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1.
2.
The ETV6-NTRK3 gene fusion, first identified in the chromosomal translocation in congenital fibrosarcoma, encodes a chimeric protein tyrosine kinase with potent transforming activity. ETV6-NTRK3-dependent transformation involves the joint action of NTRK3 signaling pathways, and aberrant cell cycle progression resulting from activation of Mek1 and Akt. The level of glutathione (GSH) was found to be markedly increased in ETV6-NTRK3-transformed NIH3T3 cells. The activities of the two GSH biosynthetic enzymes as well as of glutathione peroxidase, together with their mRNAs, were also higher in the transformed cells. The transformed cells were able to grow in the presence of GSH-depleting agents, whereas the control cells were not. L-Buthionine-(S,R)-sulfoximine (BSO) inhibited activation of Mek1 and Akt in the transformed NIH3T3 cells. These observations imply that up-regulation of GSH biosynthesis plays a central role in ETV6-NTRK3-induced transformation.  相似文献   

3.
The 12p13 ETV6 (TEL) gene is frequently targeted by chromosomal translocations in human malignancies, resulting in the formation of oncogenic ETV6 gene fusions. Many of the known partner genes encode protein tyrosine kinases (PTKs), generating fusion proteins that function as chimeric PTKs. ETV6-NTRK3 (EN), comprised of the ETV6 SAM domain fused to the NTRK3 PTK, is unique among ETV6 chimeric oncoproteins, as it is expressed in cancers of multiple lineages. We initially hypothesized that, similar to other ETV6-PTK chimeras, SAM-mediated dimerization of EN leads to constitutive activation of the PTK and downstream signaling cascades. However, when the EN SAM domain was replaced with an inducible FK506 binding protein (FKBP) dimerization system, resulting FKBP-NTRK3 chimeras failed to transform NIH 3T3 cells even though PTK activation was preserved. It was recently shown that the ETV6 SAM domain has two potential interacting surfaces, raising the possibility that this domain can mediate protein polymerization. We therefore mutated each EN SAM binding interface in a manner shown previously to abolish self-association of wild-type ETV6. Each mutation completely blocked the ability of EN to polymerize, to activate its PTK, and to transform NIH 3T3 cells. Furthermore, EN itself formed large polymeric structures within cells while mutant EN proteins were present only as monomers. Finally, we observed a dominant negative effect on the transformation of isolated SAM domains coexpressed in EN-transformed cells. Taken together, our results suggest that higher-order polymerization may be a critical requirement for the transformation activity of EN and possibly other ETV6-PTK fusion proteins.  相似文献   

4.
Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R- mouse embryo fibroblasts with R- cells engineered to reexpress IGF-IR (R+ cells). We previously showed that R- cells expressing EN (R- EN cells) are resistant to transformation but that transformation is restored in R+ cells. We now show that while R- EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R+ cells expressing EN (R+ EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R- EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R- EN cells, R+ EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis.  相似文献   

5.
p62Dok, the rasGAP-binding protein, is a common target of protein-tyrosine kinases. It is one of the major tyrosine-phosphorylated molecules in v-Src-transformed cells. Dok consists of an amino-terminal Pleckstrin homology domain, a putative phosphotyrosine binding domain, and a carboxyl-terminal tail containing multiple tyrosine phosphorylation sites. The importance and function of these sequences in Dok signaling remain largely unknown. We have demonstrated here that the expression of Dok can inhibit cellular transformation by the Src tyrosine kinase. Both the phosphotyrosine binding domain and the carboxyl-terminal tail of Dok (in particular residues 336-363) are necessary for such activity. Using a combinatorial peptide library approach, we have shown that the Dok phosphotyrosine binding domain binds phosphopeptides with the consensus motif of Y/MXXNXL-phosphotyrosine. Furthermore, Dok can homodimerize through its phosphotyrosine binding domain and Tyr(146) at the amino-terminal region. Mutations of this domain or Tyr(146) that block homodimerization significantly reduce the ability of Dok to inhibit Src transformation. Our results suggest that Dok oligomerization through its multiple domains plays a critical role in Dok signaling in response to tyrosine kinase activation.  相似文献   

6.
We recently reported that the ETV6/FLT3 fusion protein conferred interleukin-3-independent growth on Ba/F3 cells. The present study has been conducted to assess role of the juxtamembrane domain of FLT3 for signal transduction and cell transformation. The wild-type ETV6/FLT3 fusion protein in transfected cells was a constitutively activated tyrosine kinase that led to up-regulation of PIM-1 and activations of STAT5, AKT, and MAPK. Deletion of the juxtamembrane domain abrogated interleukin-3-independent growth of the transfected cells and PIM-1 up-regulation, whereas it retained compatible levels of phosphorylations of STAT5, AKT, and MAPK. Further deletion of N-terminal region of the tyrosine kinase I domain of FLT3 completely abolished these phosphorylations. Our data indicate that the juxtamembrane domain of FLT3 in ETV6/FLT3 fusion protein is critical for cell proliferation and PIM-1 up-regulation that might be independent of a requirement for signaling through STAT5, MAPK, and AKT pathways.  相似文献   

7.
The human bcr gene encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, and an SH2-binding region. However, the precise physiological functions of BCR are unknown. Coexpression of BCR with the cytoplasmic protein-tyrosine kinase encoded by the c-fes proto-oncogene in Sf-9 cells resulted in stable BCR-FES protein complex formation and tyrosine phosphorylation of BCR. Association involves the SH2 domain of FES and a novel binding domain localized to the first 347 amino acids of the FES N-terminal region. Deletion of the homologous N-terminal BCR-binding domain from v-fps, a fes-related transforming oncogene, abolished transforming activity and tyrosine phosphorylation of BCR in vivo. Tyrosine phosphorylation of BCR in v-fps-transformed cells induced its association with GRB-2/SOS, the RAS guanine nucleotide exchange factor complex. These data provide evidence that BCR couples the cytoplasmic protein-tyrosine kinase and RAS signaling pathways.  相似文献   

8.
SHP-1 is a cytoplasmic SH2 domain containing protein-tyrosine phosphatase (PTP) involved in the negative regulation of multiple signaling pathways in hematopoietic, nervous, and epithelial cells. The thyroid TRK-T3 oncogene consists of the NTRK1 tyrosine kinase domain fused in-frame with sequences of the TFG (TRK-fused gene), encoding a protein of unknown function. TFG contains a coiled-coil domain responsible for TRK-T3 oligomerization. In addition, recent analysis of the sequences outside of the coiled-coil domain suggested possible interactions with other proteins. Based on the presence of a putative SHP-1 SH2-binding site within the TFG sequences, we have investigated the role of the SHP-1 phosphatase in TRK-T3 oncoprotein signaling. In this study we show that SHP-1 interacts with and down-regulates TRK-T3. We provide evidence that SHP-1 SH2 and catalytic domains, respectively, associate with the TFG- and NTRK1-derived portions of TRK-T3. Our data contribute to the definition of cellular mechanisms involved in thyroid tumorigenesis. Moreover, it reveals TFG as a novel protein able to modulate SHP-1 activity.  相似文献   

9.
C Zong  R Yan  A August  J E Darnell  Jr    H Hanafusa 《The EMBO journal》1996,15(17):4515-4525
The proto-oncogene c-eyk, the cellular counterpart of a transforming oncogene, v-eyk, encodes a receptor protein tyrosine kinase with a distinctive extracellular region. We now demonstrate that c-Eyk can be constitutively activated through dimerization, and that the active Eyk displays a unique signaling pattern. When the kinase domain of c-Eyk was fused to the extracellular and transmembrane domains of CD8, the resulting chimera showed elevated kinase activity and caused cellular transformation. We found that the activated Eyk kinases, both v- and c-Eyk, constitutively stimulate the JAK-STAT pathway, while exerting little effect on other signaling routes such as the Ras-MAP kinase and the JNK pathways. The activated Eyk kinases specifically stimulate tyrosine phosphorylation of STAT1, STAT3 and JAK1. These downstream molecules also co-immunoprecipitate with the constitutively dimerized form of Eyk. The Eyk kinase activity is required for STAT1 stimulation. We found that the activation of STAT1 but not STAT3 correlates well with cellular transformation. In constitutively stimulating the JAK-STAT pathway, particularly STAT1, Eyk is unique in its downstream signaling and may be dependent on this pathway for cellular transformation.  相似文献   

10.
In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.  相似文献   

11.
In response to insulin, tyrosine kinase activity of the insulin receptor is stimulated, leading to autophosphorylation and tyrosine phosphorylation of proteins including insulin receptor subunit (IRS)-1, IRS-2, and Shc. Phosphorylation of these proteins leads to activation of downstream events that mediate insulin action. Insulin receptor kinase activity is requisite for the biological effects of insulin, and understanding regulation of insulin receptor phosphorylation and kinase activity is essential to understanding insulin action. Receptor tyrosine kinase activity may be altered by direct changes in tyrosine kinase activity, itself, or by dephosphorylation of the insulin receptor by protein-tyrosine phosphatases. After 1 min of insulin stimulation, the insulin receptor was tyrosine phosphorylated 8-fold more and Shc was phosphorylated 50% less in 32D cells containing both IRS-1 and insulin receptors (32D/IR+IRS-1) than in 32D cells containing only insulin receptors (32D/IR), insulin receptors and IRS-2 (32D/IR+IRS-2), or insulin receptors and a form of IRS-1 that cannot be phosphorylated on tyrosine residues (32D/IR+IRS-1F18). Therefore, IRS-1 and IRS-2 appeared to have different effects on insulin receptor phosphorylation and downstream signaling. Preincubation of cells with pervanadate greatly decreased protein-tyrosine phosphatase activity in all four cell lines. After pervanadate treatment, tyrosine phosphorylation of insulin receptors in insulin-treated 32D/IR, 32D/ IR+IRS-2, and 32D/IR+IRS-1F18 cells was markedly increased, but pervanadate had no effect on insulin receptor phosphorylation in 32D/IR+IRS-1 cells. The presence of tyrosine-phosphorylated IRS-1 appears to increase insulin receptor tyrosine phosphorylation and potentially tyrosine kinase activity via inhibition of protein-tyrosine phosphatase(s). This effect of IRS-1 on insulin receptor phosphorylation is unique to IRS-1, as IRS-2 had no effect on insulin receptor tyrosine phosphorylation. Therefore, IRS-1 and IRS-2 appear to function differently in their effects on signaling downstream of the insulin receptor. IRS-1 may play a major role in regulating insulin receptor phosphorylation and enhancing downstream signaling after insulin stimulation.  相似文献   

12.
The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.  相似文献   

13.
A novel member of the p62(dok) family of proteins, termed DOKL, is described. DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins. The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues. DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells. DOKL is phosphorylated by the Abl tyrosine kinase in vivo. In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation. Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation. The inhibitory effect requires the PTB domain of DOKL. Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl. These results suggest that DOKL may modulate Abl function.  相似文献   

14.
After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.  相似文献   

15.
Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.  相似文献   

16.
Rajala RV  Chan MD 《Biochemistry》2005,44(22):7929-7935
Recently we have shown that insulin fails to induce the phosphorylation of IRS-1 in the retina [Rajala et al. (2004) Biochemistry 43, 5637-5650], even though there is widespread expression of IRS-1 throughout the retina. These results suggest the expression of tissue-specific regulators in the retina. Yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal insulin receptor identified a novel member of the Grb7 gene family, Grb14. Phosphorylation prediction software indicated 6 out of 18 tyrosine residues were most likely to be phosphorylated. Out of six tyrosine phosphorylation sites, one of the tyrosine residues in Grb14 is present in a conserved sequence motif, FXNPXY. The NPXY motifs are recognized by proteins containing a domain known as phosphotyrosine-binding (PTB) or phosphotyrosine-interacting domain (PID). The biological function of the PTB domain is to drive recruitment of signaling adapters such as IRS-1 or Shc to NPXpY (pY stands for phosphotyrosine) on activated receptor tyrosine kinases. We have made a novel finding that the PTB domain of IRS-1 binds to the NPXY motif of Grb14 in a phosphorylation-independent manner. In addition, Grb14-IRS-1 complexes are detected in lysates prepared from retina tissues. We suggest that the Grb14 NPXY motif could be acting as a dominant negative for IRS-1 functions in the retina, and this hypothesis is consistent with the recent study that Grb14-deficient mice exhibit enhanced IRS-1 phosphorylation and activation of protein kinase B. This is the first report describing the presence of the NPXY motif in Grb14 and binding of the PTB domain of IRS-1 in a phosphorylation-independent manner.  相似文献   

17.
The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.  相似文献   

18.
19.
Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.  相似文献   

20.
C‐terminal Src kinase (Csk) that functions as an essential negative regulator of Src family tyrosine kinases (SFKs) interacts with tyrosine‐phosphorylated molecules through its Src homology 2 (SH2) domain, allowing it targeting to the sites of SFKs and concomitantly enhancing its kinase activity. Identification of additional Csk‐interacting proteins is expected to reveal potential signaling targets and previously undescribed functions of Csk. In this study, using a direct proteomic approach, we identified 151 novel potential Csk‐binding partners, which are associated with a wide range of biological functions. Bioinformatics analysis showed that the majority of identified proteins contain one or several Csk‐SH2 domain‐binding motifs, indicating a potentially direct interaction with Csk. The interactions of Csk with four proteins (partitioning defective 3 (Par3), DDR1, SYK and protein kinase C iota) were confirmed using biochemical approaches and phosphotyrosine 1127 of Par3 C‐terminus was proved to directly bind to Csk‐SH2 domain, which was consistent with predictions from in silico analysis. Finally, immunofluorescence experiments revealed co‐localization of Csk with Par3 in tight junction (TJ) in a tyrosine phosphorylation‐dependent manner and overexpression of Csk, but not its SH2‐domain mutant lacking binding to phosphotyrosine, promoted the TJ assembly in Madin‐Darby canine kidney cells, implying the involvement of Csk‐SH2 domain in regulating cellular TJs. In conclusion, the newly identified potential interacting partners of Csk provided new insights into its functional diversity in regulation of numerous cellular events, in addition to controlling the SFK activity.  相似文献   

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