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1.
Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.  相似文献   

2.
We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.  相似文献   

3.
Zhang Y  Dong Z  Bode AM  Ma WY  Chen N  Dong Z 《DNA and cell biology》2001,20(12):769-779
Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.  相似文献   

4.
The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.  相似文献   

5.
In the present study, we demonstrated that Ang II provokes a transitory enhancement of focal adhesion kinase (FAK) and paxillin phosphorylation in human umbilical endothelial cells (HUVEC). Moreover, Ang II induces a time- and dose-dependent augmentation in cell migration, but does not affect HUVEC proliferation. The effect of Ang II on FAK and paxillin phosphorylation was markedly attenuated in cells pretreated with wortmannin and LY294002, indicating that phosphoinositide 3-kinase (PI3K) plays an important role in regulating FAK activation. Similar results were observed when HUVEC were pretreated with genistein, a non-selective tyrosine kinases inhibitor, or with the specific inhibitor PP2 for Src family kinases, demonstrating the involvement of protein tyrosine kinases, and particularly Src family of tyrosine kinases, in the downstream signalling pathway of Ang II receptors. Furthermore, FAK and paxillin phosphorylation was markedly blocked after treatment of HUVEC with AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) phosphorylation. Pretreatment of cells with inhibitors of PI3K, Src family tyrosine kinases, and EGFR also decreased HUVEC migration. In conclusion, these results suggest that Ang II mediates an increase in FAK and paxillin phosphorylation and induces HUVEC migration through signal transduction pathways dependent on PI3K and Src tyrosine kinase activation and EGFR transactivation.  相似文献   

6.
Activation of the extracellular calcium-sensing receptor (CaR) stimulates mitogen-activated protein kinases to upregulate the synthesis and secretion of parathyroid hormone related peptide (PTHrP) from cells expressing the CaR heterologously or endogenously. The current experiments demonstrate that this occurs because CaR activation "transactivates" the EGF receptor (EGFR). Time dependent increases in tyrosine phosphorylation of the EGFR after addition of extracellular calcium ([Ca2+]o, 3 mM) occurred in stably CaR-transfected HEK293 cells but not in non-transfected HEK293 cells. AG1478, an EGFR kinase inhibitor, prevented the CaR-mediated increases of pERK and PTHrP release, while AG1296, a PDGFR kinase inhibitor, had no effect. Inhibitors of matrix metalloproteinase and heparin bound-EGF prevented the CaR-mediated increases of pERK and PTHrP, consistent with a "triple-membrane-spanning signaling" requirement for transactivation of the EGFR by the CaR. Proximal and distal signal transduction cascades activated by the CaR may reflect transactivation of the EGFR by the extracellular calcium-sensing receptor.  相似文献   

7.
We have previously found that bronchial epithelial cells express CCR3 whose signaling elicits mitogen-activated protein (MAP) kinase activation and cytokine production. Several investigators have focused on the signaling crosstalk between G protein-coupled receptors (GPCRs) and epidermal growth factor receptor (EGFR) in cancer cells. In this study, we investigated the role of EGFR in CCR3 signaling in the bronchial epithelial cell line NCI-H292. Eotaxin (1-100 nM) induced dose-dependent tyrosine phosphorylation of EGFR in NCI-H292 cells. Pretreatment of the cells with the EGFR inhibitor (AG1478) significantly inhibited the MAP kinase phosphorylation induced by eotaxin. Eotaxin stimulated IL-8 production, which was inhibited by AG1478. The transactivation of EGFR through CCR3 is a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells. The delineation of the signaling pathway of chemokines will help to develop a new therapeutic strategy to allergic diseases including bronchial asthma.  相似文献   

8.
Previous studies demonstrated that ionizing radiation activates the epidermal growth factor receptor (EGFR), as measured by Tyr autophosphorylation, and induces transient increases in cytosolic free [Ca2+], [Ca2+]f. The mechanistic linkage between these events has been investigated in A431 squamous carcinoma cells with the EGFR Tyr kinase inhibitor, AG1478. EGFR autophosphorylation induced by radiation at doses of 0.5-5 Gy or EGF concentrations of 1-10 ng/ml is inhibited by >75% at 100 nM AG1478. Activation of EGFR enhances IP3 production as a result of phospholipase C (PLC) activation. At the doses used, radiation stimulates Tyr phosphorylation of both, PLCgamma and erbB-3, and also mediates the association between erbB-3 and PLCgamma not previously described. The increased erbB-3 Tyr phosphorylation is to a significant extent due to transactivation by EGFR as >70% of radiation- and EGF-induced erbB-3 Tyr phosphorylation is inhibited by AG 1478. The radiation-induced changes in [Ca2+]f are dependent upon EGFR, erbB-3 and PLCgamma activation since radiation stimulated IP3 formation and Ca2+ oscillations are inhibited by AG1478, the PLCgamma inhibitor U73122 or neutralizing antibody against an extracellular epitope of erbB-3. These results demonstrate that radiation induces qualitatively and quantitatively similar responses to EGF in stimulation of the plasma membrane-associated receptor Tyr kinases and immediate downstream effectors, such as PLCgamma and Ca2+.  相似文献   

9.
Focal adhesion kinase (FAK) plays a key role in the crosstalk of growth factor- and cell adhesion-mediated signaling pathway. In this study, we found that the quantitative change of phosphorylated FAK was bell-shaped time-dependently by EGF stimulation in immortalized human keratinocyte (HaCaT). EGF enhanced FAK phosphorylation and cell spreading in adhering HaCaT cells with low-phosphorylated FAK. On the other hand, spread HaCaT cells having high-phosphorylated FAK changed to round shapes with FAK dephosphorylation 15 min after EGF stimulation. Pharmacological agents, U0126 and PD98059 (mitogen-activated protein kinases (MAPK) kinases (MEK) inhibitors), and AG1478 (an EGF receptor kinase inhibitor) blocked the cell rounding and FAK dephosphorylation. In addition, the EGFR-MAPK signaling pathway had an influence on cell migration by regulating FAK dephosphorylation of keratinocytes in response of EGF, since the MEK inhibitors and AG1478 suppressed EGF-induced cell migration. However, FAK phosphorylation and HaCaT cell spreading were inhibited only by the antagonist of EGF-EGFR binding but not by the MEK inhibitors and AG1478. Taken together, we suggest that EGF is antagonistically involved in both FAK phosphorylation and dephosphorylation with different mechanisms in a cell.  相似文献   

10.
The specific delta-opioid receptor agonist [D-Ala(2)-D-Leu(5)]enkephalin (DADLE) protects against infarction in the heart when given before ischemia. In rabbit, this protection leads to phosphorylation of the pro-survival kinases Akt and extracellular signal-regulated kinase (ERK) and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1 h starting 5 min before reperfusion and reduced infarct size from 31.0 +/- 2.3% in the control group to 14.6 +/- 1.6% (P = 0.01). This protection was abolished by cotreatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective before ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt and ERK1 and -2 in reperfused hearts (2.5 +/- 0.5, 1.6 +/- 0.2, and 2.3 +/- 0.7-fold of baseline levels, P < 0.05 vs. control). The DADLE-dependent increases in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 +/- 0.2-fold, P = 0.03 vs. control). Thus the delta-opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK and is dependent on the transactivation of the EGFR.  相似文献   

11.
Tyrphostin AG1478 is known as a specific and reversible inhibitor of TK (tyrosine kinase) activity of the EGFR [EGF (epidermal growth factor) receptor]. It is attractive as an anticancer agent for cancers with elevated EGFR TK levels. However, post‐application effects of AG1478 are not well studied. We have analysed EGFR phosphorylation after termination of AG1478 application using human epidermoid carcinoma A431 cells. It was found that AG1478 inhibitory action is fast, but not fully reversible: removal of tyrphostin resulted in incomplete restoration of the overall EGFR phosphorylation. Analysing the state of two individual autophosphorylation sites of internalized EGFR, Tyr1045 and Tyr1173, we demonstrated that phosphorylation of Tyr1173 involved in stimulation of the MAPK (mitogen‐activated protein kinase) cascade was restored much more efficiently than that in position 1045, which binds the ubiquitin ligase c‐Cbl and is necessary for targeting the receptor for lysosomal degradation. c‐Cbl association with EGFR abolished by AG1478 was not reestablished after tyrphostin cessation. As a consequence, ubiquitination‐dependent EGFR delivery to lysosomes was blocked, while phosphorylation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) was even increased. Thus, after termination of AG1478, the intracellular level of the inhibitor can be reached at which mitogenic signalling will be restored, whereas the EGFR negative regulation due to lysosomal degradation will not.  相似文献   

12.
Specific survival signals derived from extracellular matrix (ECM) and growth factors are required for mammary epithelial cell survival. We have previously demonstrated that inhibition of ECM-induced ERK1/2 MAPK pathway with PD98059 leads to apoptosis in primary mouse mammary epithelial cells. In this study, we have further investigated MAPK signal transduction in cell survival of these cells cultured on a laminin rich reconstituted basement membrane. ERK1/2 phosphorylation is activated in the absence of insulin by cell-cell substratum interactions that cause ligand-independent EGFR transactivation. Intact EGFR signal transduction is required for ECM determined cell survival as the EGFR pathway inhibitor, AG1478, induces apoptosis of these cultures. Rescue of AG1478 or PD98059 treated cultures by PTPase inhibition with vanadate restores cellular phospho-ERK1/2 levels and prevents apoptosis. These results emphasize that ERK1/2 phosphorylation and inhibition of PTPase activity are necessary for PMMEC cell survival.  相似文献   

13.
Proximal events in signaling by plasma membrane estrogen receptors   总被引:18,自引:0,他引:18  
Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.  相似文献   

14.
In mesangial cells angiotensin II (Ang II) has been shown to activate extracellular regulated kinases 1 and 2 (ERK1/2). Here, we studied the role of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) in Ang II-induced ERK1/2 activation in human mesangial cells. Ang II induced activation of ERK1/2 via the AT(1) receptor, and this response was blocked by the PDGFR-selective tyrosine kinase inhibitor AG1295, but not by AG1478, an EGFR-selective tyrosine kinase inhibitor, indicating participation of the PDGFR, but not of the EGFR in Ang II-induced ERK1/2 activation. In agreement with this assumption, Ang II caused tyrosine phosphorylation of the PDGFR and the adapter protein Shc in an AG1295-sensitive fashion. In conclusion, our data show that Ang II-induced activation of mitogenic signalling cascade in human mesangial cells involves ligand-independent activation of the PDGFR, but not of the coexpressed EGFR.  相似文献   

15.
LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor (EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNFalpha protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.  相似文献   

16.
Up-regulation of intercellular adhesion molecule-1 (ICAM-1) is frequently implicated in lung inflammation. Sphingosine-1-phosphate (S1P) has been shown to play a key role in inflammation via adhesion molecules induction, and then causes lung injury. However, the mechanisms underlying S1P-induced ICAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unclear. The effect of S1P on ICAM-1 expression was determined by Western blot and real-time PCR. The involvement of signaling pathways in these responses was investigated by using the selective pharmacological inhibitors and transfection with siRNAs. S1P markedly induced ICAM-1 expression and monocyte adhesion which were attenuated by pretreatment with the inhibitor of S1PR1 (W123), S1PR3 (CAY10444), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), PI3K (LY294002), or AP-1 (Tanshinone IIA) and transfection with siRNA of S1PR1, S1PR3, c-Src, EGFR, PDGFR, p38, p42, JNK1, c-Jun, or c-Fos. We observed that S1P-stimulated p42/p44 MAPK and p38 MAPK activation was mediated via a c-Src/EGFR and PDGFR-dependent pathway. S1P caused the c-Src/EGFR/PDGFR complex formation. On the other hand, we demonstrated that S1P induced p42/p44 MAPK and p38 MAPK-dependent Akt activation. In addition, S1P-stimulated JNK1/2 phosphorylation was attenuated by SP600125 or PP1. Finally, S1P enhanced c-Fos mRNA levels and c-Jun phosphorylation. S1P-induced c-Jun activation was reduced by PP1, AG1478, AG1296, U0126, SP600125, SB202190, or LY294002. These results demonstrated that S1P-induced ICAM-1 expression and monocyte adhesion were mediated through S1PR1/3/c-Src/EGFR, PDGFR/p38 MAPK, p42/p44 MAPK/Akt-dependent AP-1 activation.  相似文献   

17.
We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.  相似文献   

18.
Overexpression of Met is a common finding in thyroid carcinomas. Recently, we reported on overexpression and ligand-independent constitutive activation of Met in anaplastic thyroid carcinoma cells. In the present study we have investigated a putative mechanism for this phenomenon. Cell lines with constitutively activated Met expressed both TGF-alpha mRNA and protein. Western blot analysis revealed expression of receptors for epidermal growth factor (EGFR) in all carcinoma cell lines; in tumor cells with elevated levels of TGF-alpha mRNA there was a constitutive tyrosine phosphorylation of the EGFRs. Preincubation of carcinoma cells with suramin decreased EGFR activation and downregulated Met expression as well as the ligand-independent phosphorylation of Met. Similar results were obtained with a EGFR tyrosine kinase inhibitor, AG 1478. The MEK inhibitor U0126 had an even more pronounced effect compared to AG 1478, indicating a Ras/MAPK-mediated signal in the regulation of Met expression and activation. Inhibition of EGFR signaling also decreased proliferation of the anaplastic thyroid carcinoma cells. Thus, aberrant activation of EGFRs may lead to an overexpression and activation of Met, which may be of importance for the malignant phenotype of anaplastic thyroid carcinomas.  相似文献   

19.
Exposure of MDA-MB-468 cells to ionizing radiation (IR) caused biphasic activation of ERK as indicated by its phosphorylation at Thr202/Tyr204. Specific epidermal growth factor receptor (EGFR) inhibitor AG1478 and specific Src inhibitor PP2 inhibited IR-induced ERK1/2 activation but phosphatidylinositol-3 kinase inhibitor wortmannin did not. IR caused EGFR tyrosine phosphorylation, whereas it did not induce EGFR autophosphorylation at Tyr992, Tyr1045, and Tyr1068 or Src-dependent EGFR phosphorylation at Tyr845. SHP-2, which positively regulates EGFR/Ras/ERK signaling cascade, became activated by IR as indicated by its phosphorylation at Tyr542. This activation was inhibited by PP2 not by AG1478, which suggests Src-dependent activation of SHP-2. Src and PTPalpha, which positively regulates Src, became activated as indicated by phosphorylation at Tyr416 and Tyr789, respectively. These data suggest that IR-induced ERK1/2 activation involves EGFR through a Src-dependent pathway that is distinct from EGFR ligand activation.  相似文献   

20.
M Eder  J D Griffin    T J Ernst 《The EMBO journal》1993,12(4):1647-1656
The ability of the receptor for the hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to function in non-hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM-CSF receptor and a series of stable transformants were isolated that bound GM-CSF with either low (KD = 860 - > 1000 pM) or high affinity (KD = 20-80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf-1, and the transient induction of c-fos and c-myc mRNAs. The activation of protein tyrosine phosphorylation by GM-CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5-20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58-60 kDa. Some of these proteins co-migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM-CSF. In particular, p42 and p44 were identified as mitogen-activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf-1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM-CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM-CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.  相似文献   

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