JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M909932199 on April 7, 2000

J. Biol. Chem., Vol. 275, Issue 26, 19768-19777, June 30, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/26/19768    most recent
M909932199v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, S.-Y.
Right arrow Articles by Avraham, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, S.-Y.
Right arrow Articles by Avraham, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Characterization of the Tyrosine Kinases RAFTK/Pyk2 and FAK in Nerve Growth Factor-induced Neuronal Differentiation*

Shin-Young Park, Hava Avraham, and Shalom AvrahamDagger

From the Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115.

Received for publication, December 9, 1999, and in revised form, March 15, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca2+ concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca2+. Blocking of extracellular Ca2+ or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca2+-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca2+-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The related adhesion focal tyrosine kinase (RAFTK),1 also known as Pyk2 (1), CAK-beta (2), and CADTK (3), is a nonreceptor tyrosine kinase and is related to focal adhesion kinase (FAK) (4). RAFTK is implicated in the regulation of ion channel activity (1), stress responses (5), cell adhesion/cytoskeletal reorganization (6-10), and vesicle trafficking (11). RAFTK is highly expressed in the central nervous system and in neuronal cell lines, including PC12 cells (1, 4). Studies have shown activation of RAFTK upon treatment with various neuronal stimuli, such as membrane depolarization and the neuropeptide bradykinin (1), suggesting a possible role of RAFTK in neuronal cell signaling.

NGF is a neurotropic factor inducing neuronal cell growth, survival, and the differentiation of distinct populations of neurons through activation of the NGF receptor, TrkA (12-14). NGF-induced neuronal responses and neuronal differentiation have been extensively studied in a pheochromocytoma cell line (PC12), which can generate action potentials when grown in the continual presence of NGF (15, 16). Among the rapid cellular changes observed is actin-cytoskeletal reorganization upon NGF stimulation (17). NGF stimulation induces the formation of ruffles with actin redistribution within a few minutes, followed by condensation of actin bundles to several dot-like aggregates that subsequently become the growth cones (18). This implies that a variety of molecular machinery is required to accomplish the actin reorganization, such as actin polymerization, bundle formation, and actin targeting to the plasma membrane. One of the intracellular signals that might be involved in cytoskeletal reorganization during NGF stimulation is Ca2+ mobilization. NGF induces both extracellular Ca2+ influx and intracellular Ca2+ release from the endoplasmic reticulum (19, 20). Calcium has also been implicated in cell growth, survival, cell death, and neural functions including excitability, neurotransmitter release, associativity, plasticity, and gene transcription (21). However, little is known about the role of intracellular Ca2+ mobilization in PC12 cells upon NGF stimulation.

Paxillin, a cytoskeletal protein and focal adhesion molecule, becomes tyrosine-phosphorylated upon integrin engagement to the extracellular matrix and associates with focal adhesions (22). Increased tyrosine phosphorylation of paxillin has also been observed upon treatment with a variety of stimuli, and paxillin is capable of binding to another cytoskeletal protein, vinculin, as well as the signaling proteins Csk, Crk, Src, p125FAK, and RAFTK (23, 24). Analysis of the primary structure of paxillin reveals the presence of (i) three tyrosine residues within the binding motif for the Crk SH2 domain (YXXP), (ii) a proline-rich motif that could serve as an SH3 binding domain, and (iii) four motifs identified or very closely related to LIM domains (25). Thus, it was proposed that paxillin associates with cell adhesion molecules and with the cytoskeleton and recruits these molecules into a signal transduction complex, near the plasma membrane, that acts as a scaffold molecule (24). Induction of paxillin tyrosine phosphorylation and its enhanced expression during neuronal differentiation indicate a possible role of paxillin in neuronal differentiation (26). Since RAFTK activation is induced by Ca2+ mobilization and is involved in cytoskeletal reorganization (22), the signaling of RAFTK and its homologous tyrosine kinase FAK and their role as mediators of intracellular Ca2+ mobilization during NGF signaling were investigated.

Here, we report that RAFTK, but not FAK, is tyrosine-phosphorylated upon NGF stimulation of PC12 cells in a time- and concentration-dependent manner. NGF-induced RAFTK phosphorylation is dependent on PLCgamma activity, Ca2+ mobilization, an intact actin-based cytoskeleton, and partially on PI3-K activity. RAFTK associates with paxillin inducing the tyrosine phosphorylation of paxillin in a Ca2+-dependent manner and translocates from the cytoplasm to the cell periphery upon NGF stimulation, whereas FAK associates with paxillin at the plasma membrane and is involved in the regulation of cell adhesion. Furthermore, RAFTK and paxillin localize at the neurites and growth cones in differentiated PC12 cells and are tyrosine-phosphorylated upon the induction of increasing intracellular Ca2+ levels by potassium depolarization. This suggests that RAFTK may play an important role in NGF-induced cytoskeletal reorganization and in neurite and growth cone function(s).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Cell Culture-- Rat pheochromocytoma PC12 cells were obtained from ATCC and maintained in DMEM (Life Technologies, Inc.) with 10% (v/v) heat-inactivated horse serum, 5% (v/v) fetal bovine serum (FBS), 50 µg/ml penicillin, and 50 µg/ml streptomycin (complete medium). For serum starvation, confluent PC12 cells were incubated for 18 h in serum-reduced medium (DMEM with 0.5% heat-inactivated horse serum, 0.25% FBS, 50 µg/ml penicillin, and 50 µg/ml streptomycin). Stimulation of PC12 cells was performed with 50 ng/ml (unless otherwise noted) of NGF (Upstate Biotechnology, Inc., or a gift from Genentech) after starvation of PC12 cells for 18 h in serum-reduced medium. Differentiation of PC12 cells was accomplished by sustained incubation with NGF in serum-reduced medium, followed by refreshing NGF and medium every other day. 293 cells were obtained from ATCC and maintained in DMEM (Life Technologies, Inc.) with 10% (v/v) FBS, 50 µg/ml penicillin, and 50 µg/ml streptomycin (complete medium). For serum starvation, 293 cells were incubated for 6 h in serum-free medium (DMEM with 50 µg/ml penicillin and 50 µg/ml streptomycin). Stimulation of 293 cells was performed with 50 ng/ml NGF (Upstate Biotechnology, Inc., or a gift from Genentech) in the presence or absence of BAPTA/AM (50 µM; 15 min pre-incubation).

Materials-- Chelerylthrine chloride, BAPTA/AM, wortmannin, LY294002, EDTA, EGTA, cytochalasin D, colchicine, and phorbol 12-myristate 13-acetate (PMA) were obtained from Calbiochem. U73122 was purchased from Research Biochemicals, Inc. Electrophoresis reagents were obtained from Bio-Rad. gamma -32P-Labeled ATP was purchased from NEN Life Science Products. Protease inhibitors and all other reagents were purchased from Sigma. Protein G-Sepharose and recombinant Protein G-agarose were purchased from Pierce and Life Technologies, Inc., respectively. Normal rabbit and mouse sera were obtained from Sigma. Anti-phosphotyrosine antibodies (PY20) were obtained from Zymed Laboratories Inc. Anti-phosphotyrosine antibodies (4G10) were a gift from Brian J. Druker (Oregon Health Sciences University). Goat anti-GST antibody was purchased from Amersham Pharmacia Biotech. Monoclonal anti-Src antibody (clone 427) was a gift from Dr. Joan S. Brugge (Harvard Medical School, Department of Cell Biology). Rabbit anti-HA, rabbit anti-Erk1 and Erk2, rabbit anti-PI3-K p85, goat anti-Akt, and goat anti-Pyk2 antibodies were purchased from Santa Cruz Biotechnology. Rabbit anti-Trk antibody was purchased from Calbiochem. Rabbit anti-phospho-Akt (Ser-473) antibody was purchased from New England Biolabs. Erk-1-HA cDNA was a gift from Dr. J. Blenis (Harvard Medical School, Department of Cell Biology). Secondary antibodies of horseradish peroxidase-conjugated sheep anti-mouse Ig and donkey anti-rabbit Ig antibodies were obtained from Amersham Pharmacia Biotech. Secondary antibodies of horseradish peroxidase-conjugated rabbit anti-goat Ig antibodies were obtained from Santa Cruz Biotechnology. FITC-conjugated goat anti-rabbit and Texas Red-conjugated horse anti-mouse antibodies were purchased from Vector Laboratories. Cy5-labeled goat anti-mouse antibody and rhodamine-labeled phalloidin were purchased from Molecular Probes. Specific antibodies (R4250) against RAFTK were generated by immunizing New Zealand White rabbits with a bacterially expressed fusion protein consisting of GST and the COOH terminus (amino acids 681-1009) of the human RAFTK cDNA subcloned into the pGEX-2T expression vector as described (6). Anti-FAK (R4714) rabbit polyclonal antibodies were generated by immunizing New Zealand White rabbits with a bacterially expressed fusion protein consisting of GST and the COOH terminus (amino acids 681-1009) of the human FAK cDNA subcloned into the pGEX-2T expression vector as described (6).

Transient Transfection of PC12 Cells with a Wild-type or Kinase Mutant of RAFTK-- The RAFTK cDNA in the pcDNA3-neo vector was constructed as described in our previous studies (4, 6). A kinase-negative mutant of RAFTK (km) was constructed by replacing Lys-475 with an Ala residue using a site-directed mutagenesis kit (CLONTECH, Palo Alto, CA). The GFP-tagged wild-type (wt) or kinase mutant (km) RAFTK was subsequently prepared by subcloning the RAFTK constructs in a pEGFP-C2 vector according to the manufacturer's protocol (CLONTECH). PC12 cells were transiently transfected with either the GFP-tagged wild-type or kinase mutant RAFTK construct as described (6) using LipofectAMINE Plus reagent (Life Technologies, Inc.). After 72 h of transfection, the cells were stimulated with NGF (50 ng/ml, for 5 min at 37 °C). Cell lysates were prepared and immunoprecipitated with anti-RAFTK antibodies or control antibodies. The immunoprecipitates were washed and analyzed for tyrosine phosphorylation as described below.

Transient Transfection of 293 Cells-- Wild-type and mutant TrkA cDNA constructs were prepared using a Transformer Site-directed Mutagenesis Kit (CLONTECH) according to the manufacturer's protocol as described previously (27). TrkA double mutants contain tyrosine to phenylalanine mutations at the PLCgamma association site Tyr-785 and the p85/PI3-K interaction site Tyr-751 (Y785F/Y751F), at the Tyr-785 and the Shc interaction site Tyr-490 (Y785F/Y490F), or at the Tyr-751 and Tyr-490 (Y751F/Y490F). The paxillin cDNA tagged with hemagglutinin (HA) in the pRcCMV vector was kindly provided by R. Salgia and J. D. Griffin (Dana Farber Cancer Institute, Boston). Transient transfection of cDNA constructs into 293 cells was performed using the calcium phosphate precipitation method. 2 µg of TrkA cDNA and 0.5 µg of RAFTK cDNA were used for each 100-mm dish of 293 cells. Empty vector cDNA was used as a transfection control. After 48 h of transfection, the cells were starved for 6 h in serum-free medium followed by NGF (50 ng/ml, 5 min) stimulation. Cell lysates were prepared and immunoprecipitated with specific antibodies, followed by immunoblotting as described below.

Preparation of Cell Lysates, Immunoprecipitations, and Immunoblotting-- Cells were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM EDTA) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and phosphatase inhibitors (1 mM Na3VO4 and 1 mM NaF). Immunoprecipitations were performed with rabbit polyclonal antibodies of anti-RAFTK (R4250) and anti-FAK (R4714) (4), followed by protein G incubation. Immunoprecipitates were separated by either gradient (4-12%) or 8% SDS-PAGE (NOVEX, San Diego, CA) under reducing conditions, electrophoretically transferred to Immobilon polyvinylidene difluoride (Millipore, Bedford, MA), and processed for immunoblotting using the enhanced chemiluminescence technique (Amersham Pharmacia Biotech).

In Vitro Autophosphorylation Kinase Assays-- The immunoprecipitated complexes, obtained by immunoprecipitating cell lysates with RAFTK antiserum and protein G, were washed twice with modified RIPA buffer and once in kinase buffer. The immune complex was then incubated in kinase buffer containing 5 µCi of [gamma -32P]ATP at room temperature for 30 min as described (28). The reaction was stopped by adding 4× SDS sample buffer and boiling the sample for 5 min. Proteins were then separated on SDS-PAGE and detected by autoradiographs. The activities were determined by densitometry of autoradiographys.

Immunocomplex Kinase Assays-- Cells were lysed in modified RIPA buffer containing protease inhibitors and phosphatase inhibitors as described above. ERK kinase activity was determined by incubating 300 µg of cellular extracts with 1 µg of anti-ERK1 antibody for 2 h at 4 °C, followed by the addition of protein G-agarose and incubation for 1 h at 4 °C. The protein G beads were then washed thoroughly before the addition of 20 µl of kinase buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 2 mM dithiothreitol, and 25 µM ATP) containing 2 µCi of [gamma -32P]ATP and 10 µg of myelin basic protein as a substrate protein. After incubation at 30 °C for 10 min, the reactions were stopped with Laemmli buffer, analyzed by SDS-PAGE, dried, and exposed to x-ray films.

Induction of Neuronal Differentiation of PC12 Cells by NGF-- PC12 cells were stimulated with NGF (50 ng/ml) for 2 days in the presence or absence of the intracellular Ca2+ chelator BAPTA/AM (50 µM). Changes in cell morphology were observed, and images of PC12 cells were taken under an inverted light microscope (Olympus CK2) with an attached camera (Olympus SC35).

Immunocytochemistry, Confocal Microscopy, and Image Analysis-- Tissue-cultured cells were fixed with 3% paraformaldehyde for 20 min and permeabilized with 0.2% (v/v) Triton X-100 in phosphate-buffered saline for 10 min. After being blocked with 10% normal goat serum in phosphate-buffered saline for 1 h, cells were incubated with specific primary antibodies and fluorescein isothiocyanate-, rhodamine-, or Cy5-conjugated secondary antibodies after washing in phosphate-buffered saline for 1 h each. Rhodamine-conjugated phalloidin was used for actin staining. The samples were analyzed using a Bio-Rad MRC-1024 confocal microscope.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NGF Induces Phosphorylation of RAFTK, but Not of FAK, in a Time- and Dose-dependent Manner-- To test the effect of NGF on RAFTK and FAK, we examined the tyrosine phosphorylation of RAFTK and FAK upon NGF stimulation of PC12 cells. The tyrosine phosphorylation of RAFTK was induced by NGF in a time-dependent manner, peaking at 5 min (Fig. 1A), whereas that of FAK was constitutively tyrosine-phosphorylated at a high level and showed no change in phosphorylation upon NGF stimulation. This suggests that RAFTK, but not FAK, is specifically involved in NGF-mediated signaling cascades (Fig. 1B). Next, we analyzed the dose response of RAFTK phosphorylation upon stimulation with various concentrations of NGF. RAFTK tyrosine phosphorylation was dose-dependent, reaching a peak at 10 ng/ml NGF (Fig. 1C). However, FAK showed no change in phosphorylation when cells were treated with various concentrations of NGF (data not shown). RAFTK kinase activity was examined to see whether phosphorylation of RAFTK leads to its activation upon NGF stimulation (Fig. 1D). In vitro autophosphorylation of RAFTK showed a significant increase in RAFTK kinase activity (30% increase), suggesting that NGF induces RAFTK kinase activation as well as its tyrosine phosphorylation. Taken together, these data indicate that NGF-induced tyrosine phosphorylation and activation are specific to RAFTK, but not FAK, in both a time- and dose-dependent manner.


View larger version (51K):
[in this window]
[in a new window]
  		Fig. 1.   NGF induces the tyrosine phosphorylation of RAFTK, but not FAK, in a time- and dose-dependent manner. A and B, immunoprecipitates (IP) of RAFTK (A) or FAK (B) from PC12 cells after treatment with NGF (50 ng/ml) for the indicated times. PC12 cells were lysed in modified RIPA buffer, and 1 mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-RAFTK or rabbit anti-FAK antibodies. The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10). The membranes were then stripped and reblotted with rabbit anti-RAFTK (1:2500) or rabbit anti-FAK (1:2500) as indicated. Immunoprecipitates with normal rabbit serum (NRS) were used as a control. Specific bands were visualized using the ECL system (Amersham Pharmacia Biotech or NEN Life Science Products). C, immunoprecipitates of RAFTK from PC12 cells after treatment with serially diluted NGF, as indicated for 5 min. The immunocomplexes were resolved by SDS-PAGE (NOVEX), transferred to PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10), followed by reblotting with rabbit anti-RAFTK (1:2500) after membrane stripping. D, RAFTK kinase activity was determined by in vitro autophosphorylation of immunoprecipitated RAFTK after NGF stimulation in the presence of [gamma -32P]ATP. The activities were determined by densitometry of autoradiographs. IB, immunoblot.

Activation of RAFTK Is Dependent on PLCgamma Activity, Intracellular Ca2+ Increase, and Partially on Extracellular Ca2+ Influx and PI3-K Activity-- To delineate the signaling cascades leading to RAFTK activation by NGF, the tyrosine phosphorylation of RAFTK was examined in the presence of specific inhibitors of the downstream signaling molecules of the TrkA receptors. When NGF binds to its receptor TrkA, the autophosphorylated receptors associate with PLCgamma , Shc, and the p85 subunit of PI3-K through a specific interaction between the SH2 domains and their specific phosphotyrosine residues (29). In our study, the NGF-induced tyrosine phosphorylation of RAFTK was partially inhibited by a PI3-K-specific inhibitor, LY294002, or wortmannin (Fig. 2, A and E). The effectiveness of LY294002 and wortmannin, specific inhibitors of PI3-K, was shown by the inhibition of Akt phosphorylation (active form) which is downstream of PI3-K activation upon NGF stimulation (Fig. 2E). U73122, an inhibitor of phospholipase C, blocked the phosphorylation of RAFTK upon NGF stimulation (Fig. 2A). This suggests that NGF-induced RAFTK phosphorylation is mediated through PLCgamma which is known to induce intracellular Ca2+ increase and protein kinase C activation by producing inositol triphosphate (IP3) and diacylglycerol, respectively. Next, we examined whether RAFTK phosphorylation is induced through IP3-mediated intracellular Ca2+ increase from the endoplasmic reticulum and/or through diacylglycerol-mediated protein kinase C activation. Fig. 2A demonstrates that BAPTA/AM, a cell-permeable Ca2+ chelator, abolishes RAFTK phosphorylation. However, chelerylthrine chloride (CC), a protein kinase C-specific inhibitor, had no effect on RAFTK phosphorylation (Fig. 2A), indicating that RAFTK phosphorylation is mediated through PLCgamma , IP3, and intracellular [Ca2+]i increase. Fig. 2D shows that CC is effective in inhibiting the tyrosine phosphorylation of RAFTK. Since Shc is known to associate with Grb2 and transmits signals to Ras, we examined whether Shc is associated with RAFTK. However, we were not able to detect any direct association between Shc and RAFTK in PC12 cells (data not shown). Previous studies have shown that NGF induces extracellular calcium influx in several cell lines including PC12 cells (20) and that RAFTK is highly phosphorylated upon treatment of cells with various Ca2+-inducing agents (1, 30). Therefore, we examined the effect of extracellular Ca2+ influx on RAFTK phosphorylation using the Ca2+ chelator, EGTA. EGTA significantly reduced the NGF-induced tyrosine phosphorylation of RAFTK (Fig. 2A), suggesting that NGF-induced extracellular Ca2+ influx contributes to the phosphorylation of RAFTK. In comparison, FAK remains at a high level of tyrosine phosphorylation and fails to respond to NGF regardless of treatment with various inhibitors (Fig. 2B). These data demonstrate that RAFTK activation is mediated through intracellular signaling cascades of the TrkA receptor, PLCgamma , IP3, through intracellular Ca2+ increase, and partially through PI3-K and extracellular Ca2+ influx upon NGF stimulation of PC12 cells.


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 2.   The effects of various inhibitors on the NGF-induced tyrosine phosphorylation of RAFTK. A, immunoprecipitates of RAFTK from PC12 cells in the absence or presence of EGTA (3 mM), BAPTA/AM (50 µM), wortmannin (WT, 100 nM), chelerylthrine chloride (CC, 1 µM), or U73122 (10 µM) with or without treatment of NGF (50 ng/ml) for 5 min. PC12 cells were lysed in modified RIPA buffer, and 1 mg of total cell lysate from each sample was immunoprecipitated (IP) with rabbit anti-RAFTK antibodies. The immunocomplexes were resolved by 4-12 or 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10), followed by blotting with rabbit anti-RAFTK (1:2500) as indicated. Specific bands were visualized using the ECL system. B, immunoprecipitates of FAK from PC12 cells in the absence or presence of EGTA (3 mM), BAPTA/AM (50 µM), wortmannin (WT, 100 nM), chelerylthrine chloride (CC, 1 µM), U73122 (10 µM) with or without treatment of NGF (50 ng/ml) for 5 min. Sample preparation, detection of tyrosine phosphorylation and FAK immunoblotting (IB) were done the same as above but with rabbit anti-FAK antibody. C, immunoprecipitates of RAFTK from PC12 cells after treatment with NGF (50 ng/ml) for 5 min in the absence or presence of cytochalasin D (CD, 2 h preincubation at 1 µM). PC12 cells were lysed in modified RIPA buffer, and 1 mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-RAFTK antibodies or with NRS as a control. The immunocomplexes were resolved by 4-12 or 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10), followed by blotting with rabbit anti-RAFTK (1:2500) as indicated. Specific bands were visualized using the ECL system. D, immunoprecipitates of RAFTK from PC12 cells after treatment with PMA (50 ng/ml) for 3 min in the absence or presence of chelerylthrine chloride (30 min preincubation at 1 µM). PC12 cells were lysed in modified RIPA buffer and 1 mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-RAFTK antibodies followed by immunoblotting as described above. E, immunoprecipitates of RAFTK from PC12 cells after treatment with NGF (50 ng/ml) for 5 min in the presence of wortmannin (100 nM) or LY294002 (LY, 10 µM). Immunoprecipitates were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with mouse anti-phosphotyrosine (4G10) antibody followed by rabbit anti-RAFTK antibody. Total cell lysates from the same preparation were resolved by 8% SDS-PAGE and probed with rabbit anti-phospho-Akt antibody (New England BioLabs; Ser-473; 1:1000) and then probed with goat anti-Akt antibody (Santa Cruz Biotechnology; 1:1000) following membrane stripping.

NGF-induced Phosphorylation of RAFTK Is Dependent on an Intact Actin Cytoskeleton-- To investigate a possible connection between RAFTK and the actin-based cytoskeleton, cells were treated with the specific actin microfilament inhibitor cytochalasin D (1 µM) for 2 h prior to NGF stimulation. Fig. 2C shows that cytochalasin D inhibits the NGF-induced phosphorylation of RAFTK, indicating that intact actin-based cytoskeletal organization is required for this NGF-induced phosphorylation of RAFTK.

Transient Transfection of RAFTK and TrkA Shows That Tyrosine Phosphorylation of RAFTK Is Mediated through PLCgamma and PI3-K-- To confirm the signaling cascades observed in our study using the specific pharmacological inhibitors, 293 cells which express neither TrkA nor RAFTK were transiently transfected with the following: RAFTK cDNA and wild-type or mutant TrkA cDNA having double tyrosine-phenylalanine mutations at the PLCgamma and the p85/PI3-K interaction sites (Y785F/Y751F), at the PLCgamma and the Shc interaction sites (Y785F/Y490F), or at the p85/PI3-K and the Shc interaction sites (Y751F/Y490F). Our results demonstrated that wild-type TrkA induced RAFTK phosphorylation upon NGF stimulation, whereas no effects were observed with the pcDNA3 vector alone (Fig. 3). Cells transfected with TrkA double mutated at the p85- and the Shc-binding sites (Y751F/Y490F) or double mutated at the PLCgamma - and the Shc-binding sites (Y785F/Y490F) retained RAFTK phosphorylation upon NGF stimulation. However, cells transfected with TrkA double mutated at the PLCgamma - and the p85-binding sites (Y785F/Y751F) showed abolishment of most of the RAFTK phosphorylation, demonstrating that both PLCgamma and PI3-K are involved in the RAFTK phosphorylation. Blocking of intracellular Ca2+ with BAPTA/AM in cells transfected with RAFTK and TrkA double-mutated at the p85- and the Shc-binding sites (Y751F/Y490F) inhibited the NGF-induced RAFTK phosphorylation. These results demonstrate that association of TrkA with PLCgamma induces RAFTK phosphorylation through intracellular Ca2+ that might be released from the endoplasmic reticulum by IP3 (Fig. 3). The TrkA mutant with the intact p85 subunit-binding site (Y785F/Y490F) induced RAFTK phosphorylation in an intracellular Ca2+-independent manner. The TrkA mutant with the intact Shc-binding site (Y785F/Y751F) induced a substantial increase in RAFTK phosphorylation but not as much as the TrkA with the intact p85-binding site (Y785F/Y490F). We analyzed the tyrosine phosphorylation of the regulatory subunit of PI3-K (p85) induced by TrkA activation. Fig. 3B shows that the tyrosine phosphorylation of PI3-K p85 was induced only by TrkA with the intact p85-binding site (wt and Y785F/Y490F), indicating that the tyrosine phosphorylation of p85 is essential in the induction of RAFTK phosphorylation.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 3.   TrkA induces the tyrosine phosphorylation of RAFTK through association with PLCgamma and PI3-K. 293 cells were transfected (Transf.) with GFP-tagged RAFTK in a pEGFP-C2 vector and wild-type TrkA (wt) or mutant TrkA double-mutated at Tyr-785 and Tyr-751 (Y785F/Y751F-intact Shc binding site), at Tyr-785 and Tyr-490 (Y785F/Y490F-intact p85 binding site), or at Tyr-751 and Tyr-490 (Y751F/Y490F-intact PLCgamma binding site) using the calcium phosphate precipitation method. Empty pcDNA3 vector (V) was used as a transfection control. After 6 h of serum starvation, cells were stimulated with NGF (50 ng/ml; 5 min) in the presence or absence of BAPTA/AM (50 µM; 15 min preincubation). Cells were then lysed in modified RIPA buffer, and 1 mg of total cell lysate from each sample was immunoprecipitated (IP) either with rabbit anti-RAFTK (A) or rabbit PI3-K p85 antibodies (B). The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10), followed by blotting (IB) either with rabbit anti-RAFTK (1:2500) (A) or rabbit PI3-K p85 (1:1000) (B) as indicated. Total cell lysate was resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with rabbit anti-Trk (Calbiochem; 1:1000) to detect expression of the transfected TrkA receptor. Specific bands were visualized using the ECL system.

Taken together, the results obtained from the co-transfection of TrkA and RAFTK are in agreement with our results obtained using the pharmacological inhibitors (Fig. 2).

NGF-induced Ca2+ Mobilization Mediates Cell Morphogenesis and Cytoskeletal Reorganization but Not Erk/MAPK Activation or Neurite Outgrowth-- To address the functional role of NGF-induced RAFTK activation, we examined whether intracellular Ca2+ signaling, which is upstream of RAFTK phosphorylation, has any role in neurite outgrowth and/or Erk/MAPK activation upon NGF stimulation. Blocking of intracellular Ca2+ mobilization by BAPTA/AM did not affect the differentiation of single cells and the activation of Erk/MAPK upon NGF stimulation (Fig. 4); however, it did cause cell aggregation and resulted in a significant inhibition of normal PC12 cell differentiation under these conditions (Fig. 4A). These data show that intracellular Ca2+ signaling does not mediate neurite outgrowth per se but does affect morphogenesis and cytoskeletal reorganization that provide the subcellular framework for neurite outgrowth during NGF-induced neuronal differentiation.


View larger version (43K):
[in this window]
[in a new window]
  		Fig. 4.   Blocking of intracellular Ca2+ affects cell attachment and cell aggregation but not Erk/MAPK activity upon NGF stimulation. A, changes in cell shape induced by the intracellular Ca2+ chelator, BAPTA/AM, upon NGF stimulation. PC12 cells were stimulated with NGF (50 ng/ml) for 2 days in the presence or absence of BAPTA/AM (50 µM). Images of the PC12 cells were taken under an inverted light microscope (Olympus CK2) with an attached camera (Olympus SC35). B, effects of the intracellular Ca2+ chelator, BAPTA/AM, on Erk/MAPK activity upon NGF stimulation. Activity of Erk was measured by its ability to phosphorylate myelin basic protein (MBP) using an in vitro kinase assay. Immunoprecipitates of Erk1 after NGF stimulation were subjected to an immunocomplex MAPK kinase assay as described under "Experimental Procedures." IP, immunoprecipitation; IB, immunoblot.

RAFTK Is Associated with Paxillin and Induces Tyrosine Phosphorylation upon NGF Stimulation-- To characterize the function of RAFTK signaling upon NGF stimulation, RAFTK-associated molecules were investigated. Paxillin, one of the focal adhesion molecules, has been implicated in neuronal differentiation (26). Our immunoprecipitation studies demonstrated that paxillin is associated with RAFTK in PC12 cells (Fig. 5A). In addition, NGF treatment increased the tyrosine phosphorylation of paxillin that was blocked by BAPTA/AM. The induction of the tyrosine phosphorylation of paxillin in an intracellular Ca2+-dependent manner correlated with that of RAFTK, suggesting that RAFTK may mediate the tyrosine phosphorylation of paxillin upon NGF stimulation. To address whether RAFTK mediates paxillin tyrosine phosphorylation, we used transient transfection of paxillin and wild-type or kinase mutant RAFTK in 293 cells. Wild-type RAFTK induced the tyrosine phosphorylation of paxillin, whereas the kinase mutant RAFTK did not (Fig. 5C), demonstrating that RAFTK mediates paxillin phosphorylation upon NGF stimulation. The association of paxillin with FAK (see Fig. 5D), in conjunction with the high basal tyrosine phosphorylation of paxillin (Fig. 5B), suggests the existence of at least two pools of paxillin as follows: (i) paxillin regulated by FAK resulting in high basal tyrosine phosphorylation and (ii) paxillin regulated by RAFTK resulting in a Ca2+-dependent increase in tyrosine phosphorylation upon NGF stimulation. Taken together, our results indicate that activation of RAFTK leads to the tyrosine phosphorylation of paxillin in an intracellular Ca2+-dependent manner upon NGF stimulation, whereas FAK was constitutively phosphorylated.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 5.   Paxillin is associated with and is tyrosine-phosphorylated by RAFTK, upon NGF stimulation, in a Ca2+-dependent manner. A, paxillin is co-immunoprecipitated with RAFTK. After PC12 cells were stimulated with NGF (50 ng/ml) for 5 min in the presence or absence of BAPTA/AM (50 µM, 15 min preincubation), the cells were lysed in modified RIPA buffer. One mg of total cell lysate from each sample was immunoprecipitated (IP) with rabbit anti-RAFTK antibodies. The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with mouse anti-phosphotyrosine antibody (4G10), rabbit anti-RAFTK (1:2500), and with monoclonal anti-paxillin (Transduction Laboratory) after membrane stripping. Specific bands were visualized using the ECL system. B, reciprocal immunoprecipitation of paxillin shows co-immunoprecipitation of RAFTK with paxillin and also shows that NGF induces the tyrosine phosphorylation of paxillin in a Ca2+-dependent manner. PC12 cells were stimulated for 5 min with NGF (50 ng/ml) in the absence or presence of BAPTA/AM (50 µM; 15 min pre-incubation) and lysed in modified RIPA buffer. One mg of total cell lysate from each sample was immunoprecipitated with monoclonal anti-paxillin (Transduction Laboratory). The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10) and monoclonal anti-paxillin (Transduction Laboratory) after membrane stripping. Specific bands were visualized using the ECL system. C, 293 cells were co-transfected with wild-type (wt) TrkA receptor cDNA, HA-tagged paxillin cDNA, and wild-type or kinase mutant (km) RAFTK cDNA by the calcium phosphate precipitation method. Empty pcDNA3 vector (V) was used as a transfection (Transf.) control. After 6 h of serum starvation, cells were stimulated with NGF (50 ng/ml) for 5 min and harvested with modified RIPA buffer. 0.5 mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-HA antibodies to precipitate HA-tagged paxillin. The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with monoclonal anti-phosphotyrosine antibodies (4G10), followed by blotting (IB) with monoclonal anti-paxillin (Transduction Laboratory) as indicated. Total cell lysate was resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with anti-Trk and anti-RAFTK antibodies, respectively, to detect expression of the transfected proteins. Specific bands were visualized using the ECL system. D, paxillin is also co-immunoprecipitated with FAK. After PC12 cells were stimulated with NGF (50 ng/ml) for 5 min, and the cells were lysed in modified RIPA buffer. One mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-FAK antibodies or with NRS as a control. The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes, and probed with mouse anti-phosphotyrosine antibody (4G10), rabbit anti-FAK (1:2500), and with monoclonal anti-paxillin (Transduction Laboratory) after membrane stripping. Specific bands were visualized using the ECL system.

Differential Distribution of RAFTK and FAK in PC12 Cells-- To address the specific regulation of RAFTK and FAK upon NGF stimulation, we analyzed the subcellular localization of RAFTK, FAK, paxillin, and actin. PC12 cells were grown on culture chamber slides coated with an extracellular matrix protein (collagen IV) and were subjected to immunofluorescence staining for RAFTK, FAK, paxillin, and actin. RAFTK was distributed mostly in the cytoplasm, whereas paxillin showed a distinct distribution pattern, present both in the plasma membrane and in the cytoplasm, suggesting two pools of paxillin (a cell membrane-associated pool and a cytoplasmic pool) (Fig. 6A). Spread cells with a large cell surface showed staining of paxillin primarily at the plasma membrane, whereas spherical cells with a small cell surface showed mainly a cytoplasmic distribution of paxillin. These results indicate the dynamic redistribution of paxillin according to the cell adhesion state. Upon NGF stimulation (5 min), RAFTK translocated to the potential sites for neurite extension at the cell periphery (Fig. 6) and co-localized with paxillin. Since previous studies showed actin redistribution at the potential sites for neurite extension, i.e. ruffling or bundle formation upon the incubation of NGF-treated cells for 5 min (18), we examined the co-localization of actin and found that actin is also co-localized with RAFTK at the potential sites for neurite extension (Fig. 6B). In contrast, as shown in Fig. 7, FAK is mostly localized at the plasma membrane, specifically at distinct "point contacts" that are known as integrin-mediated cell adhesion sites in neuronal cells (31). Paxillin is co-localized with FAK at point contacts (Fig. 7). Actin is also co-localized with FAK and paxillin; however, this co-localization occurs without the formation of actin stress fibers (Fig. 7). In differentiated PC12 cells, FAK is localized at the edges of neurites and filopodia of growth cones, indicating the involvement of FAK and paxillin in cell adhesion. The differential distribution of RAFTK and FAK suggests that RAFTK is involved in the regulation of paxillin in the cytoplasm in response to NGF stimulation, whereas FAK may be involved in the regulation of cell adhesion both in undifferentiated and differentiated PC12 cells.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Early phase subcellular localization of RAFTK with paxillin after NGF stimulation. PC12 cells were grown on collagen type IV-coated culture chamber slides (Nalge Nunc) a day before NGF stimulation. In the absence (A) or presence (B) of NGF (50 ng/ml, 5 min) stimulation, PC12 cells were stained with rabbit anti-RAFTK and mouse anti-paxillin-specific antibodies followed by FITC-labeled goat anti-rabbit Ig antibody, rhodamine-labeled phalloidin, and Cy5-labeled goat anti-mouse Ig antibody as described under "Experimental Procedures." Images of the immunofluorescent-stained cells were collected by laser confocal microscopy (Bio-Rad MRC-1024 confocal microscope). For the staining control, PC12 cells were treated with secondary antibody alone.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 7.   Subcellular localization of FAK with paxillin at point contacts in PC12 cells. PC12 cells were grown on collagen type IV coated culture chamber slides a day before NGF stimulation. A, in the absence or (B) presence of NGF (50 ng/ml, 2 days) stimulation, PC12 cells were stained with FAK and paxillin specific antibodies followed by FITC-labeled goat anti-rabbit Ig antibody, rhodamine-labeled phalloidin, and Cy5-labeled goat anti-mouse Ig antibody as described in "Experimental Procedures." Images of the immunofluorescent stained cells were collected by laser confocal microscopy. For the staining control, PC12 cells were treated with secondary antibody alone. Scale bar = 10 µm.

Association and Phosphorylation of RAFTK and Paxillin upon Membrane Depolarization in Differentiated PC12 Cells-- In differentiated PC12 cells, RAFTK is localized at neurites and growth cones (Fig. 8A), specifically in the main body of neurites and growth cones, whereas FAK is localized at the edges of neurites and filopodia of growth cones (Fig. 7). Since Ca2+ mobilization is a key signaling modulator of neurite and growth cone activities, such as elongation, retraction, and maintenance (32), we investigated whether RAFTK and paxillin are functionally associated in Ca2+ signaling in differentiated PC12 cells. We found that RAFTK and paxillin were co-immunoprecipitated in these differentiated cells (Fig. 8, B and C). Upon KCl stimulation, there was a significant increase in both RAFTK and paxillin tyrosine phosphorylation that was blocked by the calcium chelator, BAPTA/AM. These results demonstrate that RAFTK is activated upon intracellular Ca2+ increase by depolarization and induces the tyrosine phosphorylation of paxillin in differentiated neuronal cells. Taken together, these data suggest possible roles of RAFTK and paxillin in neurite and growth cone function.


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 8.   Tyrosine phosphorylation of RAFTK and paxillin upon KCl stimulation of differentiated PC12 cells. A, subcellular localization of RAFTK and paxillin. PC12 cells were grown on poly-D-lysine coated culture chamber slides and stimulated with NGF for 4 days. After fixation and permeabilization, the cells were stained with RAFTK and paxillin specific antibodies followed by FITC-labeled goat anti-rabbit Ig antibody and Texas red-labeled goat anti-mouse Ig antibody as described in "Experimental Procedures." Scale bar = 10 µm. B, and C, after PC12 cells were differentiated with NGF (50 ng/ml) for 4 days, they were stimulated with KCl (60 mM, 3 min) in the presence or absence of BAPTA/AM (50 µM, 15 min pre-incubation) and lysed in modified RIPA buffer. One mg of total cell lysate from each sample was immunoprecipitated with rabbit anti-RAFTK (B) or monoclonal anti-paxillin (C) antibodies. The immunocomplexes were resolved by 8% SDS-PAGE (NOVEX), transferred to Immobilon-PVDF membranes and probed with monoclonal anti-phosphotyrosine antibody (4G10), and then with either rabbit anti-RAFTK (1:2500) or monoclonal anti-paxillin (Transduction Laboratory) after membrane stripping. Specific bands were visualized using the ECL system.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RAFTK is highly expressed in neuronal cells, is involved in various neuronal signaling pathways (1, 4), and is also suggested to be an important mediator of neuronal function, such as excitability (1) and excitotoxicity (33). However, little is known about the function of RAFTK and its homologous tyrosine kinase FAK in the nervous system. In this study, we address a possible role of RAFTK and FAK in neuronal differentiation.

The tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner (see Fig. 1, A and C) indicates that RAFTK is an intracellular signaling mediator during NGF-induced PC12 cell differentiation. In contrast, the high level of constitutive tyrosine phosphorylation of FAK, with no change in its tyrosine phosphorylation upon NGF stimulation (Fig. 1B), indicates that NGF-induced intracellular signaling is specific to RAFTK and not to its homologous tyrosine kinase FAK.

In our efforts to decipher the signaling pathway from the TrkA receptor to RAFTK activation, we found using pharmacological inhibitors that RAFTK phosphorylation is mediated through the TrkA receptor, PLCgamma , IP3, intracellular Ca2+ increases, and partially through extracellular Ca2+ influx and PI3-K (Fig. 2 and Fig. 9). Transient transfection of RAFTK and TrkA receptor mutants showed that both PLCgamma - and p85-binding sites in the TrkA receptor are responsible for the induction of RAFTK phosphorylation upon NGF stimulation (Fig. 3). Our experiment also showed that TrkA association with either PLCgamma or PI3-K p85 alone is strong enough to saturate the phosphorylation of RAFTK in the overexpression system (Fig. 3). This may explain why we could not see inhibition of RAFTK phosphorylation using a single point mutation of the TrkA receptor at 785, 751, or 490 in the transient transfection study (data not shown). Activation of PI3-K can be induced in two ways upon NGF stimulation. One is through the direct interaction between TrkA (PI3-K site at 751) and the regulatory subunit of PI3-K (p85) (29). The other is through the interaction between TrkA (Shc-binding site at 490) and Shc, which leads to activation of p21ras and association of ras with the catalytic subunit of PI3-K (p110), which in turn induces the activation of PI3-K (34). Therefore, the tyrosine phosphorylation of p85 is induced only by TrkA with the intact p85-binding site (wt or Y785F/Y490F) as our results show in Fig. 3B. The finding that TrkA with the intact Shc-binding site (Y785F/Y751F) induced a significantly lesser phosphorylation of RAFTK than TrkA with the intact p85-binding site (wt or Y785F/Y490F) despite PI3-K activation through the TrkA-Shc-ras complex indicates the important role of p85 in the induction of RAFTK phosphorylation. This is consistent with the partial inhibition of RAFTK phosphorylation by the PI3-K inhibitor wortmannin or LY294002, as shown in Fig. 2A. A recent study in macrophages showed co-immunoprecipitation of RAFTK with p85, supporting the importance of p85 in the signaling between RAFTK and PI3-K (35).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 9.   A model of the differential regulation of RAFTK and FAK upon NGF stimulation of PC12 cells. BAPTA/AM -cell permeable Ca2+ chelator; EGTA -extracellular Ca2+ chelator; LY294002 -PI3-K inhibitor; U73122 -Phospholipase C inhibitor; Wortmannin -PI3-K inhibitor.

It has been suggested that the duration of Erk/MAPK activation is a key signaling indicator determining the fate of PC12 cells toward cell differentiation or proliferation (36). We investigated whether NGF-induced RAFTK activation is involved in the induction of Erk/MAPK activation. Although transfection of the RAFTK kinase mutant reduced the activity of Erk/MAPK substantially (data not shown), it did not inhibit neurite outgrowth. Transfection of wild-type RAFTK neither induced nor inhibited neurite outgrowth (data not shown), suggesting that activation of RAFTK may be related to the induction of specific gene(s) or other change(s) rather than to neurite outgrowth during neuronal differentiation.

The morphological changes of the cell induced by BAPTA/AM (Fig. 4) suggest that Ca2+ signaling and downstream Ca2+-dependent RAFTK signaling may be related to cytoskeletal reorganization and cell shape changes rather than to neurite outgrowth via Erk/MAPK activation. The focal adhesion molecule paxillin is co-immunoprecipitated with RAFTK (Fig. 5), and the increase in paxillin tyrosine phosphorylation in a Ca2+-dependent manner is correlated with RAFTK phosphorylation (Fig. 2 and Fig. 5B). Furthermore, transient transfection of paxillin and RAFTK shows the induction of paxillin phosphorylation by RAFTK upon NGF stimulation (Fig. 5C). Although FAK is also associated with paxillin and is known to mediate paxillin phosphorylation (37), it is constitutively phosphorylated and shows no change in phosphorylation level upon NGF stimulation. Thus, the increase in phosphorylation of paxillin upon NGF stimulation is not attributable to FAK but to RAFTK (see Figs. 1, 2, and 5). A similar pattern of differential regulation of paxillin phosphorylation by RAFTK and FAK upon various stimuli was reported in rat liver GN4 epithelial cells (7). NGF-induced paxillin phosphorylation was also reported in a subclone of PC12 cells (38) and in B cells (39). Paxillin is a cytoskeletal protein and functions as a scaffold for the recruitment of signaling molecules during cell adhesion and cell morphogenesis (24). In neuronal cells, paxillin is implicated in the regulation of cytoskeletal reorganization and/or cell adhesion changes during neuronal differentiation (26). Therefore, it is conceivable that RAFTK association with paxillin regulates cytoskeletal reorganization and/or cell adhesion changes during NGF-induced PC12 cell differentiation. In previous studies, RAFTK has also been shown to be involved in cell adhesion and cytoskeletal organization through p130Cas (40) and paxillin (9). The involvement of RAFTK in cytoskeletal organization and the requirement of an intact actin-based cytoskeleton for RAFTK phosphorylation upon various stimuli, including NGF (Fig. 2C and (22)), strongly suggest that RAFTK is a key component of cytoskeletal organization.

Point contacts are the sites of functional cell adhesion in neuronal cells and have been well characterized in a previous report (31). In contrast to that report which detected no staining of FAK, we found the localization of FAK at point contacts in PC12 cells grown on collagen type IV (Fig. 7A) and laminin (data not shown), suggesting that FAK and paxillin are associated at the point contacts and are involved in cell adhesion. Subcellular distribution of RAFTK, FAK, paxillin, and actin suggests that there are two pools of paxillin with dynamic redistribution (see Figs. 6 and 7); one pool of paxillin, associated with FAK at the plasma membrane, is involved in cell adhesion and is regulated by FAK; the other pool, associated with RAFTK at the cytoplasm, is involved in NGF signaling and is regulated by RAFTK (Fig. 6A). Paxillin is a multidomain adaptor protein capable of interacting with several structural and signaling proteins including vinculin, FAK, RAFTK, Src, and Crk (24). There is a dynamic interaction between cell adhesion and intracellular signaling. RAFTK-mediated paxillin phosphorylation may regulate the recruitment of various molecules into a signal transduction complex leading to cell adhesion changes (inside-out signaling) during the early phase of NGF stimulation. Consistent with our findings, expression of a kinase mutant of RAFTK in a stable PC12 cell line using a tetracycline-sensitive promoter system resulted in the loss of cell-to-substrate adhesion and the formation of cell aggregates in response to NGF stimulation (41). This supports the view that RAFTK is an essential mediator of cell adhesion changes upon NGF stimulation via paxillin. Although RAFTK and FAK are related tyrosine kinases and share unique and significant amino acid sequence homology (48% identity and 65% similarity) (4), they have distinct roles in signaling, i.e. FAK is more involved in integrin-mediated signaling, whereas RAFTK is stimulated by a variety of extracellular agonists that increase intracellular Ca2+ concentrations (1, 5). The various subcellular localizations and tyrosine phosphorylations of RAFTK and FAK may explain the differential regulation of specific pools of paxillin upon various cellular stimuli of PC12 cells. In Fig. 9, we illustrate a current model of differential regulation of paxillin by RAFTK and FAK during NGF-induced PC12 cell differentiation.

Growth cones in differentiated neuronal cells play vital roles in the navigation, elongation, retraction, and maintenance of neurites, and intracellular Ca2+ is one of the key signaling components regulating this growth cone activity (32). RAFTK was found at relatively high levels in the axons of brain cells, suggesting that it might play an important role in the functions of axons or that the RAFTK-related signaling pathway is closely associated with cytoskeletal components (42). RAFTK localization at neurites and growth cones in conjunction with its tyrosine phosphorylation upon membrane depolarization suggests its possible role with paxillin in Ca2+-mediated growth cone function in differentiated neuronal cells (Fig. 8, B and C). Bradykinin, a neuropeptide activating the G protein-coupled receptor and producing intracellular Ca2+ increase, induces neurite retraction (43). We found that bradykinin also induces the tyrosine phosphorylation of RAFTK and paxillin in a Ca2+-dependent manner in differentiated PC12 cells.2 This suggests the involvement of RAFTK in neurite retraction through paxillin reorganization.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Jerome E. Groopman, Shuxian Jiang, Hiroshi Yamashita, Jinkyu Lim, Tae Kim, and Karin A. Schinkmann for advice, discussions, and support of this work. We thank Dr. Brian J. Druker, Oregon Health Sciences University, for the 4G10 antibody. NGF was a generous gift from Genentech, Inc. We also thank Janet Delahanty for editing the manuscript, and Daniel Kelley for preparation of the figures.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL55445 (to S. A.), HL51456 (to H. A.), DAMD17-98-1-8032 (to H. A.), DAMD17-99-1-9078 (to H. A.), and CA76226 (to H. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated in memory of Ronald Ansin for his friendship and support for our research program.

Dagger To whom correspondence should be addressed: Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115. Tel.: 617-667-0063; Fax: 617-975-6373; E-mail: savraham@caregroup.harvard.edu.

Published, JBC Papers in Press, April 7, 2000, DOI 10.1074/jbc.M909932199

2 K. A. Schinkmann, S.-Y. Park, and S. Avraham, personal communication.

    ABBREVIATIONS

The abbreviations used are: RAFTK, related adhesion focal tyrosine kinase; BAPTA/AM, [1,2-bis(o-amino-5-fluorophenoxy)ethane- N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester]; CC, chelerylthrine chloride; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FBS, fetal bovine serum; HA, hemagglutinin; IP3, inositol triphosphate; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NRS, normal rabbit serum; PI3-K, phosphatidylinositol 3-kinase; PLCgamma , phospholipase Cgamma ; Shc, src homology containing protein; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; DMEM, Dulbecco's modified Eagle's medium; FITC, fluorescein isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Lev, S., Moreno, H., Martinez, R., Canoll, P., Peles, E., Musacchio, J. M., Plowman, G. D., Rudy, B., and Schlessinger, J. (1995) Nature 376, 737-745
2. Sasaki, H., Nagura, K., Ishino, M., Tobioka, H., Kotani, K., and Sasaki, T. (1995) J. Biol. Chem. 270, 21206-21219
3. Yu, H., Li, X., Marchetto, G. S., Dy, R., Hunter, D., Calvo, B., Dawson, T. L., Wilm, M., Anderegg, R. J., Graves, L. M., and Earp, H. S. (1996) J. Biol. Chem. 271, 29993-29998
4. Avraham, S., London, R., Fu, Y., Ota, S., Hiregowdara, D., Li, J., Jiang, S., Pasztor, L. M., White, R. A., Groopman, J. E., and Avraham, H. (1995) J. Biol. Chem. 270, 27742-27751
5. Tokiwa, G., Dikic, I., Lev, S., and Schlessinger, J. (1996) Science 273, 792-794
6. Li, J., Avraham, H., Rogers, R. A., Raja, S., and Avraham, S. (1996) Blood 88, 417-428
7. Li, X., and Earp, H. S. (1997) J. Biol. Chem. 272, 14341-14348
8. Matsuya, M., Sasaki, H., Aoto, H., Mitaka, T., Nagura, K., Ohba, T., Ishino, M., Takahashi, S., Suzuki, R., and Sasaki, T. (1998) J. Biol. Chem. 273, 1003-1014
9. Salgia, R., Avraham, S., Pisick, E., Li, J. L., Raja, S., Greenfield, E. A., Sattler, M., Avraham, H., and Griffin, J. D. (1996) J. Biol. Chem. 271, 31222-31226
10. Schaller, M. D., and Sasaki, T. (1997) J. Biol. Chem. 272, 25319-25325
11. Andreev, J., Simon, J. P., Sabatini, D. D., Kam, J., Plowman, G., Randazzo, P. A., and Schlessinger, J. (1999) Mol. Cell. Biol. 19, 2338-2350
12. Kaplan, D. R., Martin-Zanca, D., and Parada, L. F. (1991) Nature 350, 158-160
13. Klein, R., Jing, S. Q., Nanduri, V., O'Rourke, E., and Barbacid, M. (1991) Cell 65, 189-197
14. Loeb, D. M., Tsao, H., Cobb, M. H., and Greene, L. A. (1992) Neuron 9, 1053-1065
15. Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428
16. Toledo-Aral, J. J., Brehm, P., Halegoua, S., and Mandel, G. (1995) Neuron 14, 607-611
17. Paves, H., Neuman, T., Metsis, M., and Saarma, M. (1988) FEBS Lett. 235, 141-143
18. Sano, M., and Iwanaga, M. (1992) Cell Struct. Funct. 17, 341-350
19. Pandiella-Alonso, A., Malgaroli, A., Vicentini, L. M., and Meldolesi, J. (1986) FEBS Lett. 208, 48-51
20. Nikodijevic, B., and Guroff, G. (1991) J. Neurosci. Res. 28, 192-199
21. Berridge, M. J. (1998) Neuron 21, 13-26
22. Avraham, H., Park, S., Schinkmann, K., and Avraham, S. (2000) Cell. Signal. 12, 123-133
23. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. (1994) Nature 372, 786-791
24. Turner, C. E. (1998) Int J. Biochem. Cell Biol. 30, 955-959
25. Turner, C. E., and Miller, J. T. (1994) J. Cell Sci. 107, 1583-1591
26. Leventhal, P. S., and Feldman, E. L. (1996) J. Biol. Chem. 271, 5957-5960
27. Yamashita, H., Avraham, S., Jiang, S., Dikic, I., and Avraham, H. (1999) J. Biol. Chem. 274, 15059-15065
28. Ganju, R. K., Hatch, W. C., Avraham, H., Ona, M. A., Druker, B., Avraham, S., and Groopman, J. E. (1997) J. Exp. Med. 185, 1055-1063
29. Obermeier, A., Lammers, R., Wiesmuller, K. H., Jung, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966
30. Hiregowdara, D., Avraham, H., Fu, Y., London, R., and Avraham, S. (1997) J. Biol. Chem. 272, 10804-10810
31. Arregui, C. O., Carbonetto, S., and McKerracher, L. (1994) J. Neurosci. 14, 6967-6977
32. Goldberg, D. J., and Grabham, P. W. (1999) Neuron 22, 423-425
33. Combs, C. K., Johnson, D. E., Cannady, S. B., Lehman, T. M., and Landreth, G. E. (1999) J. Neurosci. 19, 928-939
34. Kaplan, D. R., and Miller, F. D. (1997) Curr. Opin. Cell Biol. 9, 213-221
35. Hatch, W. C., Ganju, R. K., Hiregowdara, D., Avraham, S., and Groopman, J. E. (1998) Blood 91, 3967-3973
36. Marshall, C. J. (1995) Cell 80, 179-185
37. Bellis, S. L., Miller, J. T., and Turner, C. E. (1995) J. Biol. Chem. 270, 17437-17441
38. Khan, M. A., Okumura, N., and Okada, M. (1996) Eur. J. Biochem. 235, 579-584
39. Melamed, I., Turner, C. E., Aktories, K., Kaplan, D. R., and Gelfand, E. W. (1995) J. Exp. Med. 181, 1071-1079
40. Astier, A., Avraham, H., Manie, S. N., Groopman, J., Canty, T., Avraham, S., and Freedman, A. S. (1997) J. Biol. Chem. 272, 228-232
41. Zwick, E., Wallasch, C., Daub, H., and Ullrich, A. (1999) J. Biol. Chem. 274, 20989-20996
42. Mitaka, T., Shindoh, M., Mochizuki, Y., Sasaki, H., Ishino, M., Matsuya, M., Ninomiya, T., and Sasaki, T. (1997) Am. J. Pathol. 150, 267-281
43. Schindelholz, B., and Reber, B. F. X. (1997) J. Neurosci. 17, 8391-8401

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
A. Prasad, Z. Qamri, J. Wu, and R. K. Ganju
Slit-2/Robo-1 modulates the CXCL12/CXCR4-induced chemotaxis of T cells
J. Leukoc. Biol., September 1, 2007; 82(3): 465 - 476.
[Abstract] [Full Text] [PDF]