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Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita, Osaka 565-0871, Japan
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Human PVR/CD155 was originally identified as a poliovirus receptor (Mendelsohn et al. 1989; Koike et al. 1990), whereas rodent Tage4 was originally identified as the product of a gene over-expressed in rodent colon carcinoma (Chadeneau et al. 1994, 1996). PVR/CD155 is also over-expressed in many human cancer cells (Gromeier et al. 2000; Masson et al. 2001; Sloan et al. 2004). Tage4 is the rodent counterpart of human PVR/CD155 (Takai et al. 2003). This molecule with three nomenclatures is re-named nectin-like molecule-5, Necl-5 (Takai et al. 2003). We recently found that when cells do not contact other cells, Necl-5 is up-regulated by growth factors such as platelet-derived growth factor (PDGF) and fibroblast growth factor, and enhances growth factor-induced cell movement and proliferation (Ikeda et al. 2004; Kakunaga et al. 2004). The Necl-5 gene has an AP-1-responsive promoter, and Necl-5 is up-regulated by Ras-Raf-MEK-ERK-AP-1 signaling (Hirota et al. 2004). Necl-5 co-localizes with integrin 
vß3 at leading edges of moving cells, and enhances their motile activity (Ikeda et al. 2004). Necl-5 forms a complex with integrin vß3, and enhances growth factor-induced formation of lamellipodia, ruffles and focal complexes, which are necessary for effective cell movement (Y. Minami et al. unpublished data). Necl-5 enhances growth factor-induced activation of Ras-Raf-MEK-ERK signaling, causing up- and down-regulation of cell cycle regulators, including cyclins D2 and E, and p27kip1, thereby shortening the period of the G1 phase of the cell cycle (Kakunaga et al. 2004). Necl-5 does not affect PDGF-induced tyrosine phosphorylation of the PDGF receptor.
Necl-5 does not show homophilic cell–cell adhesion activity, but heterophilically trans-interacts with nectin-3, a member of the Ig-like nectin family, which is a Ca2+-independent cell–cell adhesion molecule, and cooperatively forms adherens junctions with cadherins (Ikeda et al. 2003; Takai et al. 2003). When cells contact other cells, Necl-5 is down-regulated from the cell surface by its trans-interaction with nectin-3 (Fujito et al. 2005), although when cells do not contact other cells, Necl-5 is up-regulated by growth factor-induced signaling (Hirota et al. 2004). Up-regulation of Necl-5 enhances growth factor-induced cell movement and proliferation, whereas down-regulation of Necl-5 reduces these mechanisms (Fujito et al. 2005). Thus, Necl-5 plays roles, at least partly, in contact inhibition of cell movement and proliferation, but it remains to be clarified how down-regulation of Necl-5 reduces growth factor-induced cell proliferation.
Sprouty (Spry) is a novel negative regulator of growth factor-induced signaling (Christofori 2003; Kim & Bar-Sagi 2004); it is tyrosine-phosphorylated by c-Src, of which activation is induced by many growth factors through their respective receptors (Gross et al. 2001; Hanafusa et al. 2002; Li et al. 2004; Mason et al. 2004). Tyrosine-phosphorylated Spry inhibits growth factor-induced activation of Ras and subsequent activation of Raf-MEK-ERK signaling (Gross et al. 2001; Hanafusa et al. 2002), but does not inhibit growth factor-induced tyrosine phosphorylation of its receptor or activation of c-Src or PI3-kinase (Gross et al. 2001; Li et al. 2004). This site of action of Spry in growth factor-induced signaling is apparently similar to that of Necl-5, although these two molecules show opposite roles in growth factor-induced signaling. Therefore, we examined whether Necl-5 physically and functionally associates with Spry, and regulates PDGF-induced Ras signaling through Spry.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We first confirmed previous observations (Aliaga et al. 1999; Zhang et al. 2002; Laprise et al. 2004) using NIH3T3 cells that activation levels of Ras and ERK are greater in cells cultured at a low cell density than those cultured at a high cell density in the presence of serum. The activation level of Ras was significantly greater in cells cultured at a low cell density than in cells cultured at a high cell density (Fig. 1Aa). Similarly, the activation level of ERK was greater in cells cultured at a low cell density than in cells cultured at a high cell density (Fig. 1Ab). These results indicated that Ras signaling was reduced by increasing cell density. We noted that cell density effects on the activation level of ERK were greater than those on the activation level of Ras. The exact reason for these different effects is unknown, but since ERK is activated by Ras via Raf and MEK, differences might be amplified by this signal cascade. Another possibility is that ERK activity might be additionally regulated by PAK, which is activated by Cdc42 or Rac and phosphorylates both Raf and MEK in an integrin-dependent manner (Slack-Davis et al. 2003; Edin & Juliano 2005) and/or by an unidentified up-stream factor other than Ras which might be regulated by cell density.
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We previously showed that cell surface Necl-5 was down-regulated in a cell density-dependent manner (Fujito et al. 2005), and that knockdown of Necl-5 reduced DNA synthesis in NIH3T3 cells cultured at a low cell density (Fujito et al. 2005). We examined whether activation levels of Ras and ERK are reduced in NIH3T3 cells in which Necl-5 was knocked down (Necl-5-knockdown NIH3T3 cells) cultured at a low cell density. NIH3T3 cells were co-transfected with siRNA vector and myc-Ras or EGFP-ERK and cultured at a low cell density. Myc-Ras or EGFP-ERK was monitored to differentiate between events of transfected cells and those of non-transfected cells. Activation levels of myc-Ras and EGFP-ERK were significantly reduced by knockdown of Necl-5 (Fig. 1Ba,Bb). siRNA vector against Necl-5 was functional, as estimated by Western blotting; and transfection efficiency of our experiments was 20%–40%. The expression level of endogenous Necl-5 in Necl-5-knockdown cells was less than 10% of that of control cells, as estimated by FACS analysis (data not shown). These results indicated that cell density-dependent changes in amount of cell surface Necl-5 were closely related to cell density-dependent changes in activation levels of Ras and ERK.
Physical interaction of Spry2 with Necl-5
It has been shown that Spry is a negative regulator for Ras signaling and DNA synthesis (Gross et al. 2001). In NIH3T3 cells, Spry1 and 2, but not 4, were detected by reverse transcription-PCR; and the expression level of Spry2 was much higher than that of Spry1 (data not shown), consistent with previously reported data (Gross et al. 2001). Therefore, we examined the possible physical interaction of Spry2 with Necl-5 in NIH3T3 cells. Western blotting showed that amounts of Spry2 were not different between cells cultured at high and low cell densities (Fig. 2A).
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vß3 at leading edges of moving NIH3T3 cells (Y. Minami et al. unpublished data). Immunofluorescence signal for endogenous Spry2 co-localized with that for endogenous Necl-5 at leading edges of moving NIH3T3 cells (Fig. 2Ba). This signal for Spry2 at leading edges increased and decreased in NIH3T3 cells stably expressing FLAG-Necl-5 (Necl-5-NIH3T3 cells) and Necl-5-knockdown-NIH3T3 cells, respectively (Fig. 2Bb,Bc). These results indicated that Spry2 co-localized with Necl-5, and that localization of Spry2 was affected by amount of cell surface Necl-5. We then examined the physical interaction of Spry2 with Necl-5 using a co-immunoprecipitation assay. When endogenous Spry2 was immunoprecipitated by a specific antibody from lysate of NIH3T3 cells cultured at a low or high cell density, endogenous Necl-5 was co-immunoprecipitated with endogenous Spry2 in cells cultured at a low cell density, but it was not significantly co-immunoprecipitated with endogenous Spry2 in cells cultured at a high cell density (Fig. 2C). Taken together, these results indicated that Necl-5 physically interacted with Spry2 in a cell density-dependent manner.
Functional association of Spry2 with Necl-5 in PDGF-induced Ras signaling
We then examined the functional association of Spry2 with Necl-5 using NIH3T3 cells which were stimulated with PDGF after serum starvation, because serum contains so many types of growth factors. However, amounts of endogenous Spry2 in cells cultured at low and high cell densities were markedly reduced by serum starvation (Fig. 3A). Their levels became less than 10% of original levels within 15 min after serum starvation (data not shown). Therefore, we exogenously expressed myc-Spry2 and examined its effects on PDGF-induced activations of Ras and ERK in NIH3T3 cells cultured at low and high cell densities. Over-expression of myc-Spry2 reduced PDGF-induced activation of co-transfected myc-Ras in cells cultured at a high cell density, but not at a low cell density (Fig. 3Ba). The expression level of endogenous Necl-5 in cells cultured at a high cell density was about 30% of that of cells cultured at a low cell density. Over-expression of myc-Spry2 did not significantly reduce PDGF-induced activation of co-transfected myc-Ras in Necl-5-NIH3T3 cells cultured at high and low cell densities (Fig. 3Ba). In contrast, knockdown of Necl-5 reduced PDGF-induced activation of co-transfected myc-Ras in cells cultured at a low cell density (Fig. 3Bb). Over-expression of myc-Spry2 further reduced PDGF-induced activation of co-transfected myc-Ras in Necl-5-knockdown-NIH3T3 cells cultured at a low cell density (Fig. 3Bb). Essentially the same results were obtained for PDGF-induced activation of co-transfected EGFP-ERK (Fig. 3Ca,Cb), although cell density effects on activation level of ERK were greater than those on the activation level of Ras, consistent with the results of Fig. 1. These results indicated that the inhibitory effect of exogenously expressed myc-Spry2 depended on cell density, and that Necl-5 reduced its inhibitory effect, thereby enhancing PDGF-induced Ras signaling.
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PDGF-independent physical interaction of Spry2 with Necl-5 through its cytoplasmic region
We next examined, using an immunoprecipitation assay, whether the physical interaction of Spry2 with Necl-5 is affected by PDGF-induced signaling. Necl-5-NIH3T3 cells were transfected with or without myc-Spry2, starved of serum, cultured in the presence or absence of PDGF and subjected to an immunoprecipitation assay using anti-Spry polyclonal antibody (pAb). Endogenous Spry2 was hardly immunoprecipitated from the lysate of the cells which were not transfected with myc-Spry2 and cultured in the absence of PDGF, because endogenous Spry2 was down-regulated by serum starvation (data not shown). Accordingly, FLAG-Necl-5 was hardly co-immnoprecipitated with endogenous Spry2 (Fig. 4A). When myc-Spry2 was immunoprecipitated from lysates of the cells which were transfected with myc-Spry2 and cultured in the presence and absence of PDGF, FLAG-Necl-5 was co-immunoprecipitated with myc-Spry2 from both lysates (Fig. 4A). Amounts of co-immunoprecipitated FLAG-Necl-5 were similar between both lysates. These results provided another line of evidence for the physical interaction of Spry2 with Necl-5, and indicated that this interaction was independent of PDGF-induced signaling.
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CP-NIH3T3 cells). Endogenous Spry2 was hardly immunoprecipitated from the lysate of the cells, which were not transfected with myc-Spry2 and cultured in the absence of PDGF, because endogenous Spry2 was down-regulated by serum starvation (data not shown). FLAG-Necl-5-CP was hardly co-immnoprecipitated with endogenous Spry2 (Fig. 4B). When myc-Spry2 was immunoprecipitated from lysates of the cells, which were transfected with myc-Spry2 and cultured in the presence and absence of PDGF, FLAG-Necl-5-CP was hardly co-immunoprecipitated with myc-Spry2 from both lysates (Fig. 4B). These results indicated that Spry2 interacted with Necl-5 mainly through the cytoplasmic region of Necl-5. Inhibitory effect of Necl-5 on c-Src-catalyzed tyrosine phosphorylation of Spry2
We next examined how the interaction of Spry2 with Necl-5 regulates PDGF-induced Ras signaling. It was shown that growth factor-induced c-Src-mediated tyrosine phosphorylation of Spry2 induced inhibition of growth factor-induced Ras signaling (Li et al. 2004), but it has not been demonstrated that Spry2 is tyrosine-phosphorylated by PDGF signaling (Mason et al. 2004). We examined the effect of physical interaction of Spry2 with Necl-5 on tyrosine phosphorylation of Spry2. When NIH3T3 cells were cultured at a high cell density in the presence and absence of pervanadate, a tyrosine phosphatase inhibitor, and stimulated with PDGF, tyrosine phosphorylation of myc-Spry2 was detected in the presence of pervanadate, but not in its absence (Fig. 5A). Spry2 was detected as two bands, and the lower band was tyrosine-phosphorylated. Tyrosine phosphorylation of myc-Spry2 was not detected under any conditions tested when cells were cultured in the absence of pervanadate. Therefore, we performed the following experiments in the presence of pervanadate. NIH3T3 cells were cultured at low and high cell densities in the presence of pervanadate and stimulated with PDGF. PDGF-induced tyrosine phosphorylation of myc-Spry2 in NIH3T3 cells cultured at low and high cell densities, but the level in the latter cells was greater than that in the former cells (Fig. 5Ba). This reaction was inhibited by SU6656, an inhibitor of the Src family (Blake et al. 2000) (Fig. 5Bb), suggesting that the PDGF-induced reaction was mediated by a Src family member, presumably c-Src, consistent with earlier observations (Li et al. 2004). Over-expression of FLAG-Necl-5 markedly reduced this reaction irrespective of cell density (Fig. 5Ba). Knockdown of Necl-5 increased this reaction in cells cultured at a low cell density (Fig. 5Bc). These results indicated that myc-Spry2, which is bound to Necl-5, was resistant to PDGF-induced, c-Src-catalyzed tyrosine phosphorylation and did not inhibit PDGF-induced Ras signaling.
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No effect of Necl-5 on PDGF-induced tyrosine phosphorylation of PDGF receptor and activation of c-Src
We examined the effects of Necl-5 on PDGF-induced tyrosine phosphorylation of PDGF receptor and activation of c-Src. These were indistinguishable between cells cultured at low and high cell densities (Fig. 6Aa,Ba). Over-expression of Necl-5 did not affect these events in cells cultured at both cell densities (Fig. 6Aa,Ba). In addition, knockdown of Necl-5 did not affect them (Fig. 6Ab,Bb). These results indicated that Necl-5 did not affect PDGF-induced tyrosine phosphorylation of PDGF receptor or activation of c-Src. Taken together, it is likely that c-Src is activated by the PDGF receptor in response to PDGF in a cell density-independent manner, although PDGF-induced c-Src-catalyzed tyrosine phosphorylation of Spry2 is regulated by cell density through Necl-5.
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We previously showed that cell surface Necl-5 was down-regulated by cell–cell contact-induced interaction with nectin-3 (Fujito et al. 2005). We then speculated that Spry2 might be released from Necl-5 upon its cell–cell contact-induced down-regulation and became sensitive to PDGF-induced c-Src-catalyzed tyrosine phosphorylation, causing inhibition of PDGF-induced Ras signaling. To examine this possibility, the extracellular fragment of nectin-3 fused to the Fc portion of IgG (Nef-3) was added to NIH3T3 cells to induce down-regulation of Necl-5, and PDGF-induced tyrosine phosphorylation of myc-Spry2 was measured. When down-regulation of Necl-5 was induced by Nef-3, Nef-3 increased PDGF-induced tyrosine phosphorylation of myc-Spry2 (Fig. 7). This effect of Nef-3 was inhibited by SU6656. These results indicated that cell–cell contact-induced down-regulation of Necl-5 made Spry2 sensitive to PDGF-induced c-Src-catalyzed tyrosine phosphorylation, causing inhibition of PDGF-induced Ras signaling.
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Discussion |
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Spry inhibits growth factor-induced signaling at a step down-stream of the receptor and up-stream of Ras (Gross et al. 2001; Hanafusa et al. 2002), and Spry becomes active when it is tyrosine-phosphorylated by c-Src which is activated by its growth factor receptor in response to its specific growth factor (Gross et al. 2001; Li et al. 2004). We previously showed that cell surface Necl-5 enhanced growth factor-induced signaling at a step down-stream of the receptor and up-stream of Ras (Kakunaga et al. 2004), and that amount of cell surface Necl-5 decreased with increasing cell density (Fujito et al. 2005). These two lines of earlier observations raised the possible functional relationship between Spry and Necl-5. Consistently, we showed here that endogenous Spry2 and endogenous Necl-5 co-localized at leading edges of moving NIH3T3 cells, and both proteins were physically associated with each other, as estimated by a co-immunoprecipitation assay. However, direct interaction of Spry2 with Necl-5 was not detected, as estimated by a cell-free assay system using pure recombinant proteins of full-length Spry2 and the cytoplasmic region of Necl-5 (data not shown), suggesting that modifications of either or both proteins and/or another factor(s) on the cytoplasmic or the plasma membrane sides are necessary for efficient interaction. Although the detailed mechanism of the interaction of Spry2 with Necl-5 remains unknown, these results supported the idea of a functional association of Spry2 with Necl-5 in growth factor-induced Ras signaling.
We also attempted to show the functional association of Spry2 with Necl-5. For this purpose, we used PDGF as a growth factor, because serum contains many types of growth factors. To stimulate NIH3T3 cells with PDGF, cells were starved of serum. We found that serum starvation rapidly induced down-regulation of Spry2. It has been shown that many growth factors, including EGF and FGF, induce up-regulation of Spry2 through the Ras-Raf-MEK-ERK pathway (Gross et al. 2001; Sasaki et al. 2001). It is likely that continuous stimulation of this signaling pathway is necessary for maintaining the level of Spry2. For this reason, we exogenously expressed myc-Spry2 and examined its role in PDGF-induced Ras signaling. Exogenously expressed myc-Spry2 co-localized with endogenous Necl-5 at leading edges of moving NIH3T3 cells (data not shown) and physically interacted with exogenously expressed Necl-5, as estimated by a co-immunoprecipitation assay. We showed that the activation levels of Ras and ERK increased with increasing amount of cell surface Necl-5. They were not decreased by myc-Spry2 in the presence of large amounts of cell surface Necl-5, whereas they were decreased by Spry2 in the presence of small amounts of cell surface Necl-5. In addition, in serum-starved cells in which endogenous Spry2 was markedly down-regulated, PDGF-induced Ras signaling was not affected by amount of cell surface Necl-5. Taken together, the present results indicated that endogenous Spry2 and Necl-5 cooperatively regulated PDGF-induced Ras signaling.
We also demonstrated here that the physical interaction of Spry2 with Necl-5 prevented Spry2 from being tyrosine-phosphorylated by c-Src which was activated by the PDGF receptor in response to PDGF, thereby reducing the inhibitory effect of Spry2 on PDGF-induced Ras signaling. Conversely, knockdown of endogenous Necl-5 made Spry2 sensitive to this tyrosine phosphorylation, thereby enhancing the inhibitory effect of Spry2 on PDGF-induced Ras signaling. In addition, we showed here that tyrosine phosphorylation of endogenous Spry2 was regulated by amount of cell surface Necl-5. Thus, it is likely from the present results that amount of cell surface Necl-5 regulates sensitivity of Spry2 to tyrosine phosphorylation by PDGF-induced activation of c-Src, thereby regulating PDGF-induced Ras signaling.
We previously showed that amount of cell surface Necl-5 was regulated by cell density; Necl-5 was down-regulated from the cell surface upon cell–cell contact, whereas it was up-regulated when cells didn't contact other cells; and down-regulation of Necl-5 was induced by its trans-interaction with nectin-3 upon cell–cell contact (Fujito et al. 2005). We showed here that down-regulation of Necl-5 by its trans-interaction with nectin-3 made exogenously expressed myc-Spry2 sensitive to PDGF-induced c-Src-catalyzed tyrosine phosphorylation. Free Spry2, which was not bound to Necl-5, was tyrosine-phosphorylated by c-Src. Necl-5 was not necessary for PDGF-induced tyrosine phosphorylation of PDGF receptor or activation of c-Src. Although this series of experiments was also performed using exogenously expressed Spry2 and Necl-5, it is likely that down-regulation of Necl-5 releases endogenous Spry2 from endogenous Necl-5, and free Spry2 is then tyrosine-phosphorylated by c-Src which is activated by PDGF receptor in response to PDGF, causing inhibition of PDGF-induced Ras signaling (Fig. 8).
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In summary, when cells do not contact other cells, Necl-5 is up-regulated and interacts with Spry2, preventing it from inhibiting PDGF-induced Ras signaling for cell proliferation, whereas when cells contact other cells, Necl-5 is down-regulated and Spry2 subsequently inhibits this signaling for cell proliferation (Fig. 8). Thus, regulation of PDGF-induced Ras signaling for cell proliferation by Necl-5 through Spry2 is likely to play a role, at least partly, in contact inhibition of cell proliferation.
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Experimental procedures |
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A rat monoclonal antibody (mAb) and a rabbit pAb against the extracellular region of Necl-5 were prepared as previously described (Ikeda et al. 2003; Fujito et al. 2005). Hybridoma cells (9E10) expressing a mouse anti-Myc mAb were purchased from the American Type Culture Collection. The following rabbit pAbs were purchased from commercial sources: anti-Spry (for immunoprecipitation) and anti-Spry2 (for Western blotting and immunofluorescence staining) from Upstate Biotechnology; anti-PDGFRß and anti-phospho-PDGFRß (Tyr857) from Santa Cruz; anti-c-Src and anti-ERK1/2 from Cell Signaling Technology; and anti-phospho-chick Src (Tyr418) from Biosource International. The following mouse mAbs were purchased from commercial sources: anti-Ras (RAS10) and anti-phospho-tyrosine Ab (4G10) from Upstate Biotechnology, anti-actin Ab from Chemicon and anti-phospho-ERK1/2 (E10) from Cell Signaling Technology. In addition, rat anti-GFP mAb was purchased from Nacalai Tesque, and a recombinant HRP-conjugated anti-phospho-tyrosine mAb (RC20) was purchased from BD Biosciences. Fatty acid-free BSA was purchased from Sigma. Protein A Sepharose was purchased from Amersham Biosciences. Human recombinant PDGF-BB was purchased from PEPROTECH. SU6656 was purchased from Calbiochem. Nef-3 was prepared as previously described (Honda et al. 2003) and cross-linked by a goat anti-human IgG Fc pAb (Jackson Immuno Research) before use.
Cell culture, transfection and small interfering RNA experiment
NIH3T3 cells were obtained from the American Type Culture Collection. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. NIH3T3 cells stably expressing FLAG-Necl-5-CP (FLAG-Necl-5 of which the cytoplasmic region was deleted; 30-374 amino acids) (Necl-5-CP-NIH3T3 cells) were prepared as previously described (Kakunaga et al. 2004). NIH3T3 cells stably expressing FLAG-Necl-5 (30-409 amino acids) (Necl-5-NIH3T3 cells) were obtained by transfection with pCAGIPuro-FLAG-Necl-5 (Ikeda et al. 2003) using LipofectAMINE 2000 reagent (Invitrogen) and by selection with puromycin. For cultures at a low or high cell density, cells were seeded at 5 x 103 or 4 x 104 cells/cm2, respectively. For transient expression experiments, cells were transfected with various expression vectors using LipofectAMINE 2000 reagent or the Nucleofector system (Amaxa Biosystems). Knockdown of Necl-5 using small interfering RNA (siRNA) vectors, pBS-H1-Necl-5 and pBS-H1-control, was performed as previously described (Fujito et al. 2005). The pBS-H1 vector was a gift from Dr Shibuya (Tokyo Medical and Dental University, Tokyo, Japan). To sort siRNA vector-transfected cells by FACS, the insert encoding the EGFP expression unit from pEGFP-C1 (Clontech) was subcloned into a region not related to siRNA expressions of pBS-H1-Necl-5 and pBS-H1-control, respectively (pBS-EGFP-H1-Necl-5 and pBS-EGFP-H1-control).
Assessment of activation of Ras and ERK
To assess activation of Ras, NIH3T3 or Necl-5-NIH3T3 cells were co-transfected with various combinations of pEFBOS-myc-Ki-Ras, pCS2-myc-mouse-Spry2 and/or siRNA vector, cultured for 24 h and replated at a low or high cell densities. After 24 h, cells were starved of serum for 16 h and stimulated with 3 ng/mL PDGF in DMEM containing 0.5% BSA for 2.5 min. The GTP-bound form of Ras was detected by the pull-down method as previously described (Kakunaga et al. 2004), and Myc-tagged Ki-Ras was monitored to differentiate between events of transfected cells and those of non-transfected cells. To assess activation of ERK, cells were co-transfected with various combinations of pEGFP-ERK2, pCS2-myc-mouse-Spry2 and/or siRNA vector, cultured for 24 h and replated at a low or high cell densities. After 24 h, cells were starved of serum for 16 h and stimulated with 3 ng/mL PDGF in DMEM containing 0.5% BSA for 5 min. Phosphorylated ERK was detected as previously described (Kakunaga et al. 2004), and EGFP-tagged ERK2 was monitored to differentiate between events of transfected cells and those of non-transfected cells. pCS2-myc-mouse-Spry2 and pEGFP-ERK2 were gifts from Dr Nishida (Kyoto University, Kyoto, Japan) and Dr Yoshimura (Kyusyu University, Fukuoka, Japan), respectively. The probes for the Ras pull-down method, Ras-binding and cysteine-rich domains of Raf-1 fused to GST, were gifts from Drs Matsuda and Nakamura (Osaka University, Osaka, Japan). Relative intensities of GTP-Ras or phosphorylated ERK bands, compared to those of total proteins, were determined using the NIH IMAGE 1.62 software (National Institutes of Health).
Co-localization assay
NIH3T3, Necl-5-NIH3T3 or Necl-5-knockdown-NIH3T3 cells were plated at a low cell density, cultured for 16 h and subjected to immunofluorescence microscopy (Fujito et al. 2005). For analysis of Necl-5-knockdown-NIH3T3 cells, NIH3T3 cells were transfected with pBS-H1-Necl-5 and cultured for 24 h in advance.
Co-immunoprecipitation assay
For the co-immunoprecipitation assay at the endogenous level, NIH3T3 cells were plated at a low or high cell density, and cultured in the presence of calf serum for 24 h. For the co-immunoprecipitation assay at an exogenous level, Necl-5-NIH3T3 or Necl-5-CP-NIH3T3 cells were transfected with pCS2-myc-mouse-Spry2, cultured for 24 h and replated at a high cell density. After 24 h, cells were starved of serum for 16 h and stimulated with or without 3 ng/mL PDGF in DMEM containing 0.5% BSA for 5 min. Cells were lyzed with lysis buffer [150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1 mM EDTA, 1% NP-40, 10% glycerol, 1 mM Na3VO4, 10 mM NaF, 10 µM
p-amidinophenylmethanesulfonyl fluoride, 10 µg/mL leupeptin, 2 µg/mL aprotinin and phosphatase inhibitor cocktail I (Sigma)]. Lysates were rotated at 4 °C for 30 min and subjected to centrifugation at 12 000 g for 20 min. Supernatants were precleared using protein A-Sepharose beads at 4 °C for 1 h, incubated with anti-Spry pAb at 4 °C for 1 h and incubated with protein A-Sepharose beads at 4 °C for 1 h. After the beads were extensively washed with lysis buffer, bound proteins were eluted by boiling the beads in SDS sample buffer for 5 min (Laemmli 1970) and subjected to SDS-polyacrylamide gel electrophoresis (PAGE), followed by Western blotting.
Assay for tyrosine phosphorylation of Spry2
For analysis of endogenous Spry2, NIH3T3 cells were plated at a low or high cell density and cultured in the presence of calf serum for 24 h. As for knockdown of Necl-5, NIH3T3 cells were transfected with pBS-EGFP-H1-Necl-5 or pBS-EGFP-H1-control and cultured for 24 h. Then, EGFP-positive cells were sorted by FACS, plated at a low cell density and cultured in the presence of calf serum for 24 h. For analysis of exogenous myc-Spry2, NIH3T3 or Necl-5-NIH3T3 cells were transfected with pCS2-myc-mouse-Spry2 and/or siRNA vector, cultured for 24 h and replated at a low or high cell density. After 24 h, cells were starved of serum for 16 h. Cells were treated prior to PDGF stimulation with 0.1 mM pervanadate in DMEM containing 0.5% BSA for 5 min and stimulated with 3 ng/mL PDGF in DMEM containing 0.5% BSA and 0.1 mM pervanadate for 10 min. For experiments using SU6656, cells were treated prior to PDGF stimulation with 1 µM SU6656 in DMEM containing 0.5% BSA for 1 h. For experiments using Nef-3, cells were cultured at a low cell density, starved of serum with DMEM containing 1% calf serum for 16 h and incubated with 50 µg/mL Nef-3 and/or 1 µM SU6656 in DMEM containing 1% calf serum for 1 h. Cells were lyzed with lysis buffer. Lysates were rotated at 4 °C for 30 min and subjected to centrifugation at 12 000 g for 20 min. Supernatants were subjected to immunoprecipitation using the anti-Spry pAb, Preclearing Matrix B and ExtraCruz B kit (Santa Cruz) according the manufacturer's protocols. After the beads were extensively washed with lysis buffer, bound proteins were eluted by boiling the beads in SDS sample buffer for 5 min and subjected to SDS-PAGE, followed by Western blotting using anti-phospho-tyrosine mAb (4G10 or RC20).
Assays for tyrosine phosphorylation of PDGF receptor and activation of c-Src
NIH3T3 or Necl-5-NIH3T3 cells were plated at a low or high cell density. After 24 h, cells were starved of serum for 16 h. To assay tyrosine phosphorylation of PDGF receptor, cells were stimulated with or without 3 ng/mL PDGF in DMEM containing 0.5% BSA for 5 min. To assay activation of c-Src, cells were stimulated with or without 10 ng/mL PDGF in DMEM containing 0.5% BSA for 5 min. After stimulation, cells were lyzed with prewarmed SDS sample buffer. To detect tyrosine phosphorylation of PDGF receptor and activation of c-Src, samples were assessed by Western blotting using anti-phospho-PDGFRß (Tyr857) and anti-phospho-chick Src (Tyr418) pAbs, respectively. For analysis of Necl-5-knockdown cells, NIH3T3 cells were transfected with pBS-EGFP-H1-Necl-5 or pBS-EGFP-H1-control and cultured for 24 h. Then, EGFP-positive cells were sorted by FACS, plated at a low cell density and subjected to assays.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: ytakai{at}molbio.med.osaka-u.ac.jp
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References |
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Received: 28 November 2006
Accepted: 6 December 2006
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