首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
There is evidence that extracellular nucleotides, acting through multiple P2 receptors, may play an important role in the regulation of bone metabolism by activating intracellular signaling cascades. We have studied the modulation of mitogen-activated protein kinase (MAPK) signaling pathways and its relationship to changes in intracellular calcium concentration ([Ca2+]i) induced by ATP in ROS-A 17/2.8 osteoblastic cells. ATP and UTP (10 μM) increased [Ca2+]i by cation release from intracellular stores. We have found that when the cells are subsequently subjected to mechanical stress (medium perturbation), a transient calcium influx occurs. This mechanical stress-activated calcium influx (MSACI) was not observed after ADP stimulation, indicating that P2Y2 receptor activation is required for MSACI. In addition, ERK 1/2 and p38 MAPK were activated by ATP in a dose- and time-dependent manner. This activation was almost completely blocked using neomycin (2.5 mM), an inhibitor of phosphoinositide-phospholipase C (PI-PLC), Ro 318220 (1 μM), a protein kinase C (PKC) inhibitor, and PP1 (50 μM), a potent and selective inhibitor of the Src-family tyrosine kinases. Ca2+-free extracellular medium (containing 0.5 mM EGTA) and the use of gadolinium (5 μM), which suppressed MSACI, prevented ERK 1/2 and p38 phosphorylation by ATP. Altogether, these results represent the first evidence to date suggesting that P2Y2 receptor stimulation by ATP in osteoblasts sensitizes mechanical stress activated calcium channels leading to calcium influx and a fast activation of the ERK 1/2 and p38 MAPK pathways. This effect also involves upstream mediators such as PI-PLC, PKC and Src family kinases.  相似文献   

2.
Netrin receptor DCC plays critical roles in many cellular processes, including axonal outgrowth and migration, angiogenesis, and apoptosis, but the molecular basis of DCC-mediated signaling is largely unclear. ERK2, a member of the MAPK family, is one of the few proteins known to be involved in DCC-mediated signaling. Here, we report that ERK2 directly interacts with DCC, and the ERK2-binding region was mapped to the conserved intracellular P1 domain of the receptor. The structure of ERK2 in complex with the P1 domain of DCC reveals that DCC contains a MAPK docking motif. The docking of the P1 domain onto ERK2 physically positions several phosphorylation sites of DCC in the vicinity of the kinase active site. We further show that the docking interaction between the P1 domain and ERK2 is essential for the ERK2-mediated phosphorylation of DCC. We conclude that DCC signaling is directly coupled with MAPK signaling cascades.  相似文献   

3.
We used a genetic approach to characterize features of mitogen-activated protein kinase (MAPK) activation occurring as a consequence of expression of distinct erbB receptor combinations in transformed human cells. Kinase-deficient erbB proteins reduced epidermal growth factor (EGF)-induced tyrosine phosphorylation of endogenous Shc proteins and also reduced immediate and sustained EGF-induced ERK MAPK activities in human glioblastoma cells, although basal ERK MAPK activities were unaffected. Basal and EGF-induced JNK and p38 MAPK kinase activities were equivalent in parental cancer cells and EGFR-inhibited subclones. When ectopically overexpressed in murine fibroblasts and human glioblastoma cells, a constitutively activated human EGF receptor oncoprotein (deltaEGFR) induced EGF-independent elevation of basal ERK MAPK activity. Basal JNK MAPK kinase activity was also specifically induced by deltaEGFR, which correlated with increased phosphorylation of a 54-kDa JNK2 protein observed in deltaEGFR-containing cells. The JNK activities in response to DNA damage were comparably increased in cells containing wildtype EGFR or deltaEGFR. Consistent with the notion that transforming erbB complexes induce sustained and unregulated MAPK activities, coexpression of p185(neu) and EGFR proteins to levels sufficient to transform murine fibroblasts also resulted in prolonged EGF-induced ERK in vitro kinase activation. Transforming erbB complexes, including EGFR homodimers, deltaEGFR homodimers, and p185(neu)/EGFR heterodimers, appear to induce sustained, unattenuated activation of MAPK activities that may contribute to increased transformation and resistance to apoptosis in primary human glioblastoma cells.  相似文献   

4.
Phosphorylation is the most important post-translational event at a cellular level that is regulated by protein kinases. MAPK is a key player in the important cellular signaling pathway. It has been hypothesized that phosphorylation might have a role in the induction of break tolerance against some autoantigens such as SRP72. The aim of this study was to explore the pathways of phosphorylation and overexpression of the SRP72 polypeptide, using an in vitro model of Jurkat cells stimulated by recombinant human (rh)IL-1β in the presence of MAPK inhibitors. We used Jurkat cells as a substrate stimulated with rhIL-1β in the presence of MAPK inhibitors at different concentrations in a time course in vitro experiment by immunoprecipitation, immunoprecipitation-Western blotting, and real time PCR. Our results showed that rhIL-1β causes up-regulation of protein expression and phosphorylation of SRP72 in Jurkat cells. Inhibitors of the MAPK pathway ERK1/2 or p38α/β down-regulate the expression of SRP72 autoantigen in Jurkat cells stimulated by rhIL-1β. Our results highlight the importance of studying the pathways of activation and overexpression of autoantigens. It will be necessary to perform careful research on various kinases pathways, including MAPK in dermatomyositis and other rheumatic diseases, to help to explain the routes of activation and inhibition of autoantigens. The understanding of this process may help to develop new therapies to prevent and control the loss of tolerance toward own normal proteins.  相似文献   

5.
6.
7.
G protein-coupled receptor kinases (GRKs) mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors (GPCRs). We investigate the role of GRK2 on epidermal growth factor (EGF) receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase (ERK/MAPK) activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold (P〈0.05) at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% (P〈0.01). Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.  相似文献   

8.
9.
The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr(402). This was reduced by Ca(2+) chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC alpha and -epsilon to the cell membrane and enhanced the association of Src with PKC alpha and PKC epsilon, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.  相似文献   

10.
Zhao LJ  Zhao P  Chen QL  Ren H  Pan W  Qi ZT 《Cell proliferation》2007,40(4):508-521
OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.  相似文献   

11.
We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA2 translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA2 may be a downstream target of MSK1.  相似文献   

12.
A receptor–ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type‐specific post‐receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death‐triggering factor in motoneurons through LT‐βR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT‐induced axonal elongation and branching require ERK and caspase‐9 pathways. This distinct response involves a compartment‐specific activation of LIGHT signals, with somatic activation‐inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.  相似文献   

13.
In most target cells, activation of the type 1 CRH receptor (CRH-R1) by CRH or urocortin (UCN I) leads to stimulation of the Gs-protein/adenylyl cyclase/protein kinase A cascade. Signal transduction of CRH-R1 also involves alternative pathways such as phosphorylation of ERK1/2 and p38 MAPK, two members of the MAPK family that mediate important pathophysiological responses. The intracellular pathways by which CRH-R1 activates these MAPK are only partially understood; here we characterized further signaling mechanisms and molecules involved in CRH-R1-mediated ERK1/2 and p38 MAPK activation. In human embryonic kidney 293 cells overexpressing recombinant CRH-R1alpha, UCN I induced ERK1/2 and p38 MAPK activation was dependent on signaling molecules involved in agonist-induced CRH-R1alpha trafficking and endocytosis. Furthermore, time course studies and use of selective inhibitors demonstrated that ERK1/2 activation occured within 5 min, was sustained for at least 60 min, and was dependent on both phosphatidylinositol 3-kinase (PI3-K)/Akt activation and epidermoid growth factor receptor transactivation involving matrix metelloproteinases. UCN I effect on p38 MAPK phosphorylation was more transient, returned to basal within 40 min and was dependent on epidermoid growth factor receptor transactivation, but not PI3-K/Akt activation. Overexpression of G(alpha-)transducin, showed that G(betagamma)-subunit activation is only partially required for ERK1/2 phosphorylation and does not play a role in p38 MAPK phosphorylation, whereas overexpression of a dominant-negative Ras (Ras N17) attenuated both ERK and p38 MAPK activation. In conclusion, a complex signaling network appears to mediate CRH-R1alpha-MAPK interactions; PI3-K might play a critical role in the regulation of CRH-R1alpha signaling selectivity and cellular responses.  相似文献   

14.
Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.  相似文献   

15.
16.
17.
Androgen ablation therapy is effective in treating androgen-dependent prostate tumors; however, tumors that can proliferate in castrate levels of androgen eventually arise. We previously reported that in CWR22Rv1 (Rv1) cells, the protease calpain 2 can cleave the androgen receptor (AR) into a constitutively active ∼80,000 low molecular weight (LMW) form. In this study, we further dissect the mechanisms that produce the AR LMW forms using Rv1 cells and the related CWR22-R1 (R1) cells. The 39-amino acid insertional mutation in the Rv1-AR (E3DM-AR) sensitizes this AR to calpain 2 proteolysis. R1 cells encode the same AR molecule as the parental CWR22 xenograft. Using calpain 2 small interfering RNA and calpeptin, we find that calpain 2 plays a role in the generation of the LMW-AR in R1 cells. Furthermore, LMW-AR expression is regulated by the activation of calpain 2 by ERK 1 and 2. Inhibition of ERK phosphorylation or small interfering RNA-mediated decrease of ERK expression reduces LMW-AR levels in R1 cells. Conversely, activation of the MAPK pathway results in increased ERK phosphorylation and increased levels of LMW-AR. Finally, analyses of human tumor samples found that LMW-AR levels are higher in tumors that have an increased calpain/calpastatin ratio and/or increased levels of phospho-ERK (pERK). This suggests that a higher calpain/calpastatin ratio collaborates with activated ERK to promote the generation of the LMW-AR.  相似文献   

18.
19.
It has been suggested that A(3) adenosine receptors (ARs) play a role in the pathophysiology of cerebral ischemia with dual and opposite neuroprotective and neurodegenerative effects. This could be due to a receptor regulation mediated by rapid phosphorylation and desensitization carried out by intracellular kinases. In this study, we investigated the involvement of extracellular regulated kinase (ERK 1 and 2), members of the mitogen-activated protein kinase (MAPK) family, in A(3) AR phosphorylation. A(3) AR mediated the activation of ERK 1/2 with a typical transient monophasic kinetics (5 min). The activation was not affected by hypertonic sucrose cell pre-treatment, suggesting that this effect occurred independently of receptor internalization. The involvement of MAPK cascade in the A(3) AR regulation process was evaluated using two well-characterized MAPK kinase inhibitors, PD98059 (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) and U0126 (1,4-diamino-2,3-dicyano-1,4-bis (aminophenylthio) butadiene). The exposure of cells to PD98059 prevented MAPK activation and inhibited homologous A(3) AR desensitization and internalization, impairing agonist-mediated receptor phosphorylation. PD98059 inhibited the membrane translocation of G protein-coupled receptor kinase (GRK(2)), which is involved in A(3) AR homologous phosphorylation, suggesting this kinase as a target for the MAPK cascade.On the contrary, the chemically unrelated inhibitor of the MAPK cascade, U0126, did not significantly affect GRK(2) membrane translocation or receptor internalization. Nevertheless, the inhibitor induced a significant impairment of receptor phosphorylation and desensitization. These results suggested that the MAPK cascade is involved in A(3) AR regulation by a feedback mechanism which controls GRK(2) activity and probably involves a direct receptor phosphorylation.  相似文献   

20.
Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号