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PubMed1994

Enhancement of CDP-DG:inositol transferase activity in src- and erbB2-transformed cells.

Phosphatidylinositol (PI) synthesis was activated in Rous sarcoma virus-infected NIH3T3 or activated erbB2-transformed NIH3T3 cells. The in vitro activity of CDP-DG:inositol transferase prepared from these cells was also higher than that from normal parent NIH3T3 cells, although phospholipase C and PI kinase activities were not significantly different among these cells. A tyrosine kinase inhibitor, erbstatin, inhibited the PI synthesis in cultured cells, suggesting that Src and ErbB2-associated tyrosine kinases are involved in activation of CDP-DG:inositol transferase in these cell lines.

1-Phosphatidylinositol 4-Kinase↗
PubMed2001

pp60(c-src) modulates microvascular endothelial phenotype and in vitro angiogenesis.

The tyrosine kinase c-src associates with the platelet-derived growth factor (PDGF) receptor. Overexpression of wild-type c-src, a kinase-negative c-src mutant, and v-src in microvascular endothelial cells modulated the mitogenic effect of PDGF, suggesting that c-src kinase activity inhibits PDGF signals. Analyses of cell morphology in two-dimensional culture revealed changes in cell shape and size induced by the overexpression of c-src proteins. Investigations in three-dimensional culture unveiled a modulatory role of c-src during in vitro angiogenesis. Overexpression of c-src resulted in an increased diameter of tube-like structures, and the number of branching segments was decreased. Expression of the kinase-negative c-src mutant resulted in abortive tube formation consisting of disconnected multicellular fragments. These results indicate that the c-src tyrosine kinase exerts regulatory effects on endothelial proliferation, size, and cytoskeletal organization in two-dimensional culture and on the formation of a differentiated multicellular network in three-dimensional culture.

Adipose Tissue↗
PubMed2001

UCS15A, a non-kinase inhibitor of Src signal transduction.

Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

3T3 Cells↗
PubMed1993

Functional coupling of the src-family protein tyrosine kinases p59fyn and p53/56lyn with the interleukin 2 receptor: implications for redundancy and pleiotropism in cytokine signal transduction.

The binding of interleukin 2 (IL-2) to the IL-2 receptor (IL-2R) induces a rapid increase in tyrosine phosphorylation of cellular proteins. In a previous study, we have shown that p56lck (lck), a src-family protein tyrosine kinase (src-PTK), physically and functionally associates with the IL-2R beta chain (IL-2R beta). To further investigate a role of src-PTKs in IL-2 signaling, we analyzed a mouse pro-B-cell line, in which lck is not expressed detectably. We observed that in this cell line, IL-2 induces activation of at least two src-PTKs, p59fyn (fyn) and p53/56lyn (lyn). Interestingly, stimulation of this cell line with IL-3 also induces activation of src-PTKs. The activation of fyn or lyn seems to be selective for stimulation with IL-2 or IL-3 since stimulation with IL-6 fails to activate them. Furthermore, we provide evidence for the physical association of fyn with IL-2R beta. Taken together with previous results, our current study suggests that different src-PTKs, each of which is expressed in a cell-type-specific manner, can participate in the IL-2 signal transduction.

Animals↗
PubMed2006

SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth.

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle-specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]-SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK-SHP-2-null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.

Animals↗
PubMed1992

Activation of protein serine/threonine kinases p42, p63, and p87 in Rous sarcoma virus-transformed cells: signal transduction/transformation-dependent MBP kinases.

We have used myelin basic protein immobilized in sodium dodecyl sulfate-polyacrylamide gels to identify protein kinases after gel electrophoresis, followed by protein kinase reactions. This technique has permitted us to detect three protein kinases in serum-deprived cells transformed by p60src. On induction of cellular transformation by a temperature-sensitive v-src, a p87 protein kinase is activated within 30 min and remains activated in fully transformed cells. The p63 protein kinase is not fully activated until 24 h but remains activated in transformed cells. The commonly studied p42MBPK is rapidly activated within 30 min, and its kinase activity decreases significantly by 24 h, when the p63 enzyme is fully activated. The p42MBPK, as well as the p63 and p87 enzymes, are stimulated by transforming p60c-src mutants but not normal c-src or nonmyristylated p60c-src. In addition, the kinase activity of p63 enzyme, but not of p42MBPK, can be induced in okadaic acid-treated chicken embryo fibroblasts, indicating that phosphatase 2A and/or phosphatase 1 may be involved in the regulation of its activity. Additional data indicate that either p42MBPK or p63 activity correlates with the stimulation of the protein kinase p90RSK. Thus, there may be two independent pathways leading to the activation of the RSK gene product.

Animals↗
PubMed1999

No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Animals↗
PubMed2002

Meeting at mitosis: cell cycle-specific regulation of c-Src by RPTPalpha.

Exquisite regulation is required for cells to properly enter and exit the phases of the cell cycle. The transmembrane receptor-like protein tyrosine phosphatase RPTPalpha, an important protein that participates in the transition of the cell cycle from G2 to mitosis activates the protein tyrosine kinase c-Src in vivo. Mustelin and Hunter discuss new findings that describe the highly regulated activation of RPTPalpha and c-Src that occurs just before entry into the mitotic phase. These findings also raise several questions that pertain to redistribution of RPTPalpha in the cell, and the role of phosphorylation and dimerization in regulating RPTPalpha activity.

Animals↗
PubMed2001

Estradiol (E2) elicits SRC phosphorylation in the mouse neocortex: the initial event in E2 activation of the MAPK cascade?

In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER alpha (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Tumor cells induced by the v-src oncogene are heterogeneous for expression of markers of mesenchyme differentiation.

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, alpha sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.

Actins↗
PubMed1990

Acetylcholine-activated currents in quail myotubes expressing viral oncogenes.

Acetylcholine-activated currents were recorded in cultured myotubes arising from embryonic quail myoblasts transformed by the v-src and v-ras oncogenes. In src-myotubes, the whole cell inward current decayed more slowly than in non-transformed controls. In ras-myotubes, the current had a faster decay and smaller amplitude than in the controls. The single-channel conductance and mean open times recorded from cell-attached patches were similar in transformed and control cells. However, in ras-myotubes the frequency of channel openings strongly decreased with time. It is concluded that oncogenic tyrosine-specific protein kinase (v-src product) and G-like p21 protein (v-ras product) can induce differential changes in the function of nicotinic ACh receptor, perhaps related to specific biochemical events elicited in the establishment of the transformed state.

Acetylcholine↗
PubMed1994

pp60c-src enhances the acetylcholine receptor-dependent catecholamine release in vaccinia virus-infected bovine adrenal chromaffin cells.

Secretion of catecholamines by adrenal chromaffin cells is a highly regulated process that involves serine/threonine and tyrosine phosphorylations. The nonreceptor tyrosine kinase pp60c-src is expressed at high levels and localized to plasma membranes and secretory vesicle membranes in these cells, suggesting an interaction of this enzyme with components of the secretory process. To test the hypothesis that pp60c-src is involved in exocytosis, we transiently expressed exogenous c-src cDNA using a vaccinia virus vector in primary cultures of bovine adrenomedullary chromaffin cells. Chromaffin cells infected with a c-src recombinant virus restored the diminished secretory activity accompanying infection by wild type virus alone or a control recombinant virus. The level of enhanced catecholamine release correlated directly with the time and level of exogenous c-src expression. These results could not be attributed to differences in cytopathic effects of wild type versus recombinant viruses as assessed by cell viability assays, nor to differences in norepinephrine uptake or basal release, suggesting that pp60c-src is involved in stimulus-secretion coupling in infected cells. Surprisingly, exogenous expression of an enzymatically inactive mutant c-src also restored catecholamine release, indicating that regions of the introduced c-src protein other than the kinase domain may affect catecholamine release. Secretory activity was elevated by both forms of c-src in response to either nicotine or carbachol (which activate the nicotinic and the nicotinic/muscarinic receptors, respectively). In contrast, release of catecholamines upon membrane depolarization (as elicited by 55 mM K+) or by treatment with the calcium ionophore A23187 was unaffected by either vaccinia infection or increased levels of pp60c-src. These results suggest that pp60c-src affects secretory processes in vaccinia-infected cells that are activated through ligand-gated, but not voltage-gated, ion channels.

Adrenal Glands↗
PubMed1991

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated.

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.

Age Factors↗
PubMed1993

Bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lack expression of osteoclast-reactive vacuolar proton pump.

Craniometaphyseal dysplasia (CMD) is a rare craniotubular bone dysplasia transmitted in autosomal dominant or recessive form. This disease is characterized by cranial bone hyperostosis and deformity of the metaphyses of the long bones. Using osteoclast-like cells formed from patient bone marrow cells, we investigated the pathophysiology of CMD in a 3-yr-old patient. Untreated bone marrow cells from the patient differentiated into osteoclast-like cells in vitro. These cells were shown to have vitronectin beta-receptors using a specific monoclonal antibody, i.e., 23C6 (CD51), which reacts with osteoclasts in human bone biopsy samples. However, the number of these osteoclast-like cells formed from the patient's bone marrow was only 40% of the normal controls. 1,25-dihydroxyvitamin-D3, bovine 1-34 parathyroid hormone, recombinant human interleukin-1 beta, recombinant human interleukin-6, or recombinant human macrophage colony-stimulating factor significantly increased, while salmon calcitonin significantly inhibited, the number of osteoclast-like cells. However, these cells could not resorb sperm whale dentin slices and lacked the osteoclast-reactive vacuolar proton pump as evidenced by a monoclonal antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression of the osteoclast-reactive vacuolar proton pump; and (b) a protooncogene c-src was not associated with the pathogenesis of the present CMD patient.

Blood Platelets↗
PubMed1996

Reduced phosphotyrosine binding by the v-Src SH2 domain is compatible with wild-type transformation.

The SH2 domain of v-Src binds phosphotyrosyl-proteins in vivo and in vitro. The function of this domain is necessary for transformation of Rat-2 cells and for morphologically wild-type transformation of chicken embryo fibroblasts (CEF). The phosphate group of phosphotyrosine interacts directly with a conserved arginine residue in the FLVRES motif of the SH2 domain, R175 in v-Src. To examine the role of phosphotyrosine binding in transformation by v-Src, we have characterized the effects of R175 mutations on the transforming ability of v-Src and on the interaction of the v-Src SH2 domain with phosphotyrosyl-proteins. The R175H mutation, and to a lesser extent the R175K mutation, reduced but did not eliminate the binding of phosphotyrosyl-proteins to the v-Src SH2 domain. However neither mutation affected transformation of CEF or Rat-2 cells by v-Src and neither mutation resulted in major changes in the level or pattern of protein-tyrosine phosphorylation in transformed CEF. In contrast, the R175E mutant of v-Src induced fusiform transformation of CEF and failed to transform Rat-2 cells; the mutant SH2 domain was insoluble when expressed in bacteria, suggesting that the R175E mutation disrupts the structure of the v-Src SH2 domain. We conclude that, although the Arg residue in the FLVRES motif is invariant in most if not all SH2 domains, at position 175 in the v-Src SH2 domain residues other than arginine can support the binding of phosphotyrosyl-proteins, albeit at reduced levels. Furthermore under the expression conditions normally used, that is when v-Src is expressed under the control of a retroviral LTR, the reduced binding of phosphotyrosyl-proteins is compatible with wild-type transformation.

Amino Acid Sequence↗
PubMed2003

v-Src rescues actin-based cytoskeletal architecture and cell motility and induces enhanced anchorage independence during oncogenic transformation of focal adhesion kinase-null fibroblasts.

The ability of the focal adhesion kinase (FAK) to integrate signals from extracellular matrix and growth factor receptors requires the integrity of Tyr397, a major autophosphorylation site that mediates the Src homology 2-dependent binding of Src family kinases. However, the precise roles played by FAK in specific Src-induced pathways, especially as they relate to oncogenic transformation, remain unclear. Here, we investigate the role of FAK in v-Src-induced oncogenic transformation by transducing temperature-sensitive v-Src (ts72v-Src) into p53-null FAK+/+ or FAK-/- mouse embryo fibroblasts (MEF). At the permissive temperature (PT), ts72v-Src induced abundant tyrosine phosphorylation, morphological transformation and cytoskeletal rearrangement in FAK-/- MEF, including the restoration of cell polarity, typical focal adhesion complexes, and longitudinal F-actin stress fibers. v-Src rescued the haptotactic, linear directional, and invasive motility defects of FAK-/- cells to levels found in FAK+/+ or FAK+/+-[ts72v-Src] cells, and, in the case of monolayer wound healing motility, there was an enhancement. Src activation failed to increase the high basal tyrosine phosphorylation of the Crk-associated substrate, CAS, found in FAK-/- MEF, indicating that CAS phosphorylation alone is insufficient to induce motility in the absence of FAK- or v-Src-induced cytoskeletal remodeling. Compared with FAK+/+[ts72v-Src] controls, FAK-/-[ts72v-Src] clones exhibited 7-10-fold higher anchorage-independent proliferation that could not be attributed to variations in either v-Src protein level or stability. Re-expression of FAK diminished the colony-forming activities of FAK-/-[ts72v-Src] without altering ts72v-Src expression levels, suggesting that FAK attenuates Src-induced anchorage independence. Our data also indicate that the enhanced Pyk2 level found in FAK-/- MEF plays no role in v-Src-induced anchorage independence. Overall, our data indicate that FAK, although dispensable, attenuates v-Src-induced oncogenic transformation by modulating distinct signaling and cytoskeletal pathways.

Actins↗
PubMed1995

Transformation and pp60v-src autophosphorylation correlate with SHC-GRB2 complex formation in rat and chicken cells expressing host-range and kinase-active, transformation-defective alleles of v-src.

The biochemical properties of several pp60v-src substrates believed to participate in src-mediated transformation were examined in cells expressing a kinase-active, transformation-defective v-src allele (v-src-F172 delta/Y416F) and its parental allele, v-src-F172 delta, a host-range--dependent allele that transforms chicken cells to a fusiform morphology, but does not transform rat cells. Because pp60v-src-F172 delta is dependent on autophosphorylation for transforming ability, these alleles provide a unique opportunity to examine the role of pp60v-src autophosphorylation in regulating substrate interactions. Increased pp125FAK tyrosine phosphorylation and high levels of pp60v-src-associated phosphotidylinositol-3' kinase activity were detected specifically in chicken cells exhibiting round, refractile transformation but not in cells transformed to a fusiform morphology. Increased pp125FAK kinase activity, but not increased pp125FAK tyrosine-phosphorylation correlated with pp60v-src autophosphorylation and increased anchorage-independent growth. Thus, pp125FAK and PI3'K may participate in morphological transformation by v-src. Furthermore, association of phosphorylated SHC with the adapter GRB2 correlated with increased anchorage-independent growth (and autophosphorylation) in both rat and chicken cells independent of the morphological phenotype induced. Therefore, host-range dependence for transformation may be regulated through association of SHC with GRB2, thus implicating SHC as a crucial substrate for src-dependent transformation.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma-associated proteins. Characterization of a c-Src-dependent 97-kD protein in vascular smooth muscle cells.

Stimulation of phospholipase C-gamma (PLC-gamma) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-gamma via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-gamma, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-gamma-associated proteins, RASM cells were labeled with [32P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-gamma was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-gamma-associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src-transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-gamma-associated protein that may be required for Ang II-mediated signal transduction.

Angiotensin II↗