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At least 991 records · Page 55Linked to original sources
PubMed2003

The critical role of c-Src and the Shc/Grb2/ERK2 signaling pathway in angiotensin II-dependent VSMC proliferation.

Angiotensin II promotes vascular smooth muscle cell proliferation through the actions of the G protein-coupled AT(1) receptor. Recent evidence suggest that the tyrosine kinase c-Src may mediate this proliferative response. c-Src can signal through multiple intracellular signaling pathways including (1) the Shc/Grb2/ERK2 pathway, (2) the signal transducers and activators of transcription (STATs), (3) the focal adhesion kinase (FAK) signaling pathway, and (4) the phosphatidylinositol 3-kinase (PI3K) signaling pathway. In this study, we sought to determine the extent to which c-Src mediates vascular smooth muscle cell proliferation through the Shc/Grb2/ERK2 signaling pathway. Here we demonstrate that treatment of vascular smooth muscle cells with angiotensin II results in activation of the Shc/Grb2/ERK2 signaling pathway as measured by (1) increased Shc tyrosine phosphorylation, (2) increased c-Src/Shc cellular co-localization, (3) increased Shc/Grb2 co-association, and (4) ERK2 activation. Furthermore, these events are critically dependent on c-Src as pharmacological inhibition of c-Src activity blocked all these cellular occurrences. Most importantly, angiotensin II-dependent cellular proliferation was measured in the presence and absence of c-Src and MEK pharmacological inhibitors. We found that pharmacological inhibition of either c-Src or ERK2 completely eliminated angiotensin II-dependent cellular proliferation. Thus, the data suggest that c-Src and the Shc/Grb2/ERK2 signaling pathway play a critical role in angiotensin II-mediated VSMC proliferation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Epidermal growth factor stimulation can substitute for c-Src overexpression in promoting breast carcinoma invasion.

BACKGROUND AND AIMS: Cancer progression is in large part dependent on the complex process of cell invasion, involving adhesion, motility, and enzymatic proteolysis. Overexpression of the Src proto-oncogene (c-Src), a nonreceptor tyrosine kinase, has been implicated in the progression of both colon and breast cancer. Our group has previously reported that overexpression of c-Src leads to a significant gain in invasive cell behavior in vitro. In this study, we sought to assess the relative importance of epidermal growth factor (EGF) stimulation and c-Src overexpression in conferring an invasive phenotype. METHODS: Breast carcinoma cells and colon epithelial cells which naturally express low levels of c-Src were used for these studies. The cells were transfected so that they overexpressed c-Src; the mock-transfected parent lines were used as controls. Transfectants were tested for changes in invasion patterns after Src inhibition and EGF stimulation. RESULTS: Invasion assays in both cell systems confirmed the importance of c-Src in determining invasive potential. A significant correlation was shown between c-Src kinase protein and cell invasion. Furthermore, Src inhibition significantly inhibited invasion in a dose-dependent manner. To clarify the relative contribution of EGF and c-Src to cell invasion, the ability of cells to invade through growth-factor-reduced matrigel, with or without EGF added, was compared to invasion through intact matrigel. The breast and colon cell lines behave quite differently in this regard. In the colon model, overexpressed c-Src is critical for cell invasion and stimulation with EGF is synergistic with c-Src overexpression. Conversely, the breast carcinoma cells transfected with c-Src were unable to invade without EGF stimulation and did not demonstrate the same synergistic relationship between c-Src and EGF. Instead, our results indicate that in BT474 breast carcinoma cells, EGF can substitute for c-Src in promoting breast cancer cell invasion. CONCLUSIONS: Because most breast carcinomas overexpress c-Src, it behooves one to question the extent to which reducing the amount of EGF and consequent EGFR activity will decrease invasion. In this study, the effects of EGF on cell invasion were determined in light of a single alteration in c-Src expression. Our results show that EGF enhances the impact of c-Src overexpression on invasion. In breast cancer cells, EGF is capable of inducing invasion to the same extent as c-Src overexpression. This suggests that anti-EGFR therapies will be efficacious in retarding breast carcinoma invasion and metastasis.

Animals↗
PubMed1992

Transforming properties and substrate specificities of the protein tyrosine kinase oncogenes ros and src and their recombinants.

To determine the sequences of the oncogenes src (encoded by Rous sarcoma virus [RSV]) and ros (encoded by UR2) that are responsible for causing different transformation phenotypes and to correlate those sequences with differences in substrate recognition, we constructed recombinants of the two transforming protein tyrosine kinases (PTKs) and studied their biological and biochemical properties. A recombinant with a 5' end from src and a 3' end from ros, called SRC x ROS, transformed chicken embryo fibroblasts (CEF) to a spindle shape morphology, mimicking that of UR2. Neither of the two reverse constructs, ROS x SRC I and ROS x SRC II, could transform CEF. However, a transforming variant of ROS x SRC II appeared during passages of the transfected cells and was called ROS x SRC (R). ROS x SRC (R) contains a 16-amino-acid deletion that includes the 3' half of the transmembrane domain of ros. Unlike RSV, ROS x SRC (R) also transformed CEF to an elongated shape similar to that of UR2. We conclude that distinct phenotypic changes of RSV- and UR2-infected cells do not depend solely on the kinase domains of their oncogenes. We next examined cellular proteins phosphorylated by the tyrosine kinases of UR2, RSV, and their recombinants as well as a number of other avian sarcoma viruses including Fujinami sarcoma virus Y73, and some ros-derived variants. Our results indicate that the UR2-encoded receptorlike PTK P68gag-ros and its derivatives have a very restricted substrate specificity in comparison with the nonreceptor PTKs encoded by the rest of the avian sarcoma viruses. Data from ros and src recombinants indicate that sequences both inside and outside the catalytic domains of ros and src exert a significant effect on the substrate specificity of the two recombinant proteins. Phosphorylation of most of the proteins in the 100- to 200-kDa range correlated with the presence of the 5' src domain, including the SH2 region, but not with the kinase domain in the recombinants. This corroborates the conclusion given above that the kinase domain of src or ros per se is not sufficient to dictate the transforming morphology of these two oncogenes. High-level tyrosyl phosphorylation of most of the prominent substrates of src is not sufficient to cause a round-shape transformation morphology.

Amino Acid Sequence↗
PubMed1994

Microtubule reorganization and lysosome redistribution by a viral v-src oncogene, in mouse Balb/3T3 cells expressing human EGF receptor.

The epidermal growth factor (EGF)-induced endocytosis of its receptor is an obligatory pathway for the cellular regulation of the EGF-specific receptor (EGF-R). BNER4 is a mouse Balb/3T3 cell line transfected with human EGF-R complementary DNA (cDNA). B4/src-13 and B4/src-24 are BNER4 cells transfected with a viral oncogene v-src. Indirect immunofluorescence study demonstrated that EGF-R was mostly localized at the perinuclear region in BNER4 cells at 60 min after EGF addition, whereas it was diffusely distributed throughout the cytoplasm in its v-src transfectants. Double indirect immunofluorescence study further confirmed that EGF-R was localized in lysosomes in BNER4 and B4/src-13 cells at 60 min after EGF addition. Intracellular distribution of the Golgi apparatus, clathrin-coated vesicles and early endosomes were similar in all cell lines. However, the lysosomes detected by anti-lysosomal membrane protein (LGP85) antibodies were diffusely distributed throughout the cytoplasm in the v-src transfectacts. By contrast, in the parental BNER4 cells, the lysosomes were mostly localized in the perinuclear region. The organization of microtubules, but not of actin, was markedly different between BNER4 cells and its v-src transfectants. Nocodazole, which depolymerizes microtubules, altered the distribution of the lysosomes and EGF-R in BNER4 cells. Both intracellular lysosome distribution and microtubule organization in nocodazole-treated BNER4 cells were found to be similar to those in its v-src transfectants without nocodazole treatment. These findings support the notion that changes in lysosome distribution may be correlated with microtubule reorganization by v-src in mouse Balb/3T3 cells.

3T3 Cells↗
PubMed1991

Expression of N-myc and c-src protooncogenes correlating to the undifferentiated phenotype and prognosis of primary neuroblastomas.

Genomic amplification of the N-myc protooncogene in neuroblastomas correctly predicts poor outcome for the patients. However, the prognosis for neuroblastomas with a single copy of N-myc is also poor in cases diagnosed after 1 year of age but good in infantile cases. To elucidate the different prognoses depending upon the age of the patients with neuroblastoma, we performed an analysis of the expression of protooncogenes related to neural differentiation. We examined the genomic amplification of N-myc in 26 specimens of neuroblastomas and further analyzed 22 of the 26 cases for expression of N-myc, c-src, c-Ha-ras, and c-fos. Consequently, we observed frequent overexpression of N-myc in undifferentiated neuroblastomas and enhanced expression of c-src and c-Ha-ras in infantile neuroblastomas with favorable prognosis and in neuroblastomas differentiated by chemotherapy. These findings suggest that c-src and c-Ha-ras play important roles in the neural differentiation of infantile neuroblastomas.

Blotting, Northern↗
PubMed1996

Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells.

The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene-transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c-Ha-ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung modules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v-src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel-coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors.

3T3 Cells↗
PubMed1999

Activating SRC mutation in a subset of advanced human colon cancers.

The discovery of Rous sarcoma virus (RSV) led to the identification of cellular Src (c-Src), a non-receptor tyrosine kinase, which has since been implicated in the development of numerous human cancers. c-Src has been found to be highly activated in colon cancers, particularly in those metastatic to the liver. Studies of the mechanism of c-Src regulation have suggested that c-Src kinase activity is downregulated by phosphorylation of a critical carboxy-terminal tyrosine (Tyr 530 in human c-Src, equivalent to Tyr 527 in chicken Src) and have implied the existence of activating mutations in this C-terminal regulatory region. We report here the identification of a truncating mutation in SRC at codon 531 in 12% of cases of advanced human colon cancer tested and demonstrate that the mutation is activating, transforming, tumorigenic and promotes metastasis. These results provide, for the first time, genetic evidence that activating SRC mutations may have a role in the malignant progression of human colon cancer.

3T3 Cells↗
PubMed2002

Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain.

STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3.Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation.

Animals↗
PubMed1997

Association between v-Src and protein kinase C delta in v-Src-transformed fibroblasts.

In response to the kinase activity of v-Src there is an increase in the membrane association of the novel protein kinase C (PKC) isoform PKC delta (Zang, Q., Frankel, P., and Foster, D. A. (1995) Cell Growth Differ. 6, 1367-1373). We report here that in v-Src-transformed cells PKC delta co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine-phosphorylated PKC delta had reduced enzymatic activity relative to the non-tyrosine-phosphorylated PKC delta from v-Src-transformed cells. The association between Src and PKC delta was dependent upon both an active Src kinase and membrane association. The association between c-Src Y527F and PKC delta was substantially enhanced by mutating a PKC phosphorylation site at Ser-12 in Src to Ala indicating that PKC delta phosphorylation of Src at Ser-12 destabilizes the interaction, possibly in a negative feedback loop. These data demonstrate that upon recruitment of PKC delta to the membrane in v-Src-transformed cells there is the formation of a Src.PKC delta complex in which PKC delta becomes phosphorylated on tyrosine and down-regulated.

Animals↗
PubMed2000

Selective inhibition of splicing at the avian sarcoma virus src 3' splice site by direct-repeat posttranscriptional cis elements.

The direct-repeat elements (dr1) of avian sarcoma virus (ASV) and leukosis virus have the properties of constitutive transport elements (CTEs), which facilitate cytoplasmic accumulation of unspliced RNA. It is thought that these elements represent binding sites for cellular factors. Previous studies have indicated that in the context of the avian sarcoma virus genome, precise deletion of both ASV dr1 elements results in a very low level of virus replication. This is characterized by a decreased cytoplasmic accumulation of unspliced RNA and a selective increase in spliced src mRNA. Deletion of either the upstream or downstream dr1 results in a delayed-replication phenotype. To determine if the same regions of the dr1 mediate inhibition of src splicing and unspliced RNA transport, point mutations in the upstream and downstream elements were studied. In the context of viral genomes with single dr1 elements, the effects of the mutations on virus replication and increases in src splicing closely paralleled the effects of the mutations on CTE activity. For mutants strongly affecting CTE activity and splicing, unspliced RNA but not spliced RNA turned over in the nucleus more rapidly than wild-type RNA. In the context of wild-type virus containing two dr1 elements, mutations of either element that strongly affect CTE activity caused a marked delay of virus replication and a selective increase in src splicing. However, the turnover of the mutant unspliced RNA as well as the spliced mRNA species did not differ significantly from that of the wild type. These results suggest the dr1 elements in ASV act to selectively inhibit src splicing and that both elements contribute to the fitness of the wild-type virus. However, a single dr1 element is sufficient to stabilize unspliced RNA.

Animals↗
PubMed1996

v-src interferes with the in vitro erythropoietic stimulatory ability of spleen stromal cells through repression of vascular cell adhesion molecule-1 and stem cell factor.

A mouse spleen stromal cell line, MSS62, can create an in vitro erythropoietic microenvironment in which development of erythropoietin-responsive progentor cells is stimulated by cell-cell contact via stem cell factor (SCF)/c-Kit and vascular cell adhesion molecule-1 (VCAM-1)/very late activation antigen-4 (VLA-4) interactions between stromal and erythroid cells. To find out the effect of src on the erythropoietic microenvironment, MSS62 cells were transfected with v-src oncogene, and its effect on erythropoietic stimulatory activity was measured. Transfectants with high v-Src activity showed reduction in erythropoietic stimulatory activity. A decrease in cell-surface VCAM-1 and SCF mRNA was accompanied by high v-Src activity. These results suggest that v-Src interferes with the erythropoietic stimulatory activity of the stromal cells through repression of VCAM-1 and SCF.

Animals↗
PubMed1995

Direct tumorigenic conversion of human gallbladder carcinoma cells by v-src but not by activated c-H-ras oncogene.

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-I human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-I cells might be driven directly by p60v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-I cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells.

Adenocarcinoma↗
PubMed2002

Src in cancer: deregulation and consequences for cell behaviour.

Considerable evidence now implicates elevated expression and/or activity of Src in cancer development. In cells, endogenous Src is switched from an inactive to an active state by a variety of mechanisms that simultaneously relieve constraints on the kinase and protein-interacting Src homology (SH) domains. As a result, Src is translocated to the cell periphery, often to sites of cell adhesion, where myristylation mediates attachment to the inner surface of the plasma membrane. From these peripheral sites, Src's catalytic activity initiates intracellular signal transduction pathways that influence cell growth and adhesion strength, the latter contributing to control of cell migration. De-regulation in cancer cells may therefore enhance tumour growth and/or stimulate migratory or invasive potential in cells that would normally be relatively non-motile. Evidence now exists to suggest that Src may also influence the life or death decisions that cells make during many biological processes. Thus, Src modulation in cancer cells can alter cell responses that are often perturbed in cancer. Consequently, there is optimism that drugs which inhibit Src's kinase activity, or the activity of its downstream effectors, might have profound effects on cancer cell behaviour and be useful therapeutic agents.

Animals↗
PubMed1991

The expression of the c-src+ proto-oncogene mRNA in foetal striatal tissue grafts.

We have studied by in situ hybridization the mRNA expression of the pp60c-src proto-oncogene in grafts of foetal striatal neurons implanted into the ibotenic acid lesioned adult rat neostriatum. Animals were studied at 5, 10, 15, 30 days and 4 months after transplantation. Using 35S-labelled specific oligonucleotide probes, high levels of mRNA for pp60c-src+ but not pp60c-src- were found in the normal embryonic striatum and in striatal grafts up to 1 month after implantation; no hybridization signal was obtained in the normal adult striatum or in 4-month old grafts. The data suggest that pp60c-src+ is highly expressed in developing, but not mature, neurons and may play a fundamental role in the maturation and differentiation of grafted embryonic striatal neurons.

Animals↗
PubMed1998

Overexpression of c-Src in areas of hyperproliferation in head and neck cancer, premalignant lesions and benign mucosal disorders.

To examine which proteins are responsible for the elevated protein tyrosine kinase (PTK) activity in human head and neck squamous cell carcinoma (HNSCC) and adjacent histologically normal epithelium, paraffin embedded sections of these tissues were stained for PTK c-Src. Using double labeling techniques and antibodies against both the proliferation marker Ki-67 and PTK c-Src, we have shown that c-Src is overexpressed in areas of hyperproliferation in HNSCC, dysplastic epithelium, benign papillomas and inflamed normal tissue. Our data indicate that c-Src is (one of) the protein(s) responsible for the increased PTK activity in HNSCC. We could not demonstrate that c-Src expression is responsible for the increased PTK activity in normal epithelium adjacent to tumour tissue. We assume that c-Src plays a role in the increased proliferation seen in (pre)malignant and benign epithelial lesions as well as in reactive inflammatory epithelial hyperplasia.

Adult↗
PubMed2003

Integrins regulate NMDA receptor-mediated synaptic currents.

Synapses contain high concentrations of integrins, adhesion receptors known to influence the operation of neighboring transmembrane proteins. Evidence that integrins are important for consolidation of long-term potentiation suggests that these adhesion proteins may modulate activities of synaptic glutamate receptors. The present study provides a first test of the possibility that integrins modulate synaptic N-methyl-d-aspartate (NMDA)-type glutamate receptor activities. Excitatory postsynaptic currents (EPSCs) were recorded with whole cell clamp from hippocampal slices in which AMPA-type glutamate receptors and GABA(A) receptors were pharmacologically blocked. Microperfusion of the peptide integrin ligand gly-arg-gly-asp-ser-pro (GRGDSP) caused an approximately twofold increase in the amplitude and duration of NMDA receptor-gated synaptic currents. Control peptides had no effect. Paired-pulse facilitation was unchanged, indicating that the ligand did not modify neurotransmitter release probabilities. Infusion of the Src kinase antagonist PP2 but not the control drug 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine eliminated the enhancing effect of GRGDSP. Integrins regulate Src kinases that are known to phosphorylate NMDA receptors. It is concluded that integrins act through this route to exert potent modulatory effects on the operation of NMDA receptors.

Animals↗
PubMed2005

Crk-associated substrate tyrosine phosphorylation sites are critical for invasion and metastasis of SRC-transformed cells.

Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.

Animals↗
PubMed1991

Platelet-derived growth factor receptor sequences important for binding of src family tyrosine kinases.

We previously demonstrated that src family tyrosine kinases associate with activated platelet-derived growth factor receptors. We have now investigated the requirement for tyrosine phosphorylation sites in the human platelet-derived growth factor receptor for this binding. Tyrosine 857, but not tyrosine 751, is required for efficient association. Furthermore, even though src family tyrosine kinases associate with receptors lacking tyrosine 751, they are not activated, implying that association does not invariably lead to activation.

Animals↗