Article |
Address correspondence to Ken Jacobson, Dept. of Cell and Developmental Biology, University of North Carolina, 108 Taylor Hall, CB7090 Chapel Hill, NC 27599-7090. Tel.: (919) 966-5703. Fax: (919) 966-1856. email: frap{at}med.unc.edu
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Abstract |
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Key Words: neurons; nerve growth factor; focal adhesions; neurite outgrowth; mass spectrometry
Abbreviations used in this paper: 2-D, 2-dimensional; JNK, c-jun NH2 terminus kinase; wt, wild-type.
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Introduction |
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The NH2-terminal half of paxillin also contains a large number of Ser-Pro epitopes, which are potential substrates for proline-directed protein kinases (Pearson and Kemp, 1991; Seger and Krebs, 1995). Indeed, it has been demonstrated that c-jun NH2 terminus kinase (JNK) phosphorylates paxillin at Ser178 in vitro and in vivo (Huang et al., 2003). Several protein kinases including Erk and p38MAPK, have been proposed to phosphorylate paxillin based on observations using chemical inhibitors (Vadlamudi et al., 1999; Ku and Meier, 2000; Liu et al., 2002), but due to the potential lack of specificity of these inhibitors, the results remain to be confirmed more directly. Paxillin is also a potential substrate for cdk5, a proline-directed protein kinase that is enriched in neuronal tissues and regulates neurite outgrowth (Nikolic et al., 1996, 1998; Zukerberg et al., 2000), because paxillin contains a consensus sequence (S/T)PX(K/H/R) for cdk5.
Integrin-mediated adhesions are essential for the neurite outgrowth (Ivankovic-Dikic et al., 2000; Rhee et al., 2000; Vogelezang et al., 2001). Thus, it is important to understand how signaling pathways regulate cell adhesion dynamics during the process of neurite outgrowth. Paxillin is a focal adhesionassociated adaptor protein involved in the regulation of focal adhesion dynamics (Liu et al., 1999; Schaller 2001; Hagel et al., 2002; Huang et al., 2003). It has been demonstrated that paxillin plays a key role in neurite outgrowth (Ivankovic-Dikic et al., 2000). Moreover, expression of the v-crk oncogene protein, the binding partner for tyrosine phosphorylated paxillin, in PC12 cells promotes neurite outgrowth by both NGF and EGF-dependent pathways (Hempstead et al., 1994), but tyrosine phosphorylation of paxillin does not seem to be essential for the neurite outgrowth of PC-12 cells (Ivankovic-Dikic et al., 2000). Phosphorylation of Ser 178 on paxillin by JNK has been shown to play a key role in cell migration (Huang et al., 2003). It has been found that paxillin band is shifted to higher molecular weight in SDS-PAGE when PC-12 cells are stimulated with NGF (Rhee et al., 2000), suggesting that serine phosphorylation of paxillin also increases upon NGF treatment, but the signaling pathways involved and the physiological role are unknown. In this paper, we demonstrate that paxillin is phosphorylated at Ser 85 by p38MAPK and cdk5/p35 in vitro, and p38MAPK is the major kinase responsible for the phosphorylation of Ser 85 on paxillin in NGF-stimulated PC-12 cells. Furthermore, p38MAPK-mediated phosphorylation of Ser 85 on paxillin is involved in NGF-induced neurite outgrowth of PC-12 cells.
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Results |
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Ser 85 is also phosphorylated in NGF-stimulated PC-12 cells
To learn whether Ser 85 on paxillin is also phosphorylated in NGF-stimulated PC-12 cells, the cells were transfected with EGFP-paxillin ß and labeled with [32P]orthophosphoric acid. EGFP-paxillin ß was immunoprecipitated with an anti-GFP antibody. The samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The phosphorylation of EGFP-paxillin ß was detected by autoradiography. As shown in Fig. 4 A, phosphorylation of paxillin ß was significantly enhanced by NGF stimulation (Contl and NGF 3hr). The paxillin bands from Fig. 4 A were excised, digested with trypsin, and subjected to 2-D phosphopeptide mapping analysis. The map of transfected EGFP-paxillin is somewhat different from that of endogenous paxillin from PC-12 cells (Fig. 1 B), probably due to the species difference of paxillin. Three major spots were observed on the 2-D maps of EGFP-paxillin ß, and treatment with NGF caused an increase in the intensity of the spots (Fig. 4 B). Furthermore, the spot b (Fig. 4 B, arrow) comigrated with the major spot from the 2-D map of in vitro phosphorylated paxillin ß (Fig. 4 B). Therefore, paxillin is phosphorylated at the same site in vitro and in PC-12 cells.
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Endogenous paxillin is phosphorylated at Ser 83 in NGF-stimulated PC-12 cells
To identify the major p38 phosphorylation sites on rat paxillin, the protein was isolated from a rat bladder tumor cell line (NBT-II), and phosphorylated with p38MAPK in vitro. The phosphorylated paxillin was subjected to 2-D mapping analysis. As shown in Fig. 6 A, there was one major phosphorylation spot on the map. To identify the phosphorylation site, phosphopeptides were recovered from that spot and analyzed by mass spectrometry. The parent ion was detected at m/z 2054.9556, which is identical to the calculated m/z of peptide 76-YAHQQPPSPSPIYSSSTK-93 with one phosphoryl group. This peptide is homologous to human paxillin peptide 76-FIHQQPQSSSPVYGSSAKA-93 (Fig. 6 C). A fragment ion was also detected at m/z 1956.9556, which is equivalent to the m/z of the same peptide minus 98, indicating that peptide YAHQQPPSPSPIYSSSTK contains a phosphorylation site. The observation of the loss of 98 of the b8 ion (b8-H3PO4) indicates that a phosphorylation site is located on fragment 76-YAHQQPPS-83, whereas the additional mass of the phosphoryl group was not detected at the b3-b7 ions (Fig. 6 B). Thus, Ser 83 on rat paxillin is phosphorylated by p38MAPK in vitro.
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p38MAPK-mediated phosphorylation of paxillin is involved in NGF-induced neurite extension
To explore the role of p38MAPK in neurite extension, the effect of SB203580 on NGF-induced neurite extension in PC-12 cells was examined. About 46% of control cells had neurites longer than two cell bodies and 28% of the cells had neurites longer than three cell bodies. Treatment with SB203580 inhibited neurite outgrowth. In the presence of 10 µM SB203580 only 19% of cells extended neurites longer than two cell bodies and 7% extended neurites longer than three cell body lengths. Treatment with 20 µM of SB203580 almost abolished the NGF-induced neurite extension of PC-12 cells (Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200307081/DC1).
To assess the role of p38MAPK-mediated paxillin phosphorylation in neurite extension, PC-12 cells were transfected with EGFP-paxillin ß or -paxS85A, and the NGF-induced neurite extension of these cells was examined. As shown in Fig. 7, whereas cells expressing EGFP-paxS85A exhibited 20% of cells bearing neurites longer than two cell bodies and 7% of cells producing neurites longer than three cell bodies, >50% of cells expressing wild-type (wt) EGFP-paxillin ß had neurites longer than two cell bodies and
32% of cells had neurites longer than three cell bodies. Expression of wt EGFP-paxillin had no effect on neurite extension of PC-12 cells, which is consistent with a recent report by Ivankovic-Dikic et al. (2000). Therefore, phosphorylation of paxillin is involved in NGF-induced neurite extension.
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Discussion |
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Several lines of evidence indicate that p38MAPK is essential for neurite outgrowth. First, SB203580, an inhibitor of p38MAPK, blocks neurite outgrowth in several systems (Morooka and Nishida, 1998; Iwasaki et al., 1999; Hansen et al., 2000; Ishii et al., 2001). Second, a dominant negative mutant of p38MAPK also inhibits neurite outgrowth induced by NGF and forskolin (Morooka and Nishida, 1998; Hansen et al., 2000). Third, activation of p38MAPK by expressing constitutively active MKK3/MKK6, the kinases that phosphorylate and activate p38, induces neurite outgrowth in the absence of growth factor stimulation (Iwasaki et al., 1999).
Paxillin is a substrate for p38MAPK in vitro and in NGF-stimulated cells
It has been suggested that paxillin is a substrate for Erk in hepatocyte growth factorstimulated epithelial and PMA-induced thymoma cells (Ku and Meier, 2000; Liu et al., 2002). However, it is unclear whether paxillin is a direct substrate for Erk in these cells. Here, we show that Erk2 phosphorylates paxillin in vitro at two major sites and that Ser 85 is a minor phosphorylation site for Erk in vitro (Fig. 3), whereas Ser 85 of exogenously expressed human paxillin (Ser 83 for the endogenous rat paxillin) is major site in NGF-stimulated PC-12 cells (Fig. 4). Also, treatment of PC-12 cells with PD98058 only partially inhibits NGF-induced phosphorylation of Ser 83 (Fig. 1). The partial inhibition could be due to the indirect inhibition of p38MAPK because it has reported that constitutive activation of MEK1, the upstream kinase of Erk and the target for PD98059, up-regulates p38MAPK activity in PC-12 cells (Morooka and Nishida, 1998). Thus, Erk may be upstream of paxillin, but may not be directly responsible for Ser 83 phosphorylation in NGF-stimulated PC-12 cells.
There is a consensus site (Ser 244) for cdk5 on paxillin. Also, NGF induces paxillin phosphorylation with a similar time course as cdk5 activation (Figs. 1 and 2). This had led us to suspect that paxillin could be a substrate for cdk5 in NGF-stimulated PC-12 cells. However, our experiments indicate that Ser 244, the consensus and the major phosphorylation site for cdk5 in vitro, is not phosphorylated in NGF-stimulated PC-12 cells (Fig. 4). Also, the phosphorylation of Ser 85 is not significantly inhibited by two cdk5 inhibitors (Figs. 4 and 5). Therefore, cdk5 is not involved in the phosphorylation of Ser 85 in PC-12 cells.
p38MAPK has been implicated in heregulin ß1induced paxillin phosphorylation in breast cancer cells (Vadlamudi et al., 1999). Paxillin phosphorylation induced by heregulin ß1 was partially inhibited by a p38 inhibitor SB203580 and a dominant negative mutant of p38MAPK, but not by PD98059 (Vadlamudi et al., 1999). Here, we show that p38MAPK phosphorylates human paxillin at three major sites in vitro (Fig. 3) and rat paxillin at one major site (Fig. 6). Ser 85 was unambiguously identified as one of the major p38MAPK phosphorylation sites on human paxillin in vitro and a major NGF-induced phosphorylation site of exogenous paxillin in PC-12 cells (Figs. 3 and 4). Also, Ser 83 was identified as the major p38MAPK phosphorylation site on rat paxillin in vitro and a major phosphorylation site of endogenous paxillin in PC-12 cells (Figs. 1 and 6). Because the sequence of rat paxillin around Ser 83 is homologous to that of human paxillin around Ser 85, and the Ser 83 and Ser 85 sites are so close (Fig. 6 C), suggesting that the two sites are likely comparable. Furthermore, the phosphorylation of Ser 85 is completely abolished by a p38MAPK inhibitor SB203580 in NGF-stimulated PC-12 cells (Fig. 5). Thus, p38MAPK is a kinase that directly phosphorylates paxillin in NGF-stimulated PC-12 cells.
Phosphorylation of paxillin is involved in neurite extension of PC-12 cells
The role of cell adhesions in neurite extension has been established previously (Ivankovic-Dikic et al., 2000; Rhee et al., 2000; Vogelezang et al., 2001). As an important component of adhesions, paxillin is also involved in neurite outgrowth (Ivankovic-Dikic et al., 2000). Although expression of wt paxillin does not influence the neurite outgrowth, expression of a LD4-deleted paxillin mutant significantly inhibits the neurite outgrowth of PC-12 cells (Ivankovic-Dikic et al., 2000). Expression of v-Crk, an adaptor protein that binds to tyrosine phosphorylated paxillin, promotes neurite formation (Hempstead et al., 1994). Surprisingly, expression of paxillin mutant (Y31F, Y118F, Y187F), which is deficient in tyrosine phosphorylation and unable to bind Crk, has no effect on neurite extension (Ivankovic-Dikic et al., 2000). This result indicates that tyrosine phosphorylation of paxillin is not essential for neurite outgrowth in PC-12 cells.
Recently, we demonstrated that phosphorylation of Ser 178 on paxillin by JNK regulates cell migration in several cell types (Huang et al., 2003). However, JNK is not activated and Ser178 is not phosphorylated in NGF-stimulated PC-12 cells (unpublished data). Instead, the activity of p38MAPK is up-regulated and the phosphorylation level of Ser 85 increases. Moreover, expression of a paxillin mutant paxS85A significantly retards NGF-induced neurite extension (Fig. 7), indicating the involvement of phosphorylation of Ser 85 by p38MAPK in NGF-induced neurite extension. Thus, two types of different cell motility are regulated by two related pathways through phosphorylating their common substrate-paxillin at distinct sites.
Paxillin phosphorylation by p38MAPK is also involved in cell adhesion reorganization
It has been reported that heregulin ß1 induces p38MAPK activation and serine phosphorylation on paxillin, resulting in focal adhesion disassembly (Vadlamudi et al., 1999). In another report, collagen I stimulates the activities of p38MAPK accompanying a decrease in focal adhesion in endothelial cells, without affecting MAPK kinase, focal adhesion kinase, or phosphatidylinositol 3-kinase (Sweeney et al., 2003). It is known that focal adhesions undergo reorganization when PC-12 cells are induced to differentiate by NGF (Rhee et al., 2000). Here, we show that either a p38MAPK dominant negative mutant or a paxillin mutant (paxS85A) cause clustered focal adhesions, whereas MKK3bE, a constitutively active MAPK kinase for p38MAPK, induces focal adhesion disassembly when they are expressed in NGF-treated PC-12 (Figs. 8 and 9; Fig. S3), suggesting that paxillin phosphorylation by p38MAPK is involved in focal adhesion organization.
How p38MAPK-mediated phosphorylation of paxillin modulates focal adhesions and neurite extension remains to be elucidated. The phosphorylation of paxillin may facilitate the interaction of paxillin with Pyk2, a nonreceptor protein tyrosine kinase that is involved in regulating focal adhesion dynamics and neurite extension (Ivankovic-Dikic et al., 2000; Park et al., 2000; Taniyama et al., 2003). Pyk2 may in turn phosphorylate the Arf-GTPaseactivating protein ASAP1 and 3-phosphoinositidedependent protein kinase 1, or activate Rho pathway (Kruljac-Letunic et al., 2003; Okigaki et al., 2003; Taniyama et al., 2003). All these signaling events regulate focal adhesion dynamics and may modulate neurite extension. However, other currently unknown mechanisms may also mediate the role of paxillin phosphorylation. For example, we found that a phosphorylated degradation product of paxillin (50 kD) was generated after NGF stimulation in PC-12 cells (unpublished data), implying that paxillin phosphorylation may be involved in its degradation. Further studies are required to understand the focal adhesion dynamics during NGF-induced differentiation of PC-12 cells and to fully elucidate the role of serine phosphorylation of paxillin in the regulation of focal adhesion structure.
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Materials and methods |
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Plasmid construction
To generate His-tagged p38, DNA fragments encoding p38
were amplified by pfu-based PCR using Flag-p38
as template and 5'-TTACTTGGATCCCAGGAGAGGCCCACGTTC-3' and 5'-TTTTTTCTGCAGTCAGGACTCCATTTCTTCTTGGTC-3' as primers, and subcloned into PQE-30 vector via BamHI and PstI sites. To create His-tagged paxillin ß, DNA fragments encoding paxillin ß were amplified by pfu-based PCR using 5'-CGACGACCTCGACGCCCTG-3' and 5'-AAAAAAGTCGACCTAGCAGAAGAGCTTGAGGAAGCA-3' as primers, and subcloned into pQE-30 vector through Klenow-blunt BamHI site and SalI. His-tagged paxS85A was created by pfu-based PCR using Quick-Change mutation kit and primer pair 5'-CCTCAGTCCTCAGCACCTGTGTACGGC-3' and 5'-GCCGTACACAGGTGCTGAGGACTGAGG-3'. To construct EGFP-paxillin ß and -paxS85A, DNA fragments encoding paxillin ß and paxS85A were amplified respectively by pfu-based PCR using primer pair 5'-AAAAAAGAATTCAGACGACCTCGACGCCCT-3' and 5'-AAAAAAGTCGACCTAGCAGAAGAGCTTGAGGAAGCA-3', and subcloned into pEGFP-C1 vector through EcoRI site and SalI. The DNA fragments encoding EGFP-paxillin ß and EGFP-paxS85A were cut out by sequential treatment with SalI, Klenow, and AgeI and inserted into a retroviral MGIN vector (Li et al., 2000) through AgeI and Klenow-blunt NotI sites.
Protein purification
His-tagged proteins were purified by affinity chromatography using Ni-NTA-agarose. In brief, expression was induced with 1 mM IPTG for 3.5 h, bacteria harvested, and sonicated in a lysis buffer (50 mM Tris-HCl, pH 8.1, 300 mM NaCl, 1% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 20 mM imidazole). The cleared lysate was applied to Ni-NTA-agarose column. His-tagged protein was eluted with 250 mM imidazole in 20 mM Tris-HCl, pH 8.1, 300 mM NaCl.
Preparation of viruses and cell infection
Retrovirus packaging cells (293GPG) were maintained in DME supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 µg/ml tetracycline, 2 µg/ml puromycin, and 0.3 mg/ml G418. The cells were transfected with MGIN plasmids using Lipofectamine Plus transfection reagent (Invitrogen) according to the manufacturer's protocol. To harvest viruses, the transfectants were grown in DME supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The medium of transfectants was collected at 48 and 72 h, filtered though 0.2-mm filter and applied to overnight cultures of PC-12 cells for infection. Cells stably expressing recombinant proteins were obtained by growing infected PC-12 cells in the presence of 0.7 mg/ml G418 for 1 wk.
In vitro phosphorylation
5 µg His-tagged paxillin ß was incubated with 0.5 µg of kinase in 50 µl of the kinase buffer (20 mM MOPS, pH 7.2, 10 mM MgCl2, 25 mM ßglycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT) containing 40 µM ATP (10 µCi
-[32P]ATP) for 30 min at RT. The reactions were terminated by adding SDS-sample buffer. The samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane for autoradiography.
In vivo phosphorylation
Cells were incubated with [32P]orthophosphoric acid in sodium phosphatedeficient MEM (Sigma-Aldrich). After 12 h, cells were harvested and lysed with a RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% IPEGAL, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 500 nM okadaic acid, 1 mM naphthyl acid phosphate). The lysates were immunoprecipitated with antipaxillin or anti-GFP antibodies. The immune complexes were analyzed by SDS-PAGE and transferred to nitrocellulose membrane for detection of phosphorylation by autoradiography.
Peptide mapping
Peptide mapping was performed as described previously (Boyle et al., 1991; Huang et al., 2003). In brief, protein bands were cut from nitrocellulose membrane and digested with trypsin (sequencing grade; Promega) in NH4HCO3, pH 7.8, with shaking at 37°C for 8 h. The samples were dried and washed in a speed-vac, and spotted onto cellulose plate for 2-D phosphopeptide mapping, using pH 8.9 buffer for electrophoresis and phosphochromatography buffer for TLC.
Mass spectrometry
Phosphopeptide was recovered from cellulose plates, cleaned up with a Zip-Tip (Millipore) as described previously (Raska et al., 2002) and analyzed by a MALDI-TOF/TOF (Voyager 4700) instrument purchased from Applied Biosystems with -cyano-4-hydroxy-cinnamic acid as matrix. The instrument was calibrated by external calibration using the ABI 4700 calibration mixture.
Neurite outgrowth
PC-12 cells were plated on 35-mm petri dishes precoated with 10 µg/ml collagen I and cultured in RPMI 1640 containing 10% horse serum, 5% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin for overnight, and cultured in DME supplemented with 1% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin for additional 6 h. The cells were then challenged with 100 ng/ml NGF and cultured for 4 d. The neurite outgrowth was photographed using a microscope (model IX81; Olympus) equipped with a 40x objective and a CCD camera (model C4880; Hamamatsu). Scoring for neurite outgrowth of PC-12 cells was performed as described by Dikic et al. (1994).
EGFP fluorescence, immunofluorescence, and microscopy
PC-12 cells expressing EGFP-paxillin ß or -paxS85A were plated on MatTek dishes (with a glass coverslip at the bottom) precoated with collagen and treated with 100 ng/ml NGF for 48 h. EGFP epifluorescence and TIRF images were taken at 37°C using a microscope (model IX81; Olympus) equipped with a 60x, 1.45 NA objective, a CCD camera (model C4880; Hamamatsu), and a Metamorph software (Universal Imaging Corp.). For immunofluorescence staining, the cells were fixed with 3% PFA and stained for vinculin with antivinculin mAb prelabeled with Alexa 568. Vinculin staining and EGFP fluorescence were observed and photographed at RT using the same system. The brightness of images were adjusted using Photoshop software.
Protein interaction and tyrosine phosphorylation
PC-12 cells expressing EGFP-paxillin ß or EGFP-paxS85A were plated on 100-mm petri dishes precoated with 10 µg/ml collagen I and cultured in normal RPMI 1640 culture medium overnight and cultured in DME supplemented with 1% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin for 12 h. The cells were then induced with 100 ng/ml NGF and cultured for 2 d. Cells were lysed with a buffer containing 50 mM Tris-HCl, pH 7.4, 1% IPEGAL, 100 mM NaCl, 5% glycerol, 5 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 500 nM okadaic acid, 1 mM naphthyl acid phosphate, and 1 mM Na3VO4. The lysates were directly mixed with SDS sample buffer, or were immunoprecipitated with anti-GFP antibodies. Immune complexes were solubilized in SDS sample buffer. Samples were analyzed by SDS-PAGE and transferred to nitrocellulose membrane to detect tyrosine phosphorylation and various protein interactions.
Online supplemental material
Fig. S1 shows that cdk5 directly phosphorylated Ser 244 on paxillin in vitro. To demonstrate that Ser 244 on paxillin is a phosphorylation site for cdk5, wt His-tagged paxillin ß or paxS244A were phosphorylated, respectively, with active cdk5/p35 and subjected to 2-D phosphopeptide mapping analysis as described in Peptide mapping section. Fig. S2 demonstrates that NGF-induced neurite extension was inhibited by a p38MAPK inhibitor, SB203580. To test the effect of SB203580 on NGF-induced neurite extension, PC-12 cells were plated on collagen-coated petri dishes, treated with SB203580 as indicated for 10 min, and stimulated with 100 ng/ml NGF without removing the inhibitor. Neurite outgrowth was analyzed after 96 h of NGF stimulation. Fig. S3 suggests that the p38MAPK pathway is involved in regulating focal adhesion reorganization. To explore the role of the p38MAPK pathway in focal adhesion dynamics, PC-12 cells were transiently transfected with EGFP vector, Flag-p38AF + EGFP vector, and Flag-MKK3bE + EGFP vector for 24 h, and plated on MatTek dishes precoated with 10 µg/ml collagen and treated with 100 ng/ml NGF for 36 h. The cells were fixed and stained with Alexa 568labeled antivinculin antibodies. Vinculin staining and EGFP fluorescence were viewed by epifluorescence microscopy as described above (EGFP...and microscopy). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200307081/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grant to K. Jacobson and M.D. Schaller; the Cell Migration Consortium to K. Jacobson; and National Institute for Dental and Cranial Research grant to K. Jacobson and M.D. Schaller.
Submitted: 14 July 2003
Accepted: 13 January 2004
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