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PubMed2005

The regulation of the expression of inducible nitric oxide synthase by Src-family tyrosine kinases mediated through MyD88-independent signaling pathways of Toll-like receptor 4.

Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to the expression of inflammatory gene products. Src-family tyrosine kinases (STKs) are known to be activated by LPS in monocytes/macrophages. Therefore, we determined the role of STKs in TLR4 signaling pathways and target gene expression in macrophages. The activation of NFkappaB, and p38 MAPK, and the expression of inducible nitric oxide synthase (iNOS) induced by LPS were not affected in macrophages deficient in three STKs (Lyn, Hck, and Fgr). These results suggest that the deletion of the three STKs among possibly nine STKs is not sufficient to abolish total activity of STKs possibly due to the functional redundancy of other STKs present in macrophages. However, two structurally unrelated pan-inhibitors of STKs, PP1 and SU6656, suppressed LPS-induced iNOS expression in MyD88-knockout as well as wild-type macrophages. The suppression of iNOS expression by the inhibitors was correlated with the downregulation of IFNbeta (a MyD88-independent gene) expression and subsequent decrease in STAT1 phosphorylation. Moreover, PP1 suppressed the expression of IFNbeta and iNOS induced by TRIF, a MyD88-independent adaptor of TLR4. PP1 suppressed STAT1 phosphorylation induced by LPS, but not by IFNbeta suggesting that STKs are involved in the primary downstream signaling pathways of TLR4, but not the secondary signaling pathways downstream of IFNbeta receptor. Together, these results demonstrate that STKs play a positive regulatory role in TLR4-mediated iNOS expression in a MyD88-independent (TRIF-dependent) manner. These results provide new insight in understanding the role of STKs in TLR4 signaling pathways and inflammatory target gene expression.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles.

c-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75 in the unique amino terminal c-Src-specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.

Alleles↗
PubMed1989

Relationship between c-src tyrosine kinase activity and the control of glucose transporter gene expression.

It has been shown previously that the rate of glucose transport in fibroblasts is accelerated by oncoproteins such as v-src, ras, and the transforming protein of feline sarcoma virus. This induction of glucose transport is associated with, and presumably caused by, induction of Hep-G2/rat brain glucose transporter gene expression. To determine the mechanism underlying the induction of glucose transporter gene expression by the v-src oncogene we studied cell lines that overexpress the normal counterpart of the v-src protein (c-src), or various mutants of the c-src protein. In these mutants, the tyrosines at positions 416, 527, or 519, or various combinations of these, have been replaced by phenylalanine by site directed mutagenesis, resulting in mutated c-src proteins that possess varying tyrosine kinase activity and transforming potential. Cells that overexpress the c-src protein show no changes in glucose transporter gene expression. However, when Tyr 527 in the COOH terminus of the c-src protein is replaced with Phe, the tyrosine kinase activity and transforming potential of the protein are increased and the protein acquires a potent ability to increase levels of glucose transporter mRNA and protein, as well as the rate of 2-deoxy-D-glucose uptake. This ability is abolished by the double mutation of Tyr to Phe in positions 416 and 527, which reduces the tyrosine kinase activity of the 527 single mutant. Thus, the ability of src proteins to induce expression of the glucose transport system is linked to the tyrosine kinase activity of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2000

Kinases of the Src family: structure and functions.

Tyrosine kinases of the Src family are involved in different signal transduction pathways in cells. The corresponding genes participate in such vital processes as growth, differentiation, adhesion, transcription, etc. Specific structural changes confer oncogenic properties to the Src protein. In this review, we summarize the available data on the structure, substrates, regulation mechanisms, and role of nonreceptor tyrosine kinases by the example of the src gene product (as the prototype member of this family) and a number of related proteins.

Animals↗
PubMed2000

Etk, a Btk family tyrosine kinase, mediates cellular transformation by linking Src to STAT3 activation.

Etk (also called Bmx) is a member of the Btk tyrosine kinase family and is expressed in a variety of hematopoietic, epithelial, and endothelial cells. We have explored biological functions, regulators, and effectors of Etk. Coexpression of v-Src and Etk led to a transphosphorylation on tyrosine 566 of Etk and subsequent autophosphorylation. These events correlated with a substantial increase in the kinase activity of Etk. STAT3, which was previously shown to be activated by Etk, associated with Etk in vivo. To investigate whether Etk could mediate v-Src-induced activation of STAT3 and cell transformation, we overexpressed a dominant-negative mutant of Etk in an immortalized, untransformed rat liver epithelial cell line, WB, which contains endogenous Etk. Dominant-negative inactivation of Etk not only blocked v-Src-induced tyrosine phosphorylation and activation of STAT3 but also caused a great reduction in the transforming activity of v-Src. In NIH3T3 cells, although Etk did not itself induce transformation, it effectively enhanced the transforming ability of a partially active c-Src mutant (c-Src378G). Furthermore, Etk activated STAT3-mediated gene expression in synergy with this Src mutant. Our findings thus indicate that Etk is a critical mediator of Src-induced cell transformation and STAT3 activation. The role of STAT3 in Etk-mediated transformation was also examined. Expression of Etk in a human hepatoma cell line Hep3B resulted in a significant increase in its transforming ability, and this effect was abrogated by dominant-negative inhibition of STAT3. These data strongly suggest that Etk links Src to STAT3 activation. Furthermore, Src-Etk-STAT3 is an important pathway in cellular transformation.

Animals↗
PubMed2000

Evidence in favour of ancient octaploidy in the vertebrate genome.

Vertebrate genomes are larger than invertebrates and show evidence of extensive gene duplication, including many collinear chromosomal segments. On the basis of this intra-genomic synteny, it has been proposed that two rounds of whole genome duplication (octaploidy) occurred early in the vertebrate lineage. Recently, this early vertebrate octaploidy has been challenged on the basis of gene trees. We report new linkage groups encompassing the matrilin (MATN), syndecan (SDC), Eyes Absent (EYA), HCK kinase and SRC kinase paralogous gene quartets. In contrast to other studies, the sequence trees are weakly supportive of ancient octaploidy. It is concluded that there is no strong evidence against the octaploidy, provided that consecutive genome duplication was rapid.

Animals↗
PubMed1996

Impaired development of CD4+ CD8+ thymoyctes by csk-'knock-in' into fyn locus.

p59fyn is one of the Src-family kinases thought to play an important role in signaling through T cell receptor. However, Fyn deficiency has caused no overt defects in vivo on T cell development, nor has it caused any changes in the phosphorylation status of molecules such as ZAP-70 which have been proposed as p59fyn substrates. This could be explained as being due to compensation of Fyn deficiency by other Src-family kinases. Here, we have 'knocked-in' the csk gene, a negative regulator of Src-family kinases, into fyn locus to challenge the problem of redundant functions among Src-family kinases. The csk-'knock-in' mice displayed atrophy of the thymic cortex and impaired development of CD4+ CD8+ thymocytes. This was concomitant with decrease in tyrosine phosphorylation of ZAP-70 and p120cbl.

Animals↗
PubMed1986

Immunological similarity between the insulin receptor and the protein encoded by the src oncogene.

Insulin receptors resemble receptors for certain growth factors (epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor I) in that all possess tyrosine-specific protein kinase activity. These cell surface receptors resemble protein kinases encoded by viral oncogenes in that both groups of enzymes phosphorylate proteins on tyrosine. Recently, we reported that there is immunological similarity between the insulin receptor and pp60src [the protein encoded by the src oncogene of Rous sarcoma virus (RSV)]. This is supported by the observation that anti-pp60src antiserum (TBR serum) immunoprecipitated radiolabeled insulin receptors derived from cultured human cells (IM-9 lymphoblasts and U-937 monocytes) and rabbit liver. Moreover, highly purified preparations of src protein inhibit the immunoprecipitation of insulin receptors by TBR serum, and the inhibition is correlated with the src kinase activity present in the preparation used. However, two observations suggested that there were immunological differences between pp60src and mammalian insulin receptors. 1) Even at a relatively high concentration (dilution, 1:10), TBR serum immunoprecipitated a relatively small percentage (approximately 20%) of the labeled insulin receptors. 2) Some lots of TBR serum with a high titer against pp60src failed to immunoprecipitate the insulin receptor. Viral oncogenes are thought to have been derived from proto-oncogenes in the host cell. Therefore, because the chicken is the natural host for RSV, we inquired whether there might be closer homology between pp60src and avian insulin receptors. Surprisingly, under conditions where TBR serum immunoprecipitates human insulin receptors, we could not detect immunoprecipitation of avian insulin receptors from chicken liver, chicken embryo fibroblasts, or turkey erythrocytes. The immunoprecipitation of human insulin receptor is not dependent on the method used for labeling the cells ([125I]insulin cross-linking), inasmuch as the receptor labeled by autophosphorylation with [gamma-32P]ATP could also be immunoprecipitated by TBR serum. These observations suggest that there is structural homology between pp60src and the insulin receptor (most likely the beta-subunit). Nevertheless, it seems unlikely that the insulin receptor gene is the proto-oncogene for the src gene of RSV.

Animals↗
PubMed1991

v-Src activates mitogen-responsive transcription factor Egr-1 via serum response elements.

Activating the protein-tyrosine kinase activity of v-Src in murine fibroblasts leads to increased expression of Egr-1, a mitogen-responsive transcription factor. v-Src-induced expression of Egr-1 is independent of protein synthesis and is controlled at the level of transcription. Target sequences responsive to v-Src-induced signals were investigated using deletion mutant and analysis of the Egr-1 promoter. Upstream Egr-1 promoter sequences linked to a reporter gene were cotransfected with a v-Src expression vector into NIH 3T3 cells. v-Src-enhanced gene expression from the Egr-1 promoter was dependent upon the presence of CC(A/T)6GG elements. The CC(A/T)6GG motif forms the core element of serum response elements (SREs) and is the binding site for serum response factor. The Egr-1 promoter sequences responsive to v-Src contained four SREs. Sequential deletion of these SREs reduced v-Src responsiveness to basal transcription levels. A single SRE from this region was able to confer v-Src responsiveness to a heterologous promoter, and a mutation to the CC(A/T)6GG box of this SRE abolished v-Src-enhanced gene expression. Thus, an early response of v-Src-induced intracellular signaling is the transcriptional activation of a growth factor-responsive transcription factor via an SRE.

Animals↗
PubMed1994

Molecular heterogeneity at the breakpoints of smaller 20q deletions.

Deletions of the long arm of chromosome 20 [del(20q)] are recurring abnormalities in patients with myeloid disorders. Although variable in size, these deletions are usually interstitial. With the object of defining a commonly deleted region for smaller 20q deletions, we used quantitative Southern blot analysis complemented by restriction fragment length polymorphism (RFLP) analysis to determine the copy number at 15 loci spanning 20q. The proximal breakpoints of three such deletions were found to separate HCK and the growth hormone releasing factor (GHRF) locus near the centromeric boundary of band 20q11.2. The distal breakpoints were localized to the vicinity of the D20S22 locus in band q13.1. A candidate tumor suppressor gene, RBL2, and the SRC oncogene were both located within the commonly deleted region. Six loci in terminal region q13.2-q13.3 were conserved on these del(20q) chromosomes, thereby confirming that the deletions were interstitial. Molecular heterogeneity at one and possibly both deletion breakpoints rules out the pathological involvement of loci at these sites. Instead, loss of a tumor suppressor locus from within the commonly deleted region may contribute to deregulated hemopoiesis.

Adult↗
PubMed2004

RACK1 regulates G1/S progression by suppressing Src kinase activity.

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.

Animals↗
PubMed2000

Nonreceptor tyrosine protein kinase pp60c-src in spatial learning: synapse-specific changes in its gene expression, tyrosine phosphorylation, and protein-protein interactions.

c-src is a nonreceptor tyrosine protein kinase that is highly concentrated in synaptic regions, including synaptic vesicles and growth cones. Here, we report that the mRNA signal of pp60c-src is widely distributed in the rat brain with particularly high concentrations in the hippocampus. After spatial maze learning, up-regulation of c-src mRNA was observed in the CA3 region of the hippocampus, which was accompanied by increases in pp60c-src protein in hippocampal synaptosomal preparations. Training also triggered an increase in c-src protein tyrosine kinase activity that was correlated with its tyrosine dephosphorylation in the synaptic membrane fraction. After training, pp60c-src from hippocampus showed enhanced interactions with synaptic proteins such as synapsin I, synaptophysin, and the type 2 N-methyl-d-aspartate receptor, as well as the cytoskeletal protein actin. The association of pp60c-src with insulin receptor in the synaptic membrane fraction, however, was temporally decreased after training. Furthermore, in vitro results showed that Ca(2+) and protein kinase C might be involved in the regulation of protein-protein interactions of pp60c-src. These results suggest, therefore, that pp60c-src participates in the regulation of hippocampal synaptic activity during learning and memory.

Animals↗
PubMed1997

v-src-induced cell shape changes in rat fibroblasts require new gene transcription and precede loss of focal adhesions.

The mechanism of v-src-induced morphological transformation is still obscure. We compared LA29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopted an elongated shape with reduced focal adhesions and loss of actin stress fibers. In contrast, activation of v-fps in D1025 cells had little effect on morphology. In both cells, paxillin was strongly tyrosine phosphorylated upon activation of the kinases. This indicates that paxillin phosphorylation is not required, or not sufficient, for the v-src-induced disruption of focal adhesions. As previously described by others, v-src activated the ras-MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggested that novel gene transcription was required. This notion was confirmed using actinomycin D and cycloheximide, which did not impair activation of v-src kinase activity, but completely blocked v-src-induced morphological changes, as demonstrated using image analysis. Furthermore, we observed that v-src-induced changes in cell shape occurred before the reduction in number and size of focal adhesions. We conclude that v-src-induced transformation of rat fibroblasts depends on synthesis of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions.

Actin Cytoskeleton↗
PubMed2006

The genomic analysis of the impact of steroid receptor coactivators ablation on hepatic metabolism.

Members of the steroid receptor coactivator (SRC) family, which include SRC-1 (NcoA-1/p160), SRC-2(TIF2/GRIP1/NcoA-2) and SRC-3(pCIP/RAC3/ACTR/pCIP/ AIB1/TRAM1), are critical mediators of steroid receptor action. Gene ablation studies previously identified SRC-1 and SRC-2 as being involved in the control of energy homeostasis. A more precise identification of the molecular pathways regulated by these coactivators is crucial for understanding the role of steroid receptor coactivators in the control of energy homeostasis and obesity. A genomic approach using microarray analysis was employed to identify the subsets of genes that are altered in the livers of SRC-1-/-, SRC-2-/-, and SRC-3-/- mice. Microarray analysis demonstrates that gene expression changes are specific and nonoverlapping for each SRC member in the liver. The overall pattern of altered gene expressions in the SRC-1-/- mice was up-regulation, whereas SRC-2-/- mice showed an overall down-regulation. Several key regulatory enzymes of energy metabolism were significantly altered in the liver of SRC-2-/- mice, which are consistent with the prior observation that SRC-2-/- mice have increased energy expenditure. This study demonstrates that the molecular targets of SRC-2 regulation in the murine liver stimulate fatty acid degradation and glycolytic pathway, whereas fatty acid, cholesterol, and steroid biosynthetic pathways are down-regulated.

Animals↗
PubMed1988

Genome structure of a new avian sarcoma virus PR2257 determined by restriction analysis.

The genome structure of a new avian src-containing acute transforming virus, designated PR2257, was studied by restriction analysis. PR2257 was isolated from a sarcoma grown in a 155-day-old chick inoculated intraembryonally with a transformation-defective mutant of daPR-C retaining no src sequences. The 4.4-kb genome of PR2257 consists of the following regions: 5' LTR--spliced version of chicken c-src--incomplete env gene--3' LTR. This structure clearly indicates that PR2257 was generated by recombination between a td daPR-C and chicken c-src.

Animals↗
PubMed1982

Avian sarcoma virus Y73 genome sequence and structural similarity of its transforming gene product to that of Rous sarcoma virus.

From the complete nucleotide sequence of the genome of the avian sarcoma virus Y73, we have predicted amino acid sequence of p90 gag-yes, the product of the transforming gene. Contrary to previous evidence from molecular hybridization studies p90 gag-yes was found to have much homology with the transforming gene product p60 src of Rous sarcoma virus, suggesting that the cellular counterparts of the two (c-yes and c-src) originated from a common prototype sequence.

Alpharetrovirus↗
PubMed2004

c-Src and hydrogen peroxide mediate transforming growth factor-beta1-induced smooth muscle cell-gene expression in 10T1/2 cells.

OBJECTIVE: Transforming growth factor-beta1 (TGF-beta1) controls the expression of numerous genes, including smooth muscle cell (SMC)-specific genes and extracellular matrix protein genes. Here we investigated whether c-Src plays a role in TGF-beta1 signaling in mouse embryonic fibroblast C3H10T1/2 cells. METHODS AND RESULTS: TGF-beta1 induction of the SMC contractile protein SM22alpha gene expression was inhibited by PP1 (an inhibitor of Src family kinases) or by C-terminal Src kinase (a negative regulator of c-Src). Induction of SM22alpha by TGF-beta1 was markedly attenuated in SYF cells (c-Src(-), Yes(-), and Fyn(-)) compared with Src(++) cells (c-Src(++), Yes(-), and Fyn(-)). PP1 also inhibited the TGF-beta1-induced expression of serum response factor (SRF), a transcription factor regulating the SMC marker gene expression. Confocal immunofluorescence analysis showed that TGF-beta1 stimulates production of hydrogen peroxide. Antioxidants such as catalase or NAD(P)H oxidase inhibitors such as apocynin inhibited the TGF-beta1-induced expression of SM22alpha. Furthermore, we demonstrate that TGF-beta1 induction of the plasminogen activator inhibitor-1 (PAI-1) gene, which is known to be dependent on Smad but not on SRF, is inhibited by PP1 and apocynin. CONCLUSIONS: Our results suggest that TGF-beta1 activates c-Src and generates hydrogen peroxide through NAD(P)H oxidase, and these signaling pathways lead to the activation of specific sets of genes, including SM22alpha and PAI-1. TGF-beta1 controls the expression of numerous genes, including SM22alpha and PAI-1. We investigated whether c-Src plays a role in TGF-beta1 signaling. TGF-beta1 induction of such genes was significantly reduced in Src family tyrosine kinase-deficient cells, and Csk and pharmacological inhibitors for Src family kinases or antioxidants inhibit the effects of TGF-beta1. These results indicate that c-Src and hydrogen peroxide are required for TGF-beta1 signaling.

Acetophenones↗
PubMed1991

Suppression of src transformation by overexpression of full-length GTPase-activating protein (GAP) or of the GAP C terminus.

Overexpression of the full-length GTPase-activating protein (GAP) has recently been shown to suppress c-ras transformation of NIH 3T3 cells but not v-ras transformation (36). Here, we show that focus formation induced by c-src was inhibited by approximately 80% when cotransfected with a plasmid encoding full-length GAP. In a similar assay, focus formation by the activated c-src (Tyr-527 to Phe) gene was inhibited by 33%. Cotransfection of the GAP C terminus coding sequences (which encode the GTPase-accelerating domain) with c-src or c-src527F inhibited transformation more efficiently than did the full-length GAP, while the GAP N terminus coding sequences had no effect on src transformation. When cells transformed by c-ras, c-src, c-src527F, or v-src were transfected with GAP or the GAP C terminus sequence in the presence of a selectable marker, 40 to 85% of the resistant colonies were found to be morphologically revertant. The GAP C terminus induced reversion of each src-transformed cell line more efficiently than the full-length GAP, but this was not the case for reversion of c-ras transformation. Biochemical analysis of v-src revertant subclones showed that the reversion correlated with overexpression of full-length GAP or the GAP C terminus. There was no decrease in the level of pp60src expression or the level of protein-tyrosine phosphorylation in vivo. We conclude that GAP can suppress transformation by src via inhibition of endogenous ras activity, without inhibiting in vivo tyrosine phosphorylation of cellular proteins induced by pp60src, and that src may negatively regulate GAP's inhibitory action on endogenous ras.

Animals↗