Acidic fibroblast growth factor (FGF-1) signaling inhibits peroxynitrite-induced cell death during pancreatic tumorigenesis

Previous immunohistochemical studies have demonstrated enhanced appearance of FGF-1 and nitrotyrosine, a footprint of reactive nitrogen species peroxynitrite (ONOO(-)), in human pancreatic adenocarcinoma. We have examined the consequences of constitutive exposure to FGF-1 in nontumorigenic rat ductal epithelial cells (ARIP). ARIP cells were transduced with either a secreted chimera of FGF-1, ARIP(FGF-1), or a control plasmid, 65 RIP(betag). These cells were evaluated for alteration in growth and morphology, responses to ONOO(-) (protein tyrosine nitration/phosphorylation), and in vivo tumor formation. ARIP(FGF-1) cells, in contrast to 65 RIP(betag), demonstrated a transformed morphology, a 2-fold increased growth rate, and enhanced protein tyrosine phosphorylation. Treatment with 150 microM ONOO(-) resulted in 86 and 7% (p <.01) death of ARIP(betag) and ARIP(FGF-1), respectively. Exposure of 65 RIP(betag) cells to ONOO(-) enhanced tyrosine phosphorylation and tyrosine nitration of several polypeptides. Cell signaling by FGF-1 enhanced both phosphorylation and nitration of tyrosine residues in target proteins modified by ONOO(-). ARIP(betag) cells failed to exhibit tumor formation in nude mice, but at d 7 in vivo cells were TUNEL and nitrotyrosine positive and FGF-1 negative. ARIP(FGF-1) cells readily formed tumor nodules, exhibiting features of pancreatic adenocarcinoma and demonstrating FGF-1-positive, nitrotyrosine-positive, and TUNEL-negative epithelium. These results suggest an interdependent role between FGF-1 and ONOO(-) during the development and progression of pancreatic adenocarcinoma.     

c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function

Accumulating evidence indicates that interactions between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases display synergistic increases in DNA synthesis, soft agar growth, and tumor formation in nude mice, and increased phosphorylation of the receptor substrates Shc and phospholipase gamma as compared with single overexpressors. These parameters correlated with the ability of c-Src and EGFR to form an EGF-dependent heterocomplex in vivo. Here we provide evidence that association between c-Src and EGFR can occur directly, as shown by receptor overlay experiments, and that it results in the appearance of two novel tyrosine phosphorylations on the receptor that are seen both in vitro and in vivo following EGF stimulation. Edman degradation analyses and co-migration of synthetic peptides with EGFR-derived tryptic phosphopeptides identify these sites as Tyr845 and Tyr1101. Tyr1101 lies within the carboxyl-terminal region of the EGFR among sites of receptor autophosphorylation, while Tyr845 resides in the catalytic domain, in a position analogous to Tyr416 of c-Src. Phosphorylation of Tyr416 and homologous residues in other tyrosine kinase receptors has been shown to be required for or to increase catalytic activity, suggesting that c-Src can influence EGFR activity by mediating phosphorylation of Tyr845. Indeed, EGF-induced phosphorylation of Tyr845 was increased in MDA468 human breast cancer cells engineered to overexpress c-Src as compared with parental MDA 468 cells. Furthermore, transient expression of a Y845F variant EGFR in murine fibroblasts resulted in an ablation of EGF-induced DNA synthesis to nonstimulated levels. Together, these data support the hypothesis that c-Src-mediated phosphorylation of EGFR Tyr845 is involved in regulation of receptor function, as well as in tumor progression.     

CD44 interaction with c-Src kinase promotes cortactin-mediated cytoskeleton function and hyaluronic acid-dependent ovarian tumor cell migration

In this study we have demonstrated that both CD44 (the hyaluronan (HA) receptor) and c-Src kinase are expressed in human ovarian tumor cells (SK-OV-3.ipl cell line), and that these two proteins are physically associated as a complex in vivo. Using a recombinant cytoplasmic domain of CD44 and an in vitro binding assay, we have detected a specific interaction between CD44 and c-Src kinase. Furthermore, the binding of HA to SK-OV-3.ipl cells promotes c-Src kinase recruitment to CD44 and stimulates c-Src kinase activity, which, in turn, increases tyrosine phosphorylation of the cytoskeletal protein, cortactin. Subsequently, tyrosine phosphorylation of cortactin attenuates its ability to cross-link filamentous actin in vitro. In addition, transfection of SK-OV-3.ipl cells with a dominant active form of c-Src (Y527F)cDNA promotes CD44 and c-Src association with cortactin in membrane projections, and stimulates HA-dependent/CD44-specific ovarian tumor cell migration. Finally, overexpression of a dominant-negative mutant of c-Src kinase (K295R) in SK-OV-3.ipl cells impairs the tumor cell-specific phenotype. Taken together, these findings strongly suggest that CD44 interaction with c-Src kinase plays a pivotal role in initiating cortactin-regulated cytoskeleton function and HA-dependent tumor cell migration, which may be required for human ovarian cancer progression.     

Identification of protein-tyrosine phosphatase 1B as the major tyrosine phosphatase activity capable of dephosphorylating and activating c-Src in several human breast cancer cell lines

c-Src tyrosine kinase activity is elevated in several types of human cancer, and this has been attributed to elevated c-Src expression levels, increased c-Src specific activity, and activating mutations in c-Src. We have found a number of human breast cancer cell lines with elevated c-Src specific activity that also possess elevated phosphatase activity directed against the carboxyl-terminal negative regulatory domain of Src family kinases. To identify this phosphatase, cell extracts from MDA-MB-435S cells were chromatographed and the fractions were assayed for phosphatase activity. Four peaks of phosphatase activity directed against the nonspecific substrate poly(Glu/Tyr) were detected. One peak also dephosphorylated a peptide modeled against the c-Src carboxyl-terminal negative regulatory domain and intact human c-Src. Immunoblotting and immunodepletion experiments identified the phosphatase as protein-tyrosine phosphatase 1B (PTP1B). Examination of several human breast cancer cell lines with increased c-Src activity showed elevated levels of PTP1B protein relative to normal control breast cells. In vitro c-Src reactivation experiments confirmed the ability of PTP1B to dephosphorylate and activate c-Src. In vivo overexpression of PTP1B in 293 cells caused a 2-fold increase of endogenous c-Src kinase activity. Our findings indicate that PTP1B is the primary protein-tyrosine phosphatase capable of dephosphorylating c-Src in several human breast cancer cell lines and suggests a regulatory role for PTP1B in the control of c-Src kinase activity.     

Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215

To elucidate the molecular mechanisms by which human epidermal growth factor receptor/heregulin (HER2/HRG) influence the migratory potential of breast cancer cells, we have used phospho-specific antibodies against c-Src kinase and focal adhesion kinase (FAK). This study establishes that HER2/HRG signaling selectively upregulates Tyr phosphorylation of c-Src at Tyr-215 located within the SH2 domain, increases c-Src kinase activity and selectively upregulates Tyr phosphorylation of FAK at Tyr-861. HER2-overexpressing tumors showed increased levels of c-Src phosphorylation at Tyr-215. These findings suggest that HER2/HRG influence metastasis of breast cancer cells through a novel signaling pathway involving phosphorylation of FAK tyrosine 861 via activation of c-Src tyrosine 215.     

Endothelin-1 decreases gap junctional intercellular communication by inducing phosphorylation of connexin 43 in human ovarian carcinoma cells

Endothelin-1 (ET-1) is overexpressed in ovarian carcinoma and acts as an autocrine factor selectively through the ETA receptor (ETAR) to promote tumor cell proliferation, survival, neovascularization, and invasiveness. Loss of gap junctional intercellular communication (GJIC) is critical for tumor progression by allowing the cells to escape growth control. Exposure of HEY and OVCA 433 ovarian carcinoma cell lines to ET-1 led to a 50-75% inhibition in intercellular communication and to a decrease in the connexin 43 (Cx43)-based gap junction plaques. To investigate the phosphorylation state of Cx43, ovarian carcinoma cell lysates were immunoprecipitated and transient tyrosine phosphorylation of Cx43 was detected in ET-1-treated cells. BQ 123, a selective ETAR antagonist, blocked the ET-1-induced Cx43 phosphorylation and cellular uncoupling. Gap junction closure was prevented by tyrphostin 25 and by the selective c-Src inhibitor, PP2. Furthermore, the increased Cx43 tyrosine phosphorylation was correlated with ET-1-induced increase of c-Src activity, and PP2 suppressed the ET-1-induced Cx43 tyrosine phosphorylation, indicating that inhibition of Cx43-based GJIC is mainly mediated by the Src tyrosine kinase pathway. In vivo, the inhibition of human ovarian tumor growth in nude mice induced by the potent ETAR antagonist, ABT-627, was associated with a reduction of Cx43 phosphorylation. These findings indicate that the signaling mechanisms involved in GJIC disruption on ovarian carcinoma cells depend on ETAR activation, which leads to the Cx43 tyrosine phosphorylation mediated by c-Src, suggesting that ETAR blockade may contribute to the control of ovarian carcinoma growth and progression also by preventing the loss of GJIC.     

Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity.

Amplification and overexpression of the neu (c-erbB2) proto-oncogene has been implicated in the pathogenesis of 20 to 30% of human breast cancers. Although the activation of Neu receptor tyrosine kinase appears to be a pivotal step during mammary tumorigenesis, the mechanism by which Neu signals cell proliferation is unclear. Molecules bearing a domain shared by the c-Src proto-oncogene (Src homology 2) are thought to be involved in signal transduction from activated receptor tyrosine kinases such as Neu. To test whether c-Src was implicated in Neu-mediated signal transduction, we measured the activity of the c-Src tyrosine kinase in tissue extracts from either mammary tumors or adjacent mammary epithelium derived from transgenic mice expressing a mouse mammary tumor virus promoter/enhancer/unactivated neu fusion gene. The Neu-induced mammary tumors possessed six- to eightfold-higher c-Src kinase activity than the adjacent epithelium. The increase in c-Src tyrosine kinase activity was not due to an increase in the levels of c-Src but rather was a result of the elevation of its specific activity. Moreover, activation of c-Src was correlated with its ability to complex tyrosine-phosphorylated Neu both in vitro and in vivo. Together, these observations suggest that activation of the c-Src tyrosine kinase during mammary tumorigenesis may occur through a direct interaction with activated Neu.     

Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases.

The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2). The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions. No tyrosine autophosphorylation was detected when PDGF beta was expressed alone. PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+. When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in [3H]inositol 4,5-biphosphate ([3H]IP2) and [3H]inositol 1,4,5-triphosphate [3H]IP3 production. Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as [3H]IP2 and [3H]IP3 generation. Phosphorylated PLC gamma 2 was found predominantly in membrane fractions. To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk. These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions. When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and [3H]IP2 and [3H]IP3 production increased 11.0- and 7.0-fold, respectively. Csk expressed alone with PLC gamma 2 was ineffective. Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor. In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src. These observations indicate that S. pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.     

Evidence that light modulates protein nitration in rat retina

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4-33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.     

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.     

CEACAM6 cross-linking induces caveolin-1-dependent, Src-mediated focal adhesion kinase phosphorylation in BxPC3 pancreatic adenocarcinoma cells

Despite lacking transmembrane or intracellular domains, glycosylphosphatidylinositol-anchored proteins can modulate intracellular signaling events, in many cases through aggregation within membrane "lipid raft" microdomains. CEACAM6 is a glycosylphosphatidylinositol-linked cell surface protein of importance in the anchorage-independent survival and metastasis of pancreatic adenocarcinoma cells. We examined the effects of antibody-mediated cross-linking of CEACAM6 on intracellular signaling events and anchorage-independent survival of the CEACAM6-overexpressing pancreatic ductal adenocarcinoma cell line, BxPC3. CEACAM6 cross-linking increased c-Src activation and induced tyrosine phosphorylation of p125(FAK) focal adhesion kinase. Focal adhesion kinase phosphorylation was dependent on c-Src kinase activation, for which caveolin-1 was required. CEACAM6 cross-linking induced a significant increase in cellular resistance to anoikis. These observations represent the first characterization of the mechanism through which this important cell surface oncoprotein influences intracellular signaling events and hence malignant cellular behavior.     

Identification of targets of c-Src tyrosine kinase by chemical complementation and phosphoproteomics

The cellular proto-oncogene c-Src is a nonreceptor tyrosine kinase involved in cell growth and cytoskeletal regulation. Despite being dysregulated in a variety of human cancers, its precise functions are not fully understood. Identification of the substrates of c-Src remains a major challenge, because there is no simple way to directly stimulate its activity. Here we combine the chemical rescue of mutant c-Src and global quantitative phosphoproteomics to obtain the first high resolution snapshot of the range of tyrosine phosphorylation events that occur in the cell immediately after specific c-Src stimulation. After enrichment by anti-phosphotyrosine antibodies, we identified 29 potential novel c-Src substrate proteins. Tyrosine phosphopeptide mapping allowed the identification of 382 nonredundant tyrosine phosphopeptides on 213 phosphoproteins. Stable isotope labeling of amino acids in cell culture-based quantitation allowed the detection of 97 nonredundant tyrosine phosphopeptides whose level of phosphorylation is increased by c-Src. A large number of previously uncharacterized c-Src putative protein targets and phosphorylation sites are presented here, a majority of which play key roles in signaling and cytoskeletal networks, particularly in cell adhesion. Integrin signaling and focal adhesion kinase signaling pathway are two of the most altered pathways upon c-Src activation through chemical rescue. In this context, our study revealed the temporal connection between c-Src activation and the GTPase Rap1, known to stimulate integrin-dependent adhesion. Chemical rescue of c-Src provided a tool to dissect the spatiotemporal mechanism of activation of the Rap1 guanine exchange factor, C3G, one of the identified potential c-Src substrates that plays a role in focal adhesion signaling. In addition to unveiling the role of c-Src in the cell and, specifically, in the Crk-C3G-Rap1 pathway, these results exemplify a strategy for obtaining a comprehensive understanding of the functions of nonreceptor tyrosine kinases with high specificity and kinetic resolution.     

Induction of motor neuron apoptosis by free 3-nitro-L-tyrosine

Peroxynitrite-dependent tyrosine nitration has been postulated to be involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Evidence supporting this supposition includes the appearance of both free and protein-linked 3-nitro-l-tyrosine (nitrotyrosine) in both sporadic and familial ALS, as well as of increased free nitrotyrosine levels in the spinal cord of transgenic mice expressing ALS-linked superoxide dismutase mutants at symptom onset. Here we demonstrate that incubation with clinically relevant concentrations of nitrotyrosine induced apoptosis in motor neurons cultured with trophic factors. Nitrotyrosine was bound to proteins, but it was not incorporated into alpha-tubulin, as previously demonstrated for other cell types. Neither inhibition of nitric oxide production nor scavenging of superoxide and peroxynitrite prevented increases in cell nitrotyrosine immunoreactivity or motor neuron death, suggesting that these effects are not due to the endogenous formation of reactive nitrogen species. In contrast, some populations of astrocytes incorporated nitrotyrosine into alpha-tubulin, but free nitrotyrosine had no effect on the viability and phenotype of astrocytes in culture, as evaluated by glial fibrillary acidic protein immunoreactivity, cell growth and morphology. Co-culture of motor neurons on astrocyte monolayers delayed, but did not prevent, nitrotyrosine-induced motor neuron death. These results suggest that free nitrotyrosine may play a role in the induction of motor neuron apoptosis in ALS.     

Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells

HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.