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At least 865 records · Page 48Linked to original sources
PubMed1991

Transformation of human kidney proximal tubule cells by a src-containing retrovirus.

Analysis of the cell-surface antigenic phenotypes of normal and malignant renal cells demonstrates that approximately 90% of cultured normal human kidney cells and virtually all renal cell carcinomas (RCC) derive from the proximal tubule (PT) cell of the nephron. In the present study, the v-src oncogene was introduced into cultured normal human PT cells, and the effects of the v-src-encoded protein (pp60v-src) on the biologic and genetic phenotype of these cells were determined. V-src-containing PT cells underwent a series of phenotypic changes characteristic of renal cells in vivo. These included (i) alterations in morphology, (ii) an increase in proliferative capacity, (iii) loss of contact inhibition, (iv) immortalization and (v) tumorigenicity. Moreover, v-src-infected PT cells developed non-random clonal karyo-typic abnormalities which are commonly observed in RCCs, including a deletion of chromosomal region 3p14-21, one of the most frequent deletions observed in human renal tumors. These results indicate that pp60v-src can initiate a complex process leading to the transformation of PT cells. This process includes the induction of genetic instability. Finally, these data provide experimental evidence corroborating cytogenetic and molecular data that a deletion of genes on chromosome 3p is a critical event in the transformation of the human renal cell.

Animals↗
PubMed1993

The Src family of protein tyrosine kinases: regulation and functions.

Most of the nine members of the Src family of tyrosine kinases are restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more ubiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (PDGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the PDGF receptor. To address the role of Src, Fyn and Yes in the response to PDGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to PDGF, and we therefore conclude that Src family tyrosine kinases are required in order for the PDGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in S. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain: phosphorylated tail interaction repressing Src activity. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia.

Increased synthesis of substance P (SP) in the dorsal root ganglia (DRG) and enhanced axonal transport to and secretion from the primary afferent sensory neurons might enhance pain signalling in the spinal dorsal horn by modifying pronociceptive pathways. IL-1beta increases SP synthesis by enhancing the expression of preprotachykinin (PPT) mRNA encoding for SP and other tachykinins in the DRG. Stimulation of IL-1 receptor by IL-1beta may induce the phosphorylation of tyrosine residues in many effector proteins through the activation of p60c-src kinase. The hypothesis that the synthesis of SP in and secretion from the primary sensory ganglia are regulated by the activation of p60c-src kinase induced by IL-1beta was tested. Pretreatment of DRG neurons in culture with herbimycin A, genistein or PP2, three structurally different nonreceptor tyrosine kinase inhibitors that act by different mechanisms, decreased the kinase activity of p60c-src induced by the activation of IL-1 receptor. PP3, a negative control for the Src family of tyrosine kinase inhibitor PP2 had no effect. Herbimycin A and genistein also decreased IL-1beta-induced expression of PPT mRNA-encoding transcripts and the levels of SP-li synthesized in the cells and secreted into the culture medium in a concentration-dependent manner. SB 203580 [a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor] and PD 98059 (a p44/42 MAPK kinase inhibitor) were ineffective in modulating IL-1beta-induced SP synthesis and secretion, and p60c-src kinase activity in DRG neurons. Whereas, IL-1 receptor antagonist and cycloheximide inhibited IL-1beta-evoked secretion of SP-like immunoreactivity (SP-li), actinomycin D decreased it significantly but did not entirely abolish it. These findings show that phosphorylation of specific protein tyrosine residue(s) following IL-1 receptor activation might play a key role in IL-1beta signalling to modulate PPT gene expression and SP secretion in sensory neurons. In view of the role of SP as an immunomodulator, these studies provide a new insight into neural-immune intercommunication in pain regulation in the sensory ganglia through the IL-1beta-induced p60c-src activation.

Animals↗
PubMed2002

The molecular basis of Src kinase specificity during vertebrate mesoderm formation.

Members of the Src family of non-receptor tyrosine kinases play a critical role in mesoderm formation in the frog, Xenopus laevis, acting as required mediators downstream of the fibroblast growth factor receptor. At least four members of this gene family, Src, Fyn, Yes, and Laloo, are expressed during early embryonic development. Ectopic expression of Laloo and Fyn, but not Src, induce mesoderm in ectodermal explants, indicating that these factors are non-redundant during early vertebrate development. Here we investigate the basis for the differential activity of the Src and Laloo kinases during mesoderm formation. We demonstrate that although both Src and Laloo physically interact with the substrate protein SNT-1/FRS2alpha only Laloo phosphorylates SNT-1, an event previously shown to be required for the activity of the latter and for mesoderm induction in vivo. We show that Src is enzymatically capable of stimulating mesoderm formation, as an activated Src construct both phosphorylates SNT-1 and induces mesoderm in explant cultures. However, a chimeric Laloo construct containing a Src C-terminal tail is inactive, suggesting that the early embryo contains a specific Laloo-activating, or Src-inactivating, factor. Finally, through further chimeric analysis, we provide evidence to suggest that differences in Laloo and Src activity are also mediated by the SH2, SH3, and kinase domains of these molecules.

Animals↗
PubMed1992

Analysis of conserved domains and sequence motifs in cellular regulatory proteins and locus control regions using new software tools for multiple alignment and visualization.

With the tremendous expansion of molecular sequence data in recent years, multiple alignment is arguably one of the two most important analytic techniques (the other being fast database searching). A number of useful approaches to this problem have previously been developed, but often they are limited to only a subset of multiple-alignment applications and cannot easily deal with the complex structural organization seen in an increasing number of sequences. For example, a single sequence may contain several domains of different evolutionary origins, and the multiplicities and relative ordering of these domains may be quite different among related sequences. Here we describe an integrated set of interactive Unix tools that combines several multiple-alignment techniques with traditional "dot-plot" visualization to provide a flexible environment for approaching complex sequence analysis problems. We apply these tools to the identification and characterization of "catalytic" domains in ras and rho/rac GTPase-activating proteins, to "Src homology" (SH2, SH3) domains in cytoplasmic signaling proteins, to repetitive sequence motifs in the alpha and beta subunits of protein prenyltransferases, and to regulatory DNA sequences in the locus control region of the beta-globin gene cluster.

Alkyl and Aryl Transferases↗
PubMed2005

Distinct and opposite roles for SH2 and SH3 domains of v-src in embryo survival and hemangiosarcoma formation.

The cellular proto-oncogene c-src is thought to be involved in formation, progression, and metastasis of a variety of tumor cell types, although its exact role during tumor cell genesis is not well defined. v-src, the viral oncogene counterpart of c-src, causes metastatic sarcomas, hemorrhagic disease, and hemangiosarcomas in chicken embryos and, thus, can be used as a constitutively activated form of src for experimentally-induced tumorigenesis. Here, we used retroviral vectors to express wild-type v-src or SH2 or SH3 domain-deleted forms (DeltaSH2 or DeltaSH3) to determine if different pathogenic effects resulted. Vectors were injected into early chick embryo midbrain ventricles and embryos were sacrificed at various ages up to embryonic day (E) 18. Retroviral expression of all forms of v-src resulted in transformation of pial connective tissue cells into large, rounded abnormal-appearing cells. Surprisingly, all forms of v-src were lethal. The v-src retrovirus was lethal and killed most embryos by E15 with the development of hemangiosarcomas over the injection site between E10-E12. The DeltaSH3 retrovirus was the most deadly, killing most embryos by E12, however, it never resulted in hemangiosarcoma formation. The DeltaSH2 retrovirus injected embryos survived longer than v-src or DeltaSH3 embryos, and some of these embryos also developed large hemangiosarcomas over the injection site between E13 and E18. These results demonstrate that the src SH2 domain is required to be fully lethal, whereas the presence of the SH3 domain attenuated lethality. Furthermore, the formation of hemangiosarcomas absolutely required the presence of the src SH3 domain and to some extent required the SH2 domain. This implicates distinct and opposite roles for SH2 and SH3 domains of src and their cellular binding partners in tumorigenesis and hemorrhagic disease.

Animals↗
PubMed1992

Src-related protein tyrosine kinases and T-cell receptor signalling.

Upon antigen stimulation, the T-cell receptor for antigen transduces an intracellular protein tyrosine phosphorylation signal that is critical for subsequent T-lymphocyte activation. As the antigen receptor does not possess an intrinsic protein tyrosine kinase activity, the mechanism by which it regulates protein tyrosine phosphorylation is unconventional. Evidence is increasing that the Src-related protein tyrosine kinases P56lck and p59fyn, as well as the protein tyrosine phosphatase CD45, are involved in this process.

Genes, src↗
PubMed1995

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Animals↗
PubMed1997

Constitutive activation of JAK1 in Src-transformed cells.

We have previously found that the signal transducer and activator of transcription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was assessed using autophosphorylation assays. In anti-JAK1 immunoprecipitates from v-Src-transformed NIH3T3 cells, a protein with the same migration as JAK1 showed substantially increased levels of 32P incorporation compared to immunoprecipitates from non-transformed cells. Similar results were obtained using anti-JAK2 immunoprecipitates; however, the level of JAK2 tyrosyl phosphorylation and 32P incorporation in anti-JAK2 immunoprecipitates were markedly lower than in anti-JAK1 immunoprecipitates. We conclude that JAK1, and possibly JAK2, are constitutively activated in Src-transformed cells, raising the possibility that Janus family kinases contribute to the constitutive activation of Stat3 previously observed in these cells and/or other properties of Src-transformed cells.

3T3 Cells↗
PubMed2001

Ras/MAP kinase pathway is associated with the control of myotube formation but not myofibril assembly in quail myoblasts transformed with Rous sarcoma virus.

Tyrosine kinase activity of v-Src from Rous sarcoma virus (RSV) inhibits the differentiation of quail myoblasts. To clarify the inhibitory mechanism, we focused on the signaling pathways from v-Src. When the activation of the Ras/MAP (mitogen-activated protein) kinase pathway was inhibited by a dominant-negative mutant of Ras or PD98059, a specific inhibitor of p42 MAP kinase kinase, differentiation was restored; muscle specific proteins were expressed and myotubes formed even under active conditions of v-Src. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (P13-kinase), showed no effects on the inhibition by v-Src. These findings suggest that v-Src activates the Ras/MAP kinase signaling pathway, but not the P13-kinase pathway, and inhibits the differentiation. However, the myotubes derived from the dominant-negative Ras did not form actin fibers, suggesting that myofibril assembly is regulated by other pathway(s) from v-Src.

Animals↗
PubMed1992

Slow time-dependent cellular transformation induced at restrictive temperature by ts-src mutants.

Cell lines infected with a temperature-sensitive Rous sarcoma virus have been widely used to study the temporal dynamics of various transformation parameters following downshift from the non-permissive temperature to the permissive temperature as it is considered that, at the non-permissive temperature, the infected cells exhibit the morphological and growth characteristics of normal cell whereas, at the permissive temperature, they exhibit characteristics of the transformed state. We show here that the apparently normal state in which tsPA1-infected FR3T3 cells are directed at the restrictive temperature is not a stationary stable state, but rather a transient one which continuously drifts as the cells are grown and passaged at this temperature and which eventually ends up as a new transformed state (T2) with morphological and growth properties definitely different from those belonging to the transformed state (T1) at the permissive temperature. The establishment of the transformed T2 state at the restrictive temperature occurs concomitantly with a steady accumulation of an intracellular pool of pp60v-src in the vicinity of the nucleus, whose traffic towards the plasma membrane is released following downshift to the permissive temperature, leading to the reappearance of transformation parameters characteristic of the transformed T1 state. Our finding raises the possibility that the v-src protein encoded by the tsPA1 mutant of Rous sarcoma virus may induce cellular transformation via two different pathways, leading to two different transformation states, depending on at which temperature the infected cells are grown. Various possible mechanisms that could be involved in the time-dependent establishment of a transformed state by ts-src mutants at the restrictive temperature are discussed.

Animals↗
PubMed1991

Phosphorylation of integrin in differentiating ts-Rous sarcoma virus-infected myogenic cells.

The differentiation of primary myogenic cultures requires the attachment of the cells to an extracellular matrix substrate using an integrin family receptor. These integrin receptors can be phosphorylated on both their alpha and beta chains, and it has been postulated that phosphorylation regulates the receptor function. Quail myogenic clones transformed with ts-LA24A differentiated into mature myotubes following a temperature shift to nonpermissive temperature which inactivates the viral src kinas. Phosphorylation of integrin beta-1 chain and of at least one alpha chain was detected on both serine and tyrosine. An additional alpha chain(s) with a mobility similar to alpha 5 was not phosphorylated at either temperature. Following the induction of differentiation by a temperature shift, there was a marked decrease in integrin phosphorylation of both alpha and beta integrin chains. This decrease was more prominent for serine than for tyrosine, suggesting that src could not be the only kinase involved. The drop in integrin phosphorylation correlated with the initiation of differentiation, suggesting that integrin phosphorylation could be at least part of the mechanism by which myogenic differentiation is blocked by v-src.

Animals↗
PubMed1995

Regulation of the Ras signaling pathway by GTPase-activating protein in PC12 cells.

We have investigated the role of Ras GTPase-activating protein (GAP) in NGF-induced neuronal differentiation by overexpressing both wild-type and membrane-targeted GAP in PC12 cells. Extension of neurites in response to NGF was completely blocked in cells expressing the highest level of membrane-targeted GAP and significantly inhibited in cells expressing either wild-type GAP or lower levels of membrane-targeted GAP. Overexpression of membrane-targeted GAP similarly inhibited induction of differentiation by src, but not by ras or raf oncogenes, indicating that GAP inhibits differentiation of PC12 cells by downregulating Ras function. GAP overexpression also inhibited stimulation of mitogen-activated protein (MAP) kinase and induction of immediate-early genes in response to NGF. In cells expressing wild-type GAP or lower levels of membrane-targeted GAP, the initial activation of MAP kinase and immediate-early gene expression were only partially inhibited. However, GAP expression in these cells resulted in substantial inhibition of sustained MAP kinase activity following NGF treatment, consistent with the inhibition of neurite extension in these cell lines. These results indicate that GAP acts as a negative regulation, rather than an effector, of Ras signaling in PC12 cells.

Animals↗
PubMed1980

The protein encoded by the transforming gene of avian sarcoma virus (pp60src) and a homologous protein in normal cells (pp60proto-src) are associated with the plasma membrane.

Oncogenesis by avian sarcoma virus is attributable to a single viral gene (src) which encodes a phosphoprotein (pp60src) with the enzymatic activity of a protein kinase. A closely related protein, pp60proto-src, occurs in uninfected cells from a wide variety of vertebrate species and is presumed to be the product of a cellular gene that served as progenitor for src. We explored the location of these proteins within the cell by using immunoprecipitation to analyze subcellular fractions prepared from avian sarcoma virus-transformed rat and chicken cells and from uninfected rat cells. We found that both pp60src and pp60proto-src were associated with the plasma membrane as active protein kinases and could be recovered efficiently only by disrupting the membranes with nonionic detergent. Our findings, in conjunction with those of other investigators, indicate that both proteins are embedded in the membrane by means of a hydrophobic domain(s); available evidence indicates that pp60src is not exposed on the surface of the cell but is accessible at the cytoplasmic aspect of the plasma membrane. These conclusions lend credence to two current speculations. First, pp60src and pp60proto-src may have similar or even identical functions. Second, neoplastic transformation may originate from derangements in the plasma membrane or its affiliated structures.

Animals↗
PubMed2004

Receptor-operated Ca2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini.

We investigated signal transduction between receptor-operated Ca(2+) influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca(2+) ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca(2+)](o), loading of the intracellular Ca(2+) chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca(2+) or Mn(2+) entry induced by CCK and carbachol, it did not affect intracellular Ca(2+) release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G(q) protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G(q)-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca(2+)](o)-dependent pancreatic exocytosis.

Amylases↗
PubMed1999

PKCdelta acts as a growth and tumor suppressor in rat colonic epithelial cells.

We have analysed the expression of three calcium-independent isoforms of protein kinase C (PKC), PKCdelta, PKCepsilon and PKCzeta, in an in vitro model of colon carcinogenesis consisting of the nontumorigenic rat colonic epithelial cell line D/WT, and a derivative src-transformed line D/src. While PKCzeta and PKCepsilon showed similar protein levels, PKCdelta was markedly decreased in D/src cells when compared to the D/WT line. To assess whether down-regulation of PKCdelta was causally involved in the neoplastic phenotype in D/src cells, we prepared a kinase-defective mutant of PKCdelta. Stable transfection of this sequence caused morphological and growth changes characteristic of partial transformation in D/WT cells. Moreover, to test whether PKCdelta was involved in growth control and transformation in this model, we overexpressed PKCdelta in D/src cells. Transfected cells underwent marked growth and morphological modifications toward the D/WT phenotype. In a late stage in culture, transfected cells ceased to proliferate, rounded up and degenerated into multinucleated, giant-like cells. We conclude that PKCdelta can reverse the transformed phenotype and act as a suppressor of cell growth in D/src cells. Moreover, our data show that downregulation of this isoenzyme of PKC may cooperate in the neoplastic transformation induced by the src oncogene in D/WT cells.

Animals↗
PubMed1991

Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src.

Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.

3T3 Cells↗