Src-like Protein Tyrosine Kinases Research Articles

Protein-tyrosine Phosphatase 1B Antagonized Signaling by Insulin-like Growth Factor-1 Receptor and Kinase BRK/PTK6 in Ovarian Cancer Cells*♦

Ovarian cancer, which is the leading cause of death from gynecological malignancies, is a heterogeneous disease known to be associated with disruption of multiple signaling pathways. Nevertheless, little is known regarding the role of protein phosphatases in the signaling events that underlie the disease; such knowledge will be essential to gain a complete understanding of the etiology of the disease and how to treat it. We have demonstrated that protein-tyrosine phosphatase 1B (PTP1B) was underexpressed in a panel of ovarian carcinoma-derived cell lines, compared with immortalized human ovarian surface epithelial cell lines. Stable restoration of PTP1B in those cancer cell lines substantially decreased cell migration and invasion, as well as proliferation and anchorage-independent survival. Mechanistically, the pro-survival IGF-1R signaling pathway was attenuated upon ectopic expression of PTP1B. This was due to dephosphorylation by PTP1B of IGF-1R β-subunit and BRK/PTK6, an SRC-like protein-tyrosine kinase that physically and functionally interacts with the IGF-1R β-subunit. Restoration of PTP1B expression led to enhanced activation of BAD, one of the major pro-death members of the BCL-2 family, which triggered cell death through apoptosis. Conversely, inhibition of PTP1B with a small molecular inhibitor, MSI-1436, increased proliferation and migration of immortalized HOSE cell lines. These data reveal an important role for PTP1B as a negative regulator of BRK and IGF-1Rβ signaling in ovarian cancer cells.

New therapeutic targets in thyroid carcinoma

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (δ-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for δ-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the δ-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged δ-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the δ-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr2]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the δ-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)-pyrazolo-[3,4-d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid δ-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the δ-OR protein. Evidence is presented that the δ-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Dok-1 tyrosine residues at 336 and 340 are essential for the negative regulation of Ras-Erk signalling, but dispensable for rasGAP-binding.

Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling.

SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9

The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells. Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells. Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck. While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck. The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT. In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity. By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells. Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.

Selective induction of tumor necrosis receptor factor 6/decoy receptor 3 release by bacterial antigens in human monocytes and myeloid dendritic cells.

Tumor necrosis factor (TNF) receptor 6/decoy receptor 3 (TR6/DcR3) is an antiapoptosis soluble receptor of the TNF family produced by tumor cells. In this study, TR6 expression in human immune cells was investigated. TR6 mRNA and protein were detectable in selected antigen-presenting cells. Monocytes and myeloid-derived dendritic cells (MDC) released the protein exclusively following stimulation of Toll-like receptor 2 (TLR2) and TLR4 by gram-positive and gram-negative bacterial antigens. Plasmacytoid dendritic cells, activated by bacterial antigens via TLR9 or by viral infection, did not produce the protein. Similarly, activated T cells did not release TR6. The release of TR6 by MDC was dependent on the activation of p42/p44 mitogen-activated protein kinases, Src-like protein tyrosine kinases, and phosphatidylinositol 3-kinase, signaling pathways important for MDC maturation and survival. In agreement with the in vitro data, TR6 levels in serum were significantly elevated in patients with bacterial infections. Overall, these data suggest a novel role for TR6 in immune responses to bacteria.

Src Is the Kinase of the Helicobacter pylori CagA Protein in Vitro and in Vivo

The gastric pathogen Helicobacter pylori uses a type IV secretion system to inject the bacterial CagA protein into gastric epithelial cells. Within the host cell, CagA becomes phosphorylated on tyrosine residues and initiates cytoskeletal rearrangements. We demonstrate here that Src-like protein-tyrosine kinases mediate CagA phosphorylation in vitro and in vivo. First, the Src-specific tyrosine kinase inhibitor PP2 specifically blocks CagA phosphorylation and cytoskeletal rearrangements thereby inhibiting the CagA-induced hummingbird phenotype of gastric epithelial cells. Second, CagA is in vivo phosphorylated by transiently expressed c-Src. Third, recombinant c-Src and lysates derived from c-Src-expressing fibroblasts but not lysates derived from Src-, Yes-, and Fyn-deficient cells phosphorylated CagA in vitro. Fourth, a transfected CagA-GFP fusion protein is phosphorylated in vivo in Src-positive fibroblasts but not in Src-, Yes-, and Fyn-deficient cells. Because a CagA-GFP fusion protein mutated in an EPIYA motif is not efficiently phosphorylated in any of these fibroblast cells, the CagA EPIYA motif appears to constitute the major c-Src phosphorylation site conserved among CagA-positive Helicobacter strains.

Gi-mediated activation of the Syk kinase by the receptor mimetic basic secretagogues of mast cells: role in mediating arachidonic acid/metabolites release.

Syk kinase is essential for FcepsilonRI-mediated signaling and release of inflammatory mediators from mast cells. We now show that activation of rat peritoneal mast cells by the nonimmunological, G(i)-mediated pathway also results in the activation of Syk. We show that compound 48/80 (c48/80), a receptor analogue that activates directly G proteins, activates Syk in a pertussis toxin-sensitive fashion. We further show that Syk activation by c48/80 is blocked by the protein kinase C inhibitor GF109203X, by the phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, by EGTA, and by the selective src-like kinase inhibitor PP1. These results suggest that in the nonimmunological, G(i)-mediated pathway, Syk is located downstream from phospholipase C and phosphatidylinositol 3-kinase. However, in common with the FcepsilonRI-mediated pathway, activation of Syk by c48/80 is dependent on a src-like protein tyrosine kinase. Finally, we show that in the nonimmunological pathway, Syk plays a central role in the release of arachidonic acid/eicosanoid metabolites, but not in the release of prestored mediators such as histamine.

Increased Cytokine-Induced Cytotoxicity of Pancreatic Islet Cells from Transgenic Mice Expressing the Src-like Tyrosine Kinase GTK

The loss of beta cells in type 1 diabetes may involve protein kinases because they control cell growth, differentiation, and survival. Previous studies have revealed that GTK, a Src-like protein tyrosine kinase expressed in beta cells (also named Bsk/Iyk), regulates multiple responses including growth and survival of rat insulinoma cells (RINm5F) and differentiation of neuronal PC12 cells. In the present study, we have generated a transgenic mouse expressing a kinase active GTK mutant (GTK-Y504F) under the control of the rat insulin I promoter to establish a role of GTK in beta cells. Control and GTK-transgenic CBA mice were used for determination of in vivo glucose tolerance and the relative insulin-positive area. Isolated islets from both groups were cultured in the absence and presence of cytokines and insulin secretion, viability and protein expression were assessed. The beta-cell mass of GTK-transgenic mice was increased as a consequence of a larger pancreas and an increased relative beta-cell area. Islets isolated from the transgenic animals exhibited an enhanced glucose-induced insulin release and reduced viability in response to cytokines that could not be explained by higher levels of nitric oxide (NO) compared with control islets. Extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt were all activated by cytokines, but GTK-transgenic islets contained higher basal levels of phosphorylated ERK1/2 and lower basal levels of phosphorylated p38 compared with the control islets. The total amount of activated MAPKs was, however, higher in the cytokine-stimulated transgenic islets compared with the control islets due to increased levels of phospho-ERK1/2. Moreover, the proline-rich tyrosine kinase (PYK) 2 (also named RAFTK/CAK beta/CADTK) levels were elevated in response to a 24-hr exposure to cytokines in control islets but not in the GTK-transgenic islets. These results suggest that although GTK increases the beta-cell mass, it also enhances islet cell death in response to cytokines and may thus be involved in the beta-cell damage in type 1 diabetes.

Tyrosine phosphorylation of the δ-opioid receptor: Evidence for its role in mitogen-activated protein kinase activation and receptor internalization∗

The internalization of G-protein-coupled receptors (GPCRs), including the delta opioid receptor (δ-OR), has been shown to involve the phosphorylation of serine and threonine residues. However, recent studies suggest that these residues may not be the only ones phosphorylated in response to prolonged opioid exposure. Tyrosines also appear important for δ-OR signalling, but it remains unclear whether they undergo phosphorylation. We examined whether the δ-OR, stably expressed in Chinese hamster ovary (CHO-K1) cells, was tyrosine-phosphorylated during prolonged agonist treatment. The epitope-tagged δ-OR was purified by immunoprecipitation, and the presence of phosphorylated tyrosines was detected using anti-phosphotyrosine antibodies. Tyrosine residues in the δ-OR were phosphorylated after exposure to the high-affinity agonist [d-Thr 2]-Leu-enkephalin-Thr (DTLET) in a time- and concentration-dependent manner. Tyrosine phosphorylation of the δ-OR appeared to require the actions of a Src-like protein tyrosine kinase, since the Src inhibitor 4-amino-5-(4-methylphenyl)-7-( t-butyl)-pyrazolo-[3,4- d]-pyrimidine (PP1) attenuated this response. PP1 also attenuated the DTLET-mediated activation of mitogen-activated protein kinase, as well as rapid δ-OR internalization, but not receptor down-regulation. Finally, only opioid agonists that induce receptor internalization via the clathrin-dependent endosomal pathway stimulated significant tyrosine phosphorylation of the δ-OR protein. Evidence is presented that the δ-OR is tyrosine-phosphorylated, and we suggest how this may have an active role in opioid receptor signalling and regulation.

Pleiotropic role of lyn kinase in leukotriene B4–induced eosinophil activation

The authors have examined the role of the src-family of protein tyrosine kinases in leukotriene B4(LTB4)–induced activation of guinea-pig eosinophils. Western blot analysis identified the src-like protein tyrosine kinases p53lyn, p56lyn, p56/59hck, p55fgr, and p56lck whereas p60src, p62yes, p55blk, and p59fyn were not detected. LTB4 promoted a rapid increase in p53/56lyn activity in eosinophils, which peaked at 5 seconds and remained elevated at 60 seconds; hck, fgr, and lck were not activated. A role for p53/56lyn in eosinophil activation was investigated with the use of the src-selective inhibitor PP1 (1 μmol/L to 10 μmol/L), which attenuated LTB4-stimulated p53/56lyn activity and the phosphorylation of extracellular signal-regulated kinase–2 in intact cells. At comparable concentrations, PP1 was also shown to attenuate LTB4-induced nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase activation, chemotaxis, and Ca++-dependent [3H]arachidonic acid (AA) release. Moreover, an inhibitor of mitogen-activated protein kinase kinase-1, PD 098059, significantly inhibited LTB4-induced chemotaxis but had no effect on oxidant production or [3H]AA release. Collectively, these results implicate lyn kinase in LTB4-induced eosinophil activation through the recruitment of divergent cell-signaling pathways.

Pleiotropic role of lyn kinase in leukotriene B4–induced eosinophil activation

AbstractThe authors have examined the role of the src-family of protein tyrosine kinases in leukotriene B4(LTB4)–induced activation of guinea-pig eosinophils. Western blot analysis identified the src-like protein tyrosine kinases p53lyn, p56lyn, p56/59hck, p55fgr, and p56lck whereas p60src, p62yes, p55blk, and p59fyn were not detected. LTB4 promoted a rapid increase in p53/56lyn activity in eosinophils, which peaked at 5 seconds and remained elevated at 60 seconds; hck, fgr, and lck were not activated. A role for p53/56lyn in eosinophil activation was investigated with the use of the src-selective inhibitor PP1 (1 μmol/L to 10 μmol/L), which attenuated LTB4-stimulated p53/56lyn activity and the phosphorylation of extracellular signal-regulated kinase–2 in intact cells. At comparable concentrations, PP1 was also shown to attenuate LTB4-induced nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase activation, chemotaxis, and Ca++-dependent [3H]arachidonic acid (AA) release. Moreover, an inhibitor of mitogen-activated protein kinase kinase-1, PD 098059, significantly inhibited LTB4-induced chemotaxis but had no effect on oxidant production or [3H]AA release. Collectively, these results implicate lyn kinase in LTB4-induced eosinophil activation through the recruitment of divergent cell-signaling pathways.

Tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase correlates with high proliferation rates in sublines derived from the Jurkat leukemia

A prominent tyrosine phosphorylated protein of 85 kDa (p85) was detected in highly proliferative sublines derived from the Jurkat T cell leukemia. We undertook a study to characterize the identity of this protein and its possible role in the hyperproliferative phenotypes observed. Using immunoblot and immunoprecipitation techniques, this protein was characterized as the p85 regulatory subunit of phosphatidylinositol 3-kinase. Cell proliferation and p85 tyrosine phosphorylation was not affected by tyrphostin AG-490, an inhibitor of Jak kinases, wortmannin or LY294002, inhibitors of the activity of the catalytic phosphatidylinositol 3-kinase subunit. Herbimycin-A and PP1, inhibitors of src-like protein tyrosine kinases, and genistein, a general tyrosine kinase inhibitor, inhibited p85 tyrosine phosphorylation and induced cell death in the sublines. PD98059, an inhibitor of Mek, inhibited cell growth of the sublines, but not that of the parental cells. It was concluded that tyrosine phosphorylation of p85 is associated with highly proliferative tumoral phenotypes, at least in T cell leukemias, independent of the phosphatidylinositol 3-kinase activity of the catalytic subunit.

Murine TRAIL (TNF-Related Apoptosis Inducing Ligand) Expression Induced by T Cell Activation Is Blocked by Rapamycin, Cyclosporin A, and Inhibitors of Phosphatidylinositol 3-Kinase, Protein Kinase C, and Protein Tyrosine Kinases: Evidence for TRAIL Induction via the T Cell Receptor Signaling Pathway

TRAIL (TNF-related apoptosis inducing ligand), like other members of the TNF family of proteins, is able to induce apoptosis in sensitive target cells. Recently, cell-surface TRAIL has been shown to be expressed by activated human and mouse T lymphocytes, raising the possibility that TRAIL might be involved in T cell-mediated cytotoxicity and/or immune regulation. In the present study we show by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis that activated, but not resting, mouse T cells express abundant TRAIL mRNA. TRAIL transcripts were detectable within 4 h of T cell activation. A panel of pharmacologic inhibitors was used to investigate the signal transduction pathways involved in TRAIL gene induction following T lymphocyte activation. TRAIL gene expression was sensitive to the src-like protein tyrosine kinase (PTK) inhibitor herbimycin A, as well as the more general PTK inhibitor genistein, suggesting the involvement of a src family PTK. The PKC inhibitors staurosporine and calphostin C, and the phosphatidylinositol 3-kinase (PI3-K) inhibitors wortmannin and LY294002, also prevented TRAIL mRNA transcription by activated T cells, indicating a role for PKC and PI3-K. In addition, TRAIL induction was inhibited by cyclosporin A, implicating the Ca2+/calmodulin-dependent protein phosphatase calcineurin. TRAIL expression was also blocked by rapamycin, which inhibits p70 S6 kinase involved in CD28 and interleukin (IL)-2 receptor signaling. However, TRAIL mRNA expression was not induced by IL-2, suggesting that TRAIL gene induction is not coupled to the IL-2 receptor. Data obtained by RT-PCR were confirmed at the protein level by immunoblotting with TRAIL-specific antibody. We conclude that TRAIL gene induction is initiated through a T cell receptor-associated signaling pathway similar to that responsible for the expression of cytokine genes such as IL-2.

Signaling complex formation of CD44 with src-related kinases

The complex formation of murine CD44 with the src-like protein tyrosine kinases, lck and lyn, was investigated. In accordance with previous observations, stable CD44- lck and CD44- lyn complexes were detected in nonstimulated lymphoid T- and B-cells, respectively. In addition, a direct modulation of lck and lyn by CD44 was observed as revealed by the CD44-dependent translocation of these enzymes to the Triton X-100 resistant cell fraction. To clarify which receptor domain is responsible for the association, peptide binding assays were performed. Interestingly, the synthetic peptide pCD44 (ILAVCIAVNSRRR), which corresponds to the plasma membrane–cytoplasmic interface region of murine CD44, exhibited a high capacity to bind lck and lyn. A single amino acid modification in the position of the cysteine residue completely abolished this interaction, while the truncation of the three tandem arginines significantly decreased it. Remarkably, similar sequences were found in a number of other molecules including subunits of receptors recognizing antigens, immunoglobulins, extracellular matrix components, accessory molecules, cytokines and also in certain viral gene products. Synthetic peptides corresponding to the homologous regions found in CD28 and FcεRIβ were also studied and comparable lck– lyn-binding potentials were detected. These data suggest a novel interaction between src-family kinases and CD44, CD28, FcεRIβ, and provide a simple model for the association of src-like kinases with transmembrane proteins.

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines.

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.

Regulation of DNA-dependent Protein Kinase by the Lyn Tyrosine Kinase

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcs to form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.

Lipid domain structure of the plasma membrane revealed by patching of membrane components.

Lateral assemblies of glycolipids and cholesterol, "rafts," have been implicated to play a role in cellular processes like membrane sorting, signal transduction, and cell adhesion. We studied the structure of raft domains in the plasma membrane of non-polarized cells. Overexpressed plasma membrane markers were evenly distributed in the plasma membrane. We compared the patching behavior of pairs of raft markers (defined by insolubility in Triton X-100) with pairs of raft/non-raft markers. For this purpose we cross-linked glycosyl-phosphatidylinositol (GPI)-anchored proteins placental alkaline phosphatase (PLAP), Thy-1, influenza virus hemagglutinin (HA), and the raft lipid ganglioside GM1 using antibodies and/or cholera toxin. The patches of these raft markers overlapped extensively in BHK cells as well as in Jurkat T-lymphoma cells. Importantly, patches of GPI-anchored PLAP accumulated src-like protein tyrosine kinase fyn, which is thought to be anchored in the cytoplasmic leaflet of raft domains. In contrast patched raft components and patches of transferrin receptor as a non-raft marker were sharply separated. Taken together, our data strongly suggest that coalescence of cross-linked raft elements is mediated by their common lipid environments, whereas separation of raft and non-raft patches is caused by the immiscibility of different lipid phases. This view is supported by the finding that cholesterol depletion abrogated segregation. Our results are consistent with the view that raft domains in the plasma membrane of non-polarized cells are normally small and highly dispersed but that raft size can be modulated by oligomerization of raft components.

Interferon-alpha and -gamma inhibit the growth and neoplastic potential of v-src-transformed human epithelial cells by reducing Src tyrosine kinase activity.

To investigate whether interferons (IFNs) selectively suppress the growth of solid tumor cells with elevated protein tyrosine kinase (PTK) activity, we evaluated the effect of recombinant IFN-alpha2a and IFN-gamma on the proliferative and neoplastic potentials triggered by p60v-src using v-src-transformed HAG-1 human epithelial cells. When compared with control cells harboring the pSV2neo gene, the monolayer growth of v-src-transformed cell lines was inhibited by both recombinant IFNs, in a dose-dependent manner, whereas growth of ras-transfected cell lines was not affected. Moreover, IFNs markedly reduced the clonogenic growth of v-src-transformed cells in soft-agar rather than monolayer growth, suggesting the preferential activity of IFNs on anchorage-independent growth. Pretreatment of cells with Src or the Src-like PTK inhibitor herbimycin A or radicicol, alleviated dose-dependently the growth-inhibitory activity of IFN-alpha2a against v-src-transformed cells, suggesting that IFNs may share a common inhibitory pathway with Src PTK inhibitors. Accordingly, like herbimycin A, IFNs were found to reduce tyrosine phosphorylation of p60v-src and suppressed in vitro p60v-src kinase activity in v-src-transformed cells. Our data, together with the fact that IFNs inhibit the growth potential driven by Src but not by activated Ras, suggest that inhibition of signal transduction pathway through Src to downstream transduction events may be a primary mechanism of IFN-induced anti-prolifeative and anti-tumoral activity.

CD66-mediated phagocytosis of Opa52 Neisseria gonorrhoeae requires a Src-like tyrosine kinase- and Rac1-dependent signalling pathway.

The interaction of Neisseria gonorrhoeae with human phagocytes is a hallmark of gonococcal infections. Recently, CD66 molecules have been characterized as receptors for Opa52-expressing gonococci on human neutrophils. Here we show that Opa52-expressing gonococci or Escherichia coli or F(ab) fragments directed against CD66, respectively, activate a signalling cascade from CD66 via Src-like protein tyrosine kinases, Rac1 and PAK to Jun-N-terminal kinase. The induced signal is distinct from Fcgamma-receptor-mediated signalling and is specific for Opa52, since piliated Opa- gonococci, commensal Neisseria cinerea or E.coli do not stimulate this signalling pathway. Inhibition of Src-like kinases or Rac1 prevents the uptake of Opa52 bacteria, demonstrating the crucial role of this signalling cascade for the opsonin-independent, Opa52/CD66-mediated phagocytosis of pathogenic Neisseria.

The Jak/Stat Pathway and Urokinase Receptor Signaling in Human Aortic Vascular Smooth Muscle Cells

The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53-60, 85-90, and 130-140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59(fyn), p53/56(lyn), p53/59(hck), and p55(fgr). Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-gamma activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the Src-PTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.