| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
EGF binding triggers dimerization of the EGFR, which enables the trans-autophosphorylation of EGFR tyrosine kinase required for its full activation. Multi-site phosphorylation of EGFR creates binding sites for adaptor molecules containing SH2 or phosphotyrosine binding domains, such as Shc and Grb2 (Jorissen et al. 2003). Association with these molecules stimulates several signaling pathways, among which the Ras/MAP kinase (MAPK) pathway has been best studied (Nishida & Gotoh 1993). Activation of Erk1/2 is known to be essential for the regulation of cellular proliferation and differentiation (Chang & Karin 2001). In a rat pheochromocytoma cell line, PC12, EGF triggers cellular proliferation with a transient activation of Erk1/2, whereas nerve growth factor (NGF) induces differentiation into a sympathetic neuron-like phenotype, accompanied by sustained activation of Erk1/2 (Marshall 1995). It is thus believed that the duration of Erk1/2 activation can define cell fate in response to EGF or NGF stimulation.
It has been shown that both Src and EGFR are often over-expressed in cancer cells, and functional synergism between Src and EGFR, such as direct association and transphosphorylation, has been also observed in cells that co-over-express Src and the EGFR (Haskell et al. 2001). Furthermore, accumulating evidence suggests that SFK is activated upon EGF stimulation and phosphorylates downstream signaling molecules involved in receptor trafficking, cytoskeletal reorganization and signal propagation (Jorissen et al. 2003). With respect to receptor trafficking from the plasma membrane to endosomes, some SFK substrates, including clathrin (Wilde et al. 1999) and dynamin (Ahn et al. 2002), have been identified as critical regulators of EGFR endocytosis. These observations strongly suggest that SFKs are involved in the down-regulation of EGF signaling by controlling the endocytosis of EGFR. However, whether SFKs are essential for EGFR endocytosis is still controversial (Sorkina et al. 2002).
It has been proposed that there are plasma membrane microdomains, called lipid rafts, where glycosphingolipid, cholesterol and a number of signaling molecules including SFKs accumulate, and that these rafts function as platforms for signal transduction (Galbiati et al. 2001). Previously, it was reported that a proportion of EGFR is localized in these regions, especially in caveolae, which are flask-shaped invaginations of lipid rafts containing caveolin-1 (Anderson 1998), and that, on EGF stimulation, EGFR moves into clathrin-coated pits, is internalized and sorted to lysosomes (Furuchi & Anderson 1998; Mineo et al. 1999). These findings suggest that lipid rafts play an important role in the regulation of EGF signaling, although the precise mechanism is still unclear.
In this study, we investigate the role of SFKs in EGF signaling through lipid rafts using PC12 cells, in which intracellular signaling pathways leading to proliferation and differentiation can be morphologically distinguished. We show that, although a large proportion of EGFR is already in lipid rafts, EGF binding promotes further accumulation, and that the activity of SFKs is required to facilitate the clustering of activated EGFRs on the membrane. These findings suggest that SFKs are involved in EGF signaling by regulating the movement of EGFR on the plasma membrane prior to receptor endocytosis.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
To evaluate the role of lipid rafts in EGF signaling, we first examined changes in tyrosine phosphorylated proteins at these sites in PC12 cells. Raft and non-raft fractions were separated from Triton X-100-treated lysates on a discontinuous sucrose density gradient, and the purity of these fractions was confirmed by the distinct separation of GM1 and the transferrin receptor (TfR) as raft and non-raft markers, respectively (Fig. 1A). Upon EGF stimulation, a 170 kDa protein underwent transient tyrosine phosphorylation in both fractions, but the levels of phosophorylation decreased more rapidly in the raft fraction than in the non-raft fraction (Fig. 1B). To determine the identity of this protein, the distribution of the EGFR protein was first examined. Western blotting analysis revealed that the EGFR is concentrated in the raft fraction even in unstimulated cells, and it accumulates further after EGF stimulation. Next, the EGFR was immunoprecipitated and the extent of tyrosine phosphorylation was determined with an anti-phosphotyrosine antibody. This analysis revealed that EGFR was concentrated in the raft fraction, that it was phosphorylated immediately after EGF stimulation, and that the 170 kDa protein was EGFR (Fig. 1B). The amount of Shc, an EGFR signaling partner (Sakaguchi et al. 1998), also increased in the raft fraction after EGF stimulation and then decreased gradually (Fig. 1C). These data indicate that a large proportion of the EGFR is activated in lipid rafts and transduces signal from these sites.
|
Inhibition of SFK and disruption of lipid rafts induce neurite extension of PC12 cells in the presence of EGF
To assess the contribution of SFK activity in EGF signaling, we first examined the effect of SFK inhibitors on the morphological changes of PC12 cells induced by EGF stimulation (Fig. 2A). Interestingly, we found that cells pretreated with the potent SFK inhibitor PP2 (Hanke et al. 1996) and with the more selective inhibitor SU6656 (Blake et al. 2000) were induced to undergo neuron-like morphological change even following EGF stimulation, although these inhibitors only modestly affected cell adhesion in the absence of EGF. To analyze these effects more quantitatively, the numbers of cells bearing neurites were determined. As summarized in Fig. 2B, EGF induced neurite extension in a substantial proportion of cells pretreated with PP2 (34.67 ± 1.45%) or SU6656 (26.00 ± 1.15%). Additionally, to more specifically inhibit SFK activity, we over-expressed Csk, a common negative regulator for SFK, by infecting PC12 cells with adenovirus vectors. Expression of a LacZ control only slightly enhanced EGF-induced neurite extension (18.67 ± 3.18%), but Csk over-expression had a significant effect (30.67 ± 3.84%) (data not shown). These results strongly suggest the involvement of SFK activity in the regulation of EGF signaling in PC12 cells.
|
Inhibition of SFK activity and disruption of lipid rafts induce sustained activation of Erk1/2
The above results raised the following question: why does EGF induce neuron-like morphological change, rather than proliferation, when SFK activity is inhibited or lipid raft function is attenuated? It is well established that the fate of PC12 cells is determined by the duration of activation of Erk1/2 (Marshall 1995). Indeed, Erk1/2 activation was significantly more sustained in NGF-stimulated cells than in EGF-stimulated cells (Fig. 3A,B). Although the effects were less prominent, treatments with PP2, SU6656 and nystatin all induced sustained activation of Erk1/2 up to 60 min after stimulation (Fig. 3C–H). To determine if these levels of activation of Erk1/2 are sufficient for differentiation, the Erk1/2 pathway was blocked by the MEK inhibitor U0126 (Favata et al. 1998). As shown in Fig. 4, U0126 treatment suppressed neurite outgrowth of PC12 cells induced by EGF when SFK activity was inhibited by PP2. Similarly, MEK inhibition repressed neurite extension of cells treated with SU6656 or nystatin (data not shown). These results indicate that inhibition of SFK activity or attenuation of lipid raft function extends the duration of Erk1/2 activation, which is sufficient for the induction of neuron-like morphological change.
|
|
To address the mechanism by which Erk1/2-mediated EGF signaling is sustained when SFK activity is inhibited, we first focused on the fate of the EGFR. It is known that activated EGFR is ubiquitinated by Cbl ubiquitin ligase and then routed from the early endosome to the lysosome for degradation (Carpenter 2000). To examine the role of SFK in these processes, we employed Csk–/– fibroblasts in which SFK is constitutively active. In these cells, the EGFR is expressed at levels high enough to detect protein degradation and to control SFK activity by re-expressing Csk. We found that the EGFR protein was rapidly degraded after EGF stimulation, while EGFR degradation was significantly suppressed by treatment with PP2 (Fig. 5A). Consistent with this, Erk1/2 activation was also sustained in these cells. Similar results were obtained in cells treated with SU6656 (data not shown).
|
Inhibition of SFK affects EGFR clustering
Endocytosis of the EGFR is known to be a primary step in the down-regulation of EGF signaling (Carpenter 2000). To identify the sites of SFK action in PC12 cells, we observed the movement of the activated EGFR by time-lapse imaging using Texas Red-conjugated EGF (EGF-TR). PC12 cells treated with PP2 or vehicle alone were loaded with EGF-TR at 4 °C, and EGF signaling was then activated by replacing the medium with warmed medium. In control cells, clustering of EGFRs was visible 2 min after activation, whereas very few clusters formed at the same time point in PP2-treated cells. Even 4 min after activation, cluster formation in PP2-treated cells was lower than in control cells (Fig. 6A). Quantitative analysis showed that inhibition of SFK activity delayed the rate of EGFR cluster formation up to 10 min after activation but did not affect the total numbers of EGFR clusters formed after longer incubation (Fig. 6B). These results suggest that SFK activity affects the rate of clustering of the activated EGFR on the membrane.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Here we observed the EGF-induced activation of SFKs, especially Src and Yes, in the raft fraction of PC12 cells, which was indicated by the increase in phosphorylation at the SFK autophosphorylation site, pY418. In contrast, even after EGF stimulation, there was no significant decrease in the phosphorylation of the C-terminal negative regulatory site, pY529, suggesting that dephosphorylation of pY529 does not contribute to SFK activation in this pathway. This is potentially due to an accumulation of Csk in lipid rafts through binding to the highly phosphorylated Csk adaptor proteins, such as Cbp. Thus, it is likely that the activated EGFR induces SFK activation by direct phosphorylation of the activation site of SFK, Y418, or by physical interaction that causes conformational change in SFKs (Thomas & Brugge 1997). However, since the activation occurred in only a small population of SFK molecules, we cannot rule out the possibility that there was some level of pY529 dephosphorylation but it was undetectable. We also showed that Fyn and Yes, but not Src, were mainly localized in lipid rafts in PC12 cells, because of the differential modifications of their unique N-terminal sequences (Thomas & Brugge 1997). However, Src and Yes, but not Fyn, were activated upon EGF stimulation, which may reflect previous observations that Fyn has some unique features compared with other SFK family members (Nada et al. 1993; Thomas et al. 1995). These findings suggest that there are some functional differences among the SFKs present in lipid rafts.
To verify the role of SFK activity in EGF signaling, we examined the effects of SFK inhibitors on the cell fate determination of PC12 cells. Interestingly, treatments with the widely used SFK inhibitors PP2 and SU6656 induced neurite extension rather than proliferation, even following EGF stimulation. Consistent with these effects, the activation of Erk1/2 was more sustained than in control cells, and the inhibition of Erk1/2 activation by a MEK inhibitor suppressed EGF-induced neurite extension. These findings reveal that the sustained activation of Erk1/2 caused by SFK inhibition induces neurite extension in EGF-stimulated PC12 cells (Vaudry et al. 2002; Wong et al. 2002). To exclude the possibility that these events are caused by a side-effect of SFK inhibitors, we over-expressed Csk, a natural specific inhibitor for SFK, in PC12 cells with an adenovirus expression system. Over-expression of Csk also dramatically induced the EGF-dependent neurite extension of PC12 cells, although adenovirus infection itself had some effects. Since adenovirus infection activates a number of signaling pathways including Erk (Liu & Muruve 2003), it is possible that the activation of such pathways synergistically promotes the EGF-dependent neurite extension of PC12 cells. Taken together, it is very likely that SFK activity is involved in the regulation of EGF signaling, particularly in defining the duration of EGFR activation.
To evaluate the role of lipid rafts in EGF signaling, we observed the effects of perturbing lipid raft function. As a raft-targeting reagent, methyl ß-cyclodextrin (Mß-CD), which strongly sequesters cholesterol from the cell surface, is widely used to attenuate the function of lipid rafts. However, Mß-CD has been reported to block clathrin-mediated endocytosis (Rodal et al. 1999; Subtil et al. 1999), a well established mechanism involved in the down-regulation of EGF signaling. To investigate other aspects of lipid raft functions, we used nystatin, which does not block the clathrin pathway (Ros-Baro et al. 2001). We found that nystatin treatment also induced the EGF-dependent neurite extension of PC12 cells in a manner similar to that of SFK inhibitors, suggesting that lipid rafts play a role independent of the clathrin pathway. These findings demonstrate that lipid rafts serve as a platform that is required for the mutual interaction of EGFR and SFKs.
When the EGFR is occupied by its ligand, it dimerizes or interacts with other receptors such as ErbB2 and then clusters to form coated pits that are internalized through the endocytotic pathway. Some proportion of the internalized EGFR is recycled to the plasma membrane by recycling endosomes, and others are subjected to degradation by the ubiquitination-lysosome pathways (Carpenter 2000). So far four endocytotic mechanisms have been described: clathrin-mediated endocytosis, caveolae/raft-dependent endocytosis, pinocytosis and macropinocytosis and phagocytosis (Nabi & Le 2003; Nichols 2003). It has been shown that clathrin is recruited to lipid rafts after EGF stimulation (Otsuki et al. 2003), suggestive of cross-talk between the caveolae/raft-dependent and the clathrin-mediated endocytotic pathways. Lipid rafts can assemble the molecular machinery necessary for the intracellular propagation of EGFR signals and for receptor internalization (Puri et al. 2005). Furthermore, SFKs contribute to clathrin-mediated endocytosis and ubiquitination-mediated protein degradation (Wilde et al. 1999; Carpenter 2000; Ahn et al. 2002). These lines of evidence demonstrate that lipid rafts and SFKs play important roles in the down-regulation mechanism involving the endocytotic pathway.
A ubiquitin ligase Cbl has been implicated in ubiquitination of receptor tyrosine kinases (RTKs) and regulation of RTK endocytosis via binding to the CIN85-endophilin complex (Dikic & Giordano 2003). Our preliminary observation revealed that tyrosine phosphorylated Cbl was recruited to lipid rafts upon EGF stimulation, and inhibition of SFKs repressed phosphorylation of Cbl to some extent. Therefore, it is possible that Cbl is involved in the SFK-mediated down-regulation of EGF signaling in PC12 cells. On the other hand, the Sprouty family proteins are also known as inhibitory molecules of RTK signaling (Dikic & Giordano 2003). In EGF signaling, however, it is reported that Sprouty2 functions as an inhibitor of Cbl (Wong et al. 2002) by blocking Cbl-mediated EGFR ubiquitination and association between Cbl and CIN85 (Haglund et al. 2005). To evaluate the role of these ubiquitin-mediated pathways in the down-regulation of EGF signaling in PC12 cells, it would be interesting to examine if Sprouty proteins could serve as targets of SFKs in lipid rafts.
In this study, we showed that inhibition of SFK activity by PP2 or Csk in Csk–/– fibroblasts suppresses EGFR degradation. However, in PC12 cells that express EGFR at much lower levels than in fibroblasts, suppression of EGFR degradation by the inhibition of SFKs or by the attenuation of lipid rafts was not apparent. Furthermore, treatment with nystatin, which does not attenuate clathrin-mediated endocytosis, did induce neurite extension of PC12 cells, and we observed that trafficking of some intracellular organelles such as endosomes and lysosomes was not significantly affected by SFK inhibition (data not shown). These findings led us to suspect that SFKs in PC12 cells mainly control EGF signaling at another step of the pathway.
To identify the steps that are regulated by SFKs in PC12 cells, we observed EGFR behavior by time-lapse and confocal microscopy. We found that the initial rate of EGFR clustering on the plasma membrane was substantially decreased by the inhibition of SFKs, raising the possibility that the delay in clustering reflects the total delay in the down-regulation of EGFR activation. It is known that the activated EGFR rapidly clusters and is subjected to endocytosis. However, the mechanism by which activated SFKs in lipid rafts promote receptor clustering remains thoroughly unknown. To address this, we are currently investigating the SFK targets responsible for the regulation of receptor dynamics in lipid rafts, particularly by focusing on molecules involved in the interaction between the plasma membrane and cytoskeletal architecture.
Finally, it should be noted that EGF stimulation also activates various negative-feedback mechanisms of Erk pathways, including phosphorylation of the Dok1/2 adaptor proteins, which activate rasGAP (Lock et al. 1999) or act as scaffolding molecules that processively assemble Src and Csk (Van Slyke et al. 2005), and an induction of the CL-100 phosphatase, also known as MAP kinase phosphatase-1 (MKP-1), which inactivates Erks (Cook et al. 1997). In addition to the endocytotic pathway, these complex mechanisms may also contribute to the rapid termination of signals, resulting in a transient activation of Erk sufficient to promote cell proliferation. Since most of these mechanisms have been shown to involve SFKs (Lock et al. 1999; Chandrasekharan et al. 2004; Van Slyke et al. 2005), it is likely that SFKs also play roles in these processes to fine-tune EGF signaling.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Preparation of anti-Cbp antibodies was previously described (Matsuoka et al. 2004). Anti-Src antibody (Ab-1) was purchased from Oncogene Research. Anti-Yes and anti-Shc antibodies were from Transduction Laboratories. Anti-phospho Erk1/2 antibody was from Cell Signaling Technology. Anti-Src [pY418] and Anti-Src [pY529] antibodies were from Biosource. Anti-phosphotyrosine (4G10) and anti-EGFR (06-129) antibodies were from Upstate Biotechnology. Anti-EGFR (1005), anti-Fyn (FYN3), anti-actin (C-11), and horseradish peroxidase-conjugated anti-goat IgG antibodies were from Santa Cruz Biotechnology. Anti-transferrin receptor, horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG antibody were from Zymed Laboratories, Inc. Cholera Toxin B Subunit Peroxidase conjugated, nystatin and U0126 were from Sigma-Aldrich. EGF was from Peprotech. PP2, PP3 and SU6656 were from Calbiochem. Texas Red-labeled EGF was from Molecular Probes. Protein G Sepharose 4 Fast Flow was from Amersham Biosciences.
Cell culture and adenovirus infection
PC12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Nissui) supplemented with 5% fetal bovine serum (FBS; Equitech-Bio, Inc.) and 5% horse serum (HS; Gibco Life Technology) on dishes coated with Collagen Type I (BD Biosciences). Csk–/– fibroblasts were kind gifts from Dr Akira Imamoto (Thomas et al. 1995) and were cultured in DMEM supplemented with 10% FBS. Csk–/– fibroblasts were infected with Ax1CAT-lacZ, Ax1CATcsk K+ and Ax1CATcsk K-adenoviruses, which were obtained as previously described (Takayama et al. 1999). After 24 h, adenovirus was removed by changing the medium, and the infected cells were used for experiments.
Growth factor stimulation and assay of neurite outgrowth
To examine the effects of EGF stimulation for short periods (0–6 h), cells were serum-starved overnight and then stimulated for the indicated time at 37 °C with 100 ng/mL EGF. PC12 cells were incubated with 1 µM PP2, PP3 or DMSO for 30 min, or with 10 µM SU6656 or DMSO for 1 h, prior to EGF stimulation. PC12 cells were incubated with 20 µg/mL nystatin for 1 h, prior to EGF stimulation. Csk–/– fibroblasts were incubated with 5 µM PP2 for 30 min. To examine the effects of EGF stimulation for longer periods (0–5 days), PC12 cells were starved overnight in DMEM containing 0.5% FBS and 0.5% HS, and maintained in this medium while they were treated with other reagents (1 µM PP2, PP3 and SU6656; 5 µM U0126; 20 µg/mL nystatin; 100 ng/mL EGF and NGF). Half of the medium was changed every 2 days. The number of cells bearing neurites was counted after 5 days. A neurite was identified as a process with a length greater than the diameter of a cell body. The percentage of cells with neurites was calculated by counting 100 cells. The data from three independent experiments were subsequently averaged and standard errors were calculated. Cell proliferation assay was performed using cell proliferation reagent WST-1 (Roche). PC12 cells were cultured on 96-well microtiter plates in the presence of various reagents, and the viable cells were stained with WST-1 after stimulation with EGF for 1–4 days.
Preparation of raft fractions and total cell lysates
To prepare raft fractions, cells were washed twice with ice-cold PBS, lyzed in buffer A (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL trypsin inhibitor, 1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 50 mM NaF, 5 mM 2-mercaptoethanol) supplemented with 0.25% Triton X-100, and stirred for 1 h at 4 °C. The lysate was adjusted to 40% sucrose in buffer A. In an ultracentrifuge tube the 2.5 mL sample was sequentially overlaid with 2 mL 35% sucrose solution and 1 mL 5% sucrose solution and centrifuged at 100 000 g, in a Beckman SW50.1 for 16 h at 4 °C. The gradient was separated into 12 fractions from the top, and fraction No. 3, at the interface between 35% and 5% sucrose, was used as the raft fraction. Fraction No. 11 was used as the non-raft fraction. To solubilize raft proteins, 1/10 volume of buffer A supplemented with 20% N-octyl-ß-D-glucoside (ODG) and 10% Nonidet P40 (NP40) was added to the raft fraction, and the sample was cleared by centrifugation at 15 000 r.p.m. for 30 min. To prepare total cell lysates, cells were washed twice with ice-cold PBS, lyzed in buffer A containing 5% glycerol, 2% ODG, and 1% NP40, and centrifuged at 15 000 r.p.m. at 4 °C for 15 min, and the supernatants were collected. The protein contents of the raft and non-raft fractions and of total cell lysates were measured by the Bradford method using bovine serum albumin (BSA) as the standard.
Immunoprecipitation
After addition of IP buffer (50 mM Tris-HCl [pH 7.4], 250 mM NaCl, 1 mM EDTA, 2% ODG, 1% NP40, 5% glycerol, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL trypsin inhibitor, 1 mM PMSF, 1 mM sodium orthovanadate, 50 mM NaF, 5 mM 2-mercaptoethanol), the raft fractions or total cell lysates were incubated with protein G Sepharose at 4 °C for 30 min, and centrifuged at 15 000 r.p.m. at 4 °C for 1 min, and supernatant was collected. Antibodies were added, and the mixtures were incubated on ice for 30 min. Protein G Sepharose was then added, and the mixtures were incubated at 4 °C for 1 h. The immunoprecipitates were washed 5 times with IP buffer and subjected to immunoblot analysis.
Immunoblot analysis
Raft and non-raft fractions were mixed with 4 x SDS sample buffer (250 mM Tris-HCl [pH 6.8], 8% SDS, 20% 2-mercaptoethanol, 0.008% Bromophenol blue). Total cell lysates and immunoprecipitates were mixed with 2 x SDS sample buffer containing 10% sucrose. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 (T-TBS), T-TBS containing 1% BSA or 5% skim milk, probed with primary antibodies and further incubated with an HRP-conjugated secondary antibody. Antibody binding was visualized with a chemiluminescence system (Perkin Elmer Life Sciences).
EGFR imaging
Time-lapse images were obtained by culturing PC12 cells on a 35 mm-diameter glass base dish coated with collagen, serum-starving overnight and incubating with 1 µM PP2 or DMSO. The cells were then chilled to 4 °C and treated with 100 ng/mL EGF conjugated to Texas Red (EGF-TR) at 4 °C for 1 h. Cells were washed twice with ice-cold DMEM (phenol red-free), warmed by replacing with medium at 37 °C, and subjected to time-lapse imaging. Cells were visualized on an Olympus IX71 microscope equipped with a CoolSNAP HQ (Roper Scientific, Trenton, NJ, USA), controlled by MetaMorph software (Universal Imaging, West Chester, PA, USA). Images were recorded every 15 s. To obtain confocal images, PC12 cells were cultured on cover glasses coated with collagen, serum-starved overnight and incubated with 10 µM SU6656 or DMSO. Cells were then chilled to 4 °C and treated with 100 ng/mL EGF-TR at 4 °C for 1 h. Cells were warmed by transferring the cover glasses to warmed medium, incubated for 0–30 min, fixed with ice-cold 4% paraformaldehyde for 20 min and washed twice with PBS. The cells were observed using an OLYMPUS IX81 confocal microscope controlled by Fluoview FV1000 software.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
* Correspondence: E-mail: okadam{at}biken.osaka-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Anderson, R.G. (1998) The caveolae membrane system. Annu. Rev. Biochem. 67, 199–225.[CrossRef][Medline]
Blake, R.A., Broome, M.A., Liu, X., et al. (2000) SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling. Mol. Cell. Biol.
20, 9018–9027.
Brdicka, T., Pavlistova, D., Leo, A., et al. (2000) Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation. J. Exp. Med.
191, 1591–1604.
Brown, M.T. & Cooper, J.A. (1996) Regulation, substrates and functions of src. Biochim. Biophys. Acta 1287, 121–149.[Medline]
Carpenter, G. (2000) The EGF receptor: a nexus for trafficking and signaling. Bioessays 22, 697–707.[CrossRef][Medline]
Chandrasekharan, U.M., Yang, L., Walters, A., Howe, P. & DiCorleto, P.E. (2004) Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation. J. Biol. Chem.
279, 46678–46685.
Chang, L. & Karin, M. (2001) Mammalian MAP kinase signalling cascades. Nature 410, 37–40.[CrossRef][Medline]
Cook, S.J., Beltman, J., Cadwallader, K.A., McMahon, M. & McCormick, F. (1997) Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells. J. Biol. Chem.
272, 13309–13319.
Dikic, I. & Giordano, S. (2003) Negative receptor signalling. Curr. Opin. Cell Biol. 15, 128–135.[CrossRef][Medline]
Favata, M.F., Horiuchi, K.Y., Manos, E.J., et al. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem.
273, 18623–18632.
Furuchi, T. & Anderson, R.G. (1998) Cholesterol depletion of caveolae causes hyperactivation of extracellular signal-related kinase (ERK). J. Biol. Chem.
273, 21099–21104.
Galbiati, F., Razani, B. & Lisanti, M.P. (2001) Emerging themes in lipid rafts and caveolae. Cell 106, 403–411.[CrossRef][Medline]
Haglund, K., Schmidt, M.H., Wong, E.S., Guy, G.R. & Dikic, I. (2005) Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation. EMBO Rep. 6, 635–641.[CrossRef][Medline]
Hanke, J.H., Gardner, J.P., Dow, R.L., et al. (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J. Biol. Chem.
271, 695–701.
Haskell, M.D., Slack, J.K., Parsons, J.T. & Parsons, S.J. (2001) c-Src tyrosine phosphorylation of epidermal growth factor receptor, P190 RhoGAP, and focal adhesion kinase regulates diverse cellular processes. Chem. Rev. 101, 2425–2440.[CrossRef][Medline]
Jorissen, R.N., Walker, F., Pouliot, N., Garrett, T.P., Ward, C.W. & Burgess, A.W. (2003) Epidermal growth factor receptor: mechanisms of activation and signalling. Exp. Cell Res. 284, 31–53.[CrossRef][Medline]
Kawabuchi, M., Satomi, Y., Takao, T., et al. (2000) Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases. Nature 404, 999–1003.[CrossRef][Medline]
Kim, K.B., Kim, S.I., Choo, H.J., Kim, J.H. & Ko, Y.G. (2004) Two-dimensional electrophoretic analysis reveals that lipid rafts are intact at physiological temperature. Proteomics 4, 3527–3535.[CrossRef][Medline]
Liu, Q. & Muruve, D.A. (2003) Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther. 10, 935–940.[CrossRef][Medline]
Lock, P., Casagranda, F. & Dunn, A.R. (1999) Independent SH2-binding sites mediate interaction of Dok-related protein with RasGTPase-activating protein and Nck. J. Biol. Chem.
274, 22775–22784.
Marshall, C.J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179–185.[CrossRef][Medline]
Matsuoka, H., Nada, S. & Okada, M. (2004) Mechanism of Csk-mediated down-regulation of Src family tyrosine kinases in epidermal growth factor signaling. J. Biol. Chem.
279, 5975–5983.
Mineo, C., Gill, G.N. & Anderson, R.G. (1999) Regulated migration of epidermal growth factor receptor from caveolae. J. Biol. Chem.
274, 30636–30643.
Nabi, I.R. & Le, P.U. (2003) Caveolae/raft-dependent endocytosis. J. Cell Biol.
161, 673–677.
Nada, S., Okada, M., MacAuley, A., Cooper, J.A. & Nakagawa, H. (1991) Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature 351, 69–72.[CrossRef][Medline]
Nada, S., Yagi, T., Takeda, H., et al. (1993) Constitutive activation of Src family kinases in mouse embryos that lack Csk. Cell 73, 1125–1135.[CrossRef][Medline]
Nichols, B. (2003) Caveosomes and endocytosis of lipid rafts. J. Cell Sci.
116, 4707–4714.
Nishida, E. & Gotoh, Y. (1993) The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem. Sci. 18, 128–131.[CrossRef][Medline]
Otsuki, M., Itoh, T. & Takenawa, T. (2003) Neural Wiskott-Aldrich syndrome protein is recruited to rafts and associates with endophilin A in response to epidermal growth factor. J. Biol. Chem.
278, 6461–6469.
Pike, L.J., Han, X. & Gross, R.W. (2005) EGF receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study. J. Biol. Chem.
280, 26796–26804.
Puri, C., Tosoni, D., Comai, R., et al. (2005) Relationships between EGFR Signaling-competent and Endocytosis-competent Membrane Microdomains. Mol. Biol. Cell
16, 2704–2718.
Rodal, S.K., Skretting, G., Garred, O., Vilhardt, F., van
Deurs, B. & Sandvig, K. (1999) Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol. Biol. Cell
10, 961–974.
Ros-Baro, A., Lopez-Iglesias, C., Peiro, S., et al. (2001) Lipid rafts are required for GLUT4 internalization in adipose cells. Proc. Natl. Acad. Sci. USA
98, 12050–12055.
Sakaguchi, K., Okabayashi, Y., Kido, Y., et al. (1998) Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol. Endocrinol.
12, 536–543.
Sargiacomo, M., Sudol, M., Tang, Z. & Lisanti, M.P. (1993) Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J. Cell Biol.
122, 789–807.
Simons, K. & Toomre, D. (2000) Lipid rafts and signal transduction. Nature Rev. Mol. Cell Biol. 1, 31–39.[CrossRef][Medline]
Smart, E.J., Ying, Y.S., Mineo, C. & Anderson, R.G. (1995) A detergent-free method for purifying caveolae membrane from tissue culture cells. Proc. Natl. Acad. Sci. USA
92, 10104–10108.
Sorkina, T., Huang, F., Beguinot, L. & Sorkin, A. (2002) Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor. J. Biol. Chem.
277, 27433–27441.
Subtil, A., Gaidarov, I., Kobylarz, K., Lampson, M.A., Keen, J.H. & McGraw, T.E. (1999) Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc. Natl. Acad. Sci. USA
96, 6775–6780.
Takayama, Y., Tanaka, S., Nagai, K. & Okada, M. (1999) Adenovirus-mediated overexpression of C-terminal Src kinase (Csk) in type I astrocytes interferes with cell spreading and attachment to fibronectin. Correlation with tyrosine phosphorylations of paxillin and FAK. J. Biol. Chem.
274, 2291–2297.
Thomas, S.M. & Brugge, J.S. (1997) Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol. 13, 513–609.[CrossRef][Medline]
Thomas, S.M., Soriano, P. & Imamoto, A. (1995) Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature 376, 267–271.[CrossRef][Medline]
Van
Slyke, P., Coll, M.L., Master, Z., Kim, H., Filmus, J. & Dumont, D.J. (2005) Dok-R mediates attenuation of epidermal growth factor-dependent mitogen-activated protein kinase and Akt activation through processive recruitment of c-Src and Csk. Mol. Cell. Biol.
25, 3831–3841.
Vaudry, D., Stork, P.J., Lazarovici, P. & Eiden, L.E. (2002) Signaling pathways for PC12 cell differentiation: making the right connections. Science
296, 1648–1649.
Wilde, A., Beattie, E.C., Lem, L., et al. (1999) EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake. Cell 96, 677–687.[CrossRef][Medline]
Wong, E.S., Fong, C.W., Lim, J., et al. (2002) Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis, and consequently enhances Ras/ERK signalling. EMBO J. 21, 4796–4808.[CrossRef][Medline]
Received: 17 July 2005
Accepted: 23 September 2005
This article has been cited by other articles:
|
|
 |
|
  N.-P. Tsai, Y.-L. Lin, Y.-C. Tsui, and L.-N. Wei Dual action of epidermal growth factor: extracellular signal-stimulated nuclear-cytoplasmic export and coordinated translation of selected messenger RNA J. Cell Biol., February 8, 2010; 188(3): 325 - 333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Baba, K. Akagi, M. Takayanagi, J. G. Flanagan, T. Kobayashi, and M. Hattori Fyn Tyrosine Kinase Regulates the Surface Expression of Glycosylphosphatidylinositol-linked Ephrin via the Modulation of Sphingomyelin Metabolism J. Biol. Chem., April 3, 2009; 284(14): 9206 - 9214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Vacaresse, B. Moller, E. M. Danielsen, M. Okada, and J. Sap Activation of c-Src and Fyn Kinases by Protein-tyrosine Phosphatase RPTP{alpha} Is Substrate-specific and Compatible with Lipid Raft Localization J. Biol. Chem., December 19, 2008; 283(51): 35815 - 35824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Sandilands, V. G. Brunton, and M. C. Frame The membrane targeting and spatial activation of Src, Yes and Fyn is influenced by palmitoylation and distinct RhoB/RhoD endosome requirements J. Cell Sci., August 1, 2007; 120(15): 2555 - 2564. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
