present in bacteria isolated from individuals with asymptomatic infection. Mutations in a number of PAI genes abolish tyrosine phosphorylation and IL-8 synthesis but not the cytoskeletal rearrangements. Kinase inhibition studies suggest there are two distinct pathways operative in stimulating IL-8 release from host cells and one of these H. pylori pathways is independent of the tyrosine phosphorylation step.     

CSF-1 stimulation induces the formation of a multiprotein complex including CSF-1 receptor, c-Cbl, PI 3-kinase, Crk-II and Grb2

Recently c-Cbl has been reported to be phosphorylated upon CSF-1 stimulation. The product of the c-cbl proto-oncogene (c-Cbl) is a 120 kDa protein harboring several docking sites for Src homology 2 (SH2) domain containing proteins and proline-rich regions that have been shown to allow its constitutive association with the SH3 domains of Grb2. We demonstrate here that CSF-1 exposure of stable transfectant CHO cells expressing the CSF-1 receptor induced the sustained tyrosine phosphorylation of c-Cbl and its subsequent association with Crk-II and the p85 kDa subunit of the PI 3-kinase, while it constitutively associates with Grb2. We demonstrate by in vitro experiments that these associations require the SH2 domain of Crk-II and both the C- and N-terminal SH2 domains of the p85 subunit of the PI 3-kinase. cCbl is the major PI 3-kinase-containing protein in c-Fms expressing CHO cells upon CSF-1 stimulation. Thus c-Cbl behaves as a core protein, allowing the formation of a quaternary complex including, Crk-II, PI 3-kinase and Grb2. We provide evidence that this multiprotein complex can interact with the tyrosine phosphorylated CSF-1 receptor through the unoccupied SH2 domain of Grb2.     

The Epstein-Barr virus latent membrane protein 1 induces expression of the epidermal growth factor receptor

The Epstein-Barr virus (EBV)-encoded LMP1 protein is an important component of the process of transformation by EBV. LMP1 is essential for transformation of B lymphocytes, most likely because of its profound effects on cellular gene expression. Although LMP1 is expressed in the majority of nasopharyngeal carcinoma (NPC) tumors, the effect of LMP1 on cellular gene expression and its contribution to the development of malignancy in epithelial cells is largely unknown. In this study the effects of LMP1 on the expression and tyrosine kinase activity of the epidermal growth factor receptor (EGFR) were investigated in C33A human epithelial cells. Stable or transient expression of LMP1 in C33A cells increased expression of the EGFR at both the protein and mRNA levels. In contrast, expression of the EGFR was not induced by LMP1 in EBV-infected B lymphocytes. Stimulation of LMP1-expressing C33A cells with epidermal growth factor (EGF) caused rapid tyrosine phosphorylation of the EGFR (pp170) as well as several other proteins, including pp120, pp85, pp75, and pp55, indicating that the EGFR induced by LMP1 is functional. LMP1 also induced expression of the A20 gene in C33A epithelial cells. In C33A cells, LMP1 expression increased the proliferative response to EGF, as LMP1-expressing C33A cells continued to increase in number when plated in serum-free media supplemented with EGF, while the neo control cells exhibited very low levels of viability and did not proliferate. Immunoblot analysis of protein extracts from nude mouse-passaged NPC tumors also demonstrated that the EGFR is overexpressed in primary NPC tumors as well as those passaged in nude mice. This study suggests that the alteration in the growth patterns of C33A cells expressing LMP1 is a result of increased proliferative signals due to enhanced EGFR expression, as well as protection from cell death due to LMP1-induced A20 expression. The induction of EGFR and A20 by LMP1 may be an important component of EBV infection in epithelial cells and could contribute to the development of epithelial malignancies such as NPC.     

Implication of Ca(2+)-dependent protein tyrosine phosphorylation in carbachol-induced phospholipase D activation in rat pheochromocytoma PC12 cells

The mechanism for carbachol (CCh)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands (111, 91, 84, 74, 65-70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111-, 91-, 84-, and 65-70-kDa proteins peaked within 1 min, and their time-dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65-70 kDa) in a preincubation time- and concentration-dependent fashion. In Ca(2+)-free buffer, CCh-induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65-70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh-induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111-kDa protein. These results suggest that Ca(2+)-dependent protein tyrosine phosphorylation is closely implicated in CCh-induced PLD activation in PC12 cells.     

Novel mechanism by which hemoglobin induces constriction of cerebral arteries

Since oxyhemoglobin (OxyHb) is implicated in the pathogenesis of cerebral vasospasm, we have investigated the role of protein tyrosine phosphorylation in OxyHb-mediated signalling in canine cerebral arteries and cultured canine cerebrovascular smooth muscle cells. OxyHb produced a contraction of basilar artery preparations, which was reversed by genistein, an inhibitor of tyrosine kinases, and PD098059, an inhibitor of mitogen-activated protein kinase. In cerebrovascular smooth muscle cells, OxyHb induced tyrosine phosphorylation of 42, 46, 54-60 and 80-100 kDa proteins with a time-course which paralleled the contractile action of OxyHb, suggesting that these events might be functionally linked. The 42 and 60 kDa proteins were immunologically related to the mitogen-activated protein kinase, extracellular signal regulated protein kinase (ERK2), and to p60c-Src (c-Src), respectively. The increase in protein tyrosine phosphorylation was attenuated by genistein, and the phosphorylation of the 42 kDa protein (ERK2) was inhibited by PD098059. These results suggest that OxyHb-mediated signalling utilizes a protein tyrosine kinase-based mechanism.     

Characterization of p59fyn-mediated signal transduction on T cell activation

Protein tyrosine kinase p59fyn is associated with the TCR-CD3 complex and is suggested to play a role in T cell activation. To determine the molecular mechanism of p59fyn-mediated signal transduction in T cell activation, we established murine T cell hybridoma lines that expressed an elevated amount of wild-type or mutant fyns. Clones that expressed high levels of normal p59fyn and active p59fyn, encoded by wild-type and f-14 mutant fyn respectively, showed enhanced IL-2 production upon stimulation by anti-CD3 antibodies or natural antigen. On the other hand, clones that expressed kinase negative p59fyn and p59fyn with an SH2 (Src-homology 2) deletion encoded by t-1 mutant fyn showed little induction of IL-2 production upon stimulation. These data suggest that p59fyn is important in T cell signaling and that the SH2 sequence plays a critical role in the reaction. Induction of tyrosine phosphorylation of multiple proteins upon antigenic stimulation was augmented similarly in the cells that respectively expressed wild-type and f-14 mutant fyns at elevated levels. The proteins that became highly tyrosine-phosphorylated included phospholipase C (PLC-gamma 1), p95vav, ZAP-70, the MAP kinase, CD3 zeta and unidentified proteins of 120, 100 and 80 kDa. Tyrosine phosphorylation of the 120, 95 and 68 kDa proteins associated with PLC-gamma 1 was also observed in these cells upon stimulation. In contrast, only the 100 kDa protein and the MAP kinase were increasingly tyrosine phosphorylated in the antigen-stimulated cells expressing t-1 fyn. These data suggest that PLC-gamma 1, PLC-gamma 1 associated molecules, p95vav, the 80 kDa protein, ZAP-70 and the CD3 zeta chain may be substrates of p59fyn or of other tyrosine kinases regulated by p59fyn and be important in T cell signaling.     

Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. We have previously shown that autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. In this report, we describe the identification of six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. We demonstrate that autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation.