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PubMed2003

c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts.

Activated fibroblast growth factor receptor 1 (FGFR1) propagates FGF signals through multiple intracellular pathways via intermediates FRS2, PLCgamma, and Ras. Conflicting reports exist concerning the interaction between FGFR1 and Src family kinases. To address the role of c-Src in FGFR1 signaling, we compared proliferative responses of murine embryonic fibroblasts (MEF) deficient in c-Src, Yes, and Fyn to MEF expressing either endogenous levels or overexpressing c-Src. MEF with endogenous c-Src had significantly greater FGF-induced DNA synthesis and proliferation than cells lacking or overexpressing c-Src. This was related directly to c-Src expression by analysis of c-Src-deficient cells transfected with and sorted for varying levels of a c-Src expression vector. This suggests an "optimal" quantity of c-Src expression for FGF-induced proliferation. To determine if this was a general phenomenon for growth factor signaling pathways utilizing c-Src, responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) were examined. As for FGF, responses to EGF were clearly inhibited when c-Src was absent or overexpressed. In contrast, varying levels of c-Src had little effect on responses to PDGF or LPA. The data show that mitogenic pathways activated by FGF-1 and EGF are regulated by c-Src protein levels and appear to differ significantly from those activated by PDGF and LPA.

Animals↗
PubMed2005

c-Src: bridging the gap between phosphorylation- and acidification-induced gap junction channel closure.

Gap junctions are a unique type of intercellular junction that mediate the direct exchange of small molecules between neighboring cells and play critical roles in the normal function of numerous organs. Mutations in the connexin proteins that make up gap junctions have been implicated in numerous human skin and neurosensory disorders. The ability of gap junctions to transmit molecules between cells is regulated by intracellular pH, the phosphorylation state of connexin, and the interaction of connexin with other cellular proteins. This Perspective focuses on the novel and complex events initiated by intracellular acidification resulting from tissue ischemia or hypoxia that lead to the interruption of intercellular communication between astrocytes. These events include alterations in connexin43 (Cx43) phosphorylation, disruption of beta-actin binding to Cx43, and the induced interaction of Cx43 with the c-Src tyrosine kinase, extracellular signal-regulated kinase 1 and 2, and mitogen-activated protein kinase phosphatase 1.

Animals↗
PubMed1992

An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.

Animals↗
PubMed1994

Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.

Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.

Amino Acid Sequence↗
PubMed2001

The thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor.

The mitogen-activated protein kinase signaling cascade is used by many G protein-coupled receptors to initiate functional events. In this study, activation of the Gq/G11-coupled thromboxane A2 (TxA2) receptor (TP) by the TxA2 mimetic IBOP in ECV304 cells was found to induce extracellular regulated kinase (ERK) phosphorylation and tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), which were inhibited by the TP antagonist SQ29548, the EGFR kinase inhibitor AG1478, the Src family kinase inhibitor PP1, the Gi/o protein inhibitor pertussis toxin (PTX), or the protein kinase C (PKC) inhibitor calphostin C. TP activation also increased Src kinase activity, which was blocked by PTX, PP1, and calphostin C, but not by AG1478, indicating that Src activation occurs before phosphorylation of EGFR. Blockade of Src activity by expression of dominant negative mutant of Src inhibits mitogen-activated protein kinase (MAPK) activation induced by TxA2. ERK activation induced by the PKC activator phorbol myristate acetate was inhibited by PTX, PP1, AG1478, and calphostin C. In contrast, activation of ERK by lysophosphatidic acid, a Gi-coupled receptor activator, was inhibited by PTX, PP1, and AG1478, but not by calphostin C. Thus, TP-stimulated ERK activation requires Gi, which in turn requires PKC activation. Immunoprecipitation of Galphai showed increased association of Galphai with TPalpha following PKC activation. In conclusion, TPalpha is directly coupled to the Gi protein by a PKC-regulated mechanism; Gi coupling causes Src-dependent transactivation of the EGFR, which is the dominant pathway in TP-mediated ERK activation.

Cell Line↗
PubMed1992

Phosphorylation of connexin43 in cells containing mutant src oncogenes.

Disruption of gap junctional communication (measured by intercellular dye transfer) in cultured fibroblasts by pp60v-src is correlated with phosphorylation of the gap junction protein, connexin43 (cx43), on tyrosine. In this report, we examine the functional relevance of these observations by studying cx43 phosphorylation in cells containing kinase-active, non-myristylated pp60(2A527F) or pp60v-src temperature sensitive (ts) for transformation. Non-transformed cells expressing pp60(2A527F) transferred fluorescent dye at high levels and contained cx43 that was phosphorylated predominately on serine. In contrast, cells transformed by kinase-active, myristylated pp60(527F) did not transfer dye and contained cx43 proteins which were phosphorylated on serine and tyrosine. Additionally, activation of ts pp60v-src tyrosine kinase activity upon shift of cells to the permissive temperature was correlated with a rapid increase in the phosphorylated tyrosine content of cx43 proteins and loss of gap junctional communication. These combined results suggested that cx43 is a substrate of pp60v-src whose phosphorylation on tyrosine may be involved in the disruption of gap junctional communication observed in Rous sarcoma virus (RSV)-transformed cells.

Animals↗
PubMed1998

An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasms.

In previous work, we established a new monoclonal antibody that specifically recognizes the active form of c-Src tyrosine kinase (Kawakatsu et al, 1996). To determine whether c-Src is active in colorectal tumorigenesis, we examined the expression of an active form of c-Src in human normal mucosa, hyperplastic polyps, adenomas, and adenocarcinomas. The tissue distribution of the active form of c-Src was studied by immunohistochemistry using this antibody, termed Clone 28. Among 66 cases of adenoma tested, 61 (92%) showed positive staining (adenoma with mild atypia, 3 of 3; adenoma with moderate atypia, 38 of 42; adenoma with severe atypia, 20 of 21). In contrast to the frequent and intense staining in adenomas, adenocarcinoma showed weak staining with less frequency in 4 of 16 (25%) cases. The number of specimens with positive staining in well- and moderately differentiated adenocarcinomas was limited to an early stage. The active form of c-Src mainly localized to the nuclear membrane and the perinuclear region. These results provide the first direct evidence that the activation of c-Src appears to be an early event in colonic carcinogenesis in situ. The findings of the present study thus allow us to propose a molecular mechanism involving c-Src activation in the process of malignant transformation of the human colonic neoplastic cells.

Antibodies, Monoclonal↗
PubMed2002

Effects of src-deficiency on the expression of in vivo toxicity of TCDD in a strain of c-src knockout mice procured through six generations of backcrossings to C57BL/6 mice.

The effect of TCDD was studied in c-src-deficient C57BL6-src(tm1sor) (N6 src -/- and -/+) mice, and their wild-type littermate mice (N6 src +/+). The former was created from the original strain of B6, 129-src(tm1sor) mice through six generations of backcrossings with C57BL6 mice. The results of a high dose TCDD toxicity tests in male mice indicated that N6 src-/+ mice were significantly less responsive to the toxic action of TCDD (115 microg/kg single i.p. injection) than N6 src+/+ mice in terms of reduced % body weight gain, the increase in the liver to body weight ratio, and the decrease in the adipose tissue to liver weight ratio and in the weight of pancreas. To understand the cause for these differential effects of TCDD we studied TCDD-induced changes in several biochemical parameters at day 10 and found that most drastically affected ones were glycogen depletion and phosphoenolpyruvate carboxykinase (PEPCK) downregulation. In addition, the degree of triglyceride accumulation in liver was less pronounced in N6-/+ than in N6+/+ mice. These findings suggest that the absence of c-src expression indeed affects the development of selected, TCDD-induced toxic endpoints that are related to wasting syndrome.

Animals↗
PubMed2002

Chemical modification of reveromycin A and its biological activities.

Various derivatives of reveromycin A, a novel inhibitor of eukaryotic cell growth, were prepared and their inhibitory effects on both isoleucyl-tRNA synthetase activity and in vitro protein synthesis, and activities on the morphological reversion of src(ts)-NRK cells were assayed. The C5 hydroxyl group and C24 carboxyl group are particularly important for these activities.

Animals↗
PubMed2001

Lack of mutation at codon 531 of SRC in advanced colorectal cancers from Italian patients.

A truncating mutation (C to T transition) at codon 531 of the human protooncogene c-src, possibly accounting for the activation of c-src tyrosine kinase, has been recently identified in a subset of advanced colorectal cancer from North-American patients. However, two subsequent studies have failed to confirm the occurrence of SRC 531 mutation in colorectal cancers from North-European and Asiatic patients, raising the hypothesis that the genetic activation of src in colon cancer might be restricted to patients belonging to specific ethnic groups. We investigated a large series of colorectal cancers from Italian patients (155 cases) with a high prevalence of liver metastasis (43%). Using a PCR-RFLP assay, the occurrence of a SRC 531 mutation was ruled out in all the investigated specimens of primary tumours and/or metastases. Our results demonstrate that SRC Gln531AMB plays no role in the development or in the progression of colorectal cancer among Italian patients.

Base Sequence↗
PubMed2001

Cloning of the proto-oncogene c-src from rat testis.

The cellular homolog of the oncogene v-src, the proto-oncogene c-src, was cloned from rat testis using a high stringency polymerase chain reaction. Rat c-src cDNA shared identity with chicken and mouse, and Rous sarcoma virus c-src and v-src, respectively. Rat c-Src protein was 98% homologous to both human and mouse c-Src. Interestingly, rat Src contained one extra amino acid compared to the mouse protein. As expected, the rat testis Src lacked the six extra residues common to the neuronal Src identified in human and mouse. Reporting of the cDNA sequence for non-neuronal, rat c-src should facilitate experimentation into cell growth and transformation using rat tissues as models of human disease.

Amino Acid Sequence↗
PubMed2000

Identification of cis-acting promoter elements that support expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) in v-src transformed Madin-Darby canine kidney cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) expressed in tumor cells is believed to be important for the pericellular degradation of extracellular matrices during invasion and metastasis. To analyze the mechanism by which MT1-MMP becomes expressed in cancer cells, we assessed the MT1-MMP promoter region for the presence of cis-acting promoter elements that support transcription in transformed cells. Our tumor model consisted of Madin-Darby canine kidney (MDCK) cells transformed by v-src (src4 cells). MT1-MMP mRNA was only faintly detected in parental cells but was strongly expressed in the src4 cells. In parallel, src4 cells invaded into collagen gels, whereas MDCK cells did not. When MDCK and src4 cells were transiently transfected with a plasmid containing of -3000 to -99 nt from the upstream region of the MT1-MMP gene, the promoter activity was 2.6-fold higher in src4 cells than in MDCK cells. Furthermore, the region between -399 and -356 nt was found to contain the src4-specific enhancer element(s). Tandem Sp1 binding sites were also found to be essential in promoting transcription. An Egr-1 site that partially overlaps with the Sp1 sites was found to cooperate with the src4-specific enhancer and to also contribute weakly to the basal promoter activity. The presence of transcription factors that bind to the src4-specific enhancer site was detected by mobility-shift assays in src4 cell nuclear extracts but only weakly in MDCK extracts. Thus, we have identified a novel enhancer element that acts specifically in the transformed cells to enhance MT1-MMP expression.

Animals↗
PubMed1999

Dissociation of CDK2 from cyclin A in response to the topoisomerase II inhibitor etoposide in v-src-transformed but not normal NIH 3T3 cells.

Our previous work has demonstrated that treatment of NIH 3T3 cells with etoposide (VP16), an inhibitor of DNA topoisomerase II and widely used anticancer agent, results in G2/M-phase arrest, whereas treatment of cells transformed by v-src, v-ras, or v-raf results in an S-phase blockage. The present studies describe the mechanistic aspects of this selective S-phase arrest in the v-src-transformed cells. The S-phase arrest in these cells was found to be coupled with depletion of cyclin A-dependent kinase activity. This decrease could not be explained by changes in the overall level of cyclin A, CDK2, p27, or p21 proteins. Rather, it was associated with a time-dependent reduction of CDK2 protein complexed with cyclin A following VP16 treatment. It was further shown that the decrease of cyclin A-associated CDK2 was linked to an increase of CDK2 protein in cyclin E immunocomplexes, which suggests that CDK2 might become redistributed following treatment with VP16. Thus, oncogenic transformation by v-src can trigger separation of CDK2 protein from cyclin A in response to VP16. This might contribute to the depletion of cyclin A-dependent kinase activity and the selective S-phase arrest by VP16 in v-src-transformed cells.

3T3 Cells↗
PubMed1994

The lack of v-src involvement in tumorigenicity of marmoset cells transformed in vitro with Rous sarcoma virus.

The transformation of nonhuman primate marmoset cells by Rous sarcoma virus of Schmidt-Ruppin strain (RSV-SR) generates transformants which lack tumorigenicity in allo- and xenogeneic hosts. Marmoset cells acquire this property when they are transformed by RSV rescued from non-tumorigenic allogeneic cells. One of the rescued RSV, when used to infect marmoset kidney cells in vitro, yielded transformants which became tumorigenic in adult allogeneic hosts. Cytogenetic and molecular analyses of transformants revealed progressive genetic changes from cell diploidy to aneuploidy and from the presence to the loss of v-src during propagation in vitro. The loss of v-src in transformed cells coincided with the evolution of aneuploid cell clone with specific marker chromosome (M1). Although both early passage diploid and late passage aneuploid transformants were tumorigenic, the induced tumors originated from different type of cells. Tumors induced by diploid- and v-src-positive transformants were derived predominantly from the host cells, while tumors induced by late-passage transformants and with deleted v-src originated from an aneuploid cell clone that contained a rearranged M1 marker chromosome. These results suggest that besides the v-src oncogene, proviral integration can facilitate chromosomal rearrangements that contribute to tumorigenic transformation of nonhuman primate cells in vitro.

Aneuploidy↗
PubMed1992

Substrate-specific modulation of Src-mediated phosphorylation of Ras and caseins by sphingosines and other substrate modulators.

It is important for the understanding of protein kinase action to differentiate between regulation at the enzyme and at the substrate levels. For example, the inhibitors dinitrophenol-tyrosine and tyrphostins act at the enzyme level to inhibit phosphorylation of all substrates by c-Src and v-Src kinases. In contrast, polylysine acts at the substrate level to stimulate Src-mediated phosphorylation of beta-casein but to inhibit phosphorylation of alpha-casein. Here we demonstrate novel enzyme-specific and substrate-specific modulations of Src kinase activity of potential physiological significance. At the enzyme level, we observed that c-Src kinase preferentially phosphorylates alpha-casein, while the v-Src kinase prefers beta-casein. At the substrate level we observed substrate-specific modulation by physiological factors including sphingosine, sphingosine derivatives and the ganglioside GM3. Galactosyl-sphingosine (psychosine) was more effective in stimulating phosphorylation of beta-casein and poly(E1A1Y1) than sphingosine. Glucosyl- and lactosyl-sphingosine were ineffective. Rat was extensively phosphorylated by c-Src in the presence of polylysine, and to a lesser extent in the sphingosine and galactosyl-sphingosine. These unexpected differences point out another potential mechanism for regulation of c-Src and v-Src kinase activities and may help to explain some of the pleotyptic manifestations of protein tyrosine kinase actions.

Animals↗
PubMed2006

Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFalpha secretion in macrophages.

As tyrosine kinases are indispensable in lipopolysaccharide (LPS)-induced macrophage activation, the myeloid-specific Src members (i.e. Lyn, Fgr and Hck) are speculated to play important roles in this process. However, the normal LPS responsiveness in lyn(-/-)fgr(-/-)hck(-/-) macrophages implicates the presence of an elusive, compensating tyrosine kinase(s). In this study, we demonstrate the upregulation of c-Src in Raw264.7 and peritoneal macrophages (PEMs) by LPS, which is inhibited by PP2 (an inhibitor for Src family kinases), pyrrolidinedithiocarbamate (PDTC; NF-kappaB inhibitor) and LY294002 (PI3K inhibitor). And this LPS-mediated c-Src induction is also observed in macrophages recovered from LPS-challenged rats. Intriguingly, PP2 attenuates the ability of PEMs to elicit COX-2 expression and nitric oxide production in response to LPS. Similar results are also observed when macrophages recovered from rats receiving either LPS alone or LPS and PP2 both are compared. Furthermore, administration of PP2 in Raw264.7 and animal models of sepsis greatly suppresses TNFalpha secretion and serum TNFalpha level, respectively. Therefore, we conclude that c-Src, with its LPS induction, has an unperceived role in transmitting LPS signaling in macrophages.

Animals↗
PubMed2004

CEACAM6 is a determinant of pancreatic adenocarcinoma cellular invasiveness.

Pancreatic adenocarcinoma is among the most aggressively invasive malignancies. The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is emerging as an important determinant of the malignant phenotype in a range of cancers. We sought to define the role of CEACAM6 in pancreatic adenocarcinoma cellular invasiveness. CEACAM6 was stably overexpressed in Capan2 cells, which inherently express low levels of CEACAM6. Retrovirally mediated RNA interference was used to silence CEACAM6 expression in BxPC3 cells, which inherently overexpress CEACAM6. Cellular invasiveness was quantified using a modified Boyden chamber assay. Overexpression of CEACAM6 increased Capan2 cellular invasiveness, whereas CEACAM6 knockdown attenuated BxPC3 invasiveness. A role for the c-Src tyrosine kinase in mediating CEACAM6-dependent invasiveness was defined using constitutively active and dominant-negative c-Src expression constructs. c-Src-dependent modulation of matrix metalloproteinase-9 activity contributes significantly to the increased cellular invasiveness induced by CEACAM6 overexpression. Levels of CEACAM6 expression can modulate pancreatic adenocarcinoma cellular invasiveness in a c-Src-dependent manner. This pathway warrants further investigation as a target for therapy.

Adenocarcinoma↗
PubMed1999

Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini.

We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini. Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting. When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected. CCK (10 pM-10 nM) and carbachol (0.1-100 microM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min. High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor. Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol. The Gq protein inhibitor G protein antagonist-2A (10 microM) and the phospholipase C (PLC) inhibitor U-73122 (10 microM) markedly reduced RhoA bands in response to CCK. The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 microM). The pp60(c-src) inhibitor herbimycin A (6 microM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 microM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 microM) had no effect. RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src. Increases in mass of this complex were observed with CCK stimulation. In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml. We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol. RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC.

ADP Ribose Transferases↗