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Originally published In Press as doi:10.1074/jbc.M511205200 on June 15, 2006

J. Biol. Chem., Vol. 281, Issue 33, 23611-23619, August 18, 2006
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Physical and Functional Interaction of Fyn Tyrosine Kinase with a Brain-enriched Rho GTPase-activating Protein TCGAP*

Hui Liu1, Takanobu Nakazawa1, Tohru Tezuka, and Tadashi Yamamoto2

From the Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Received for publication, October 14, 2005 , and in revised form, June 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fyn, a member of the Src family of tyrosine kinases, is implicated in both brain development and adult brain function. In the present study, we identified a Rho GTPase-activating protein (GAP), TCGAP (Tc10/Cdc42 GTPase-activating protein), as a novel Fyn substrate. TCGAP interacted with Fyn and was phosphorylated by Fyn, with Tyr-406 in the GAP domain as a major Fyn-mediated phosphorylation site. Fyn suppressed the GAP activity of wild-type TCGAP but not the Y406F mutant of TCGAP in a phosphorylation-dependent manner, suggesting that Fyn-mediated Tyr-406 phosphorylation negatively regulated the TCGAP activity. In situ hybridization analyses showed that TCGAP mRNA was expressed prominently in both immature and adult mouse brain, with high levels in cortex, corpus striatum, hippocampus, and olfactory bulb. Overexpression of wild-type TCGAP in PC12 cells suppressed nerve growth factor-induced neurite outgrowth, whereas a GAP-defective mutant of TCGAP enhanced the neurite outgrowth. Nerve growth factor enhanced tyrosine phosphorylation of TCGAP through activation of Src family kinases. These results suggest that TCGAP is involved in Fyn-mediated regulation of axon and dendrite outgrowth.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fyn, a member of the Src family of non-receptor tyrosine kinases, plays important roles in brain development and mature brain functions. Fyn-knock-out mice have various neural defects, including defective long term potentiation, impaired spatial memory, hypomyelination, abnormal dendrite orientation, uncoordinated hippocampal structure, increased fearfulness, and increased ethanol sensitivity (1–6). These multiple effects of Fyn deficiency in the brain indicate that Fyn is involved in several neural signaling pathways. Recent studies have identified several Fyn-binding proteins and substrates in the brain that include CNR, mDab1, N-WASP, PSD-95, p190RhoGAP, and NR2B. These molecules are important for neural migration, synaptic plasticity, oligodendrocyte differentiation, and axon growth and guidance (7–13). However, the molecular mechanisms by which Fyn regulates these processes remain elusive.

The Rho family of small GTPases (Rho GTPases), such as RhoA, Rac1, and Cdc42, play important roles in cytoskeletal remodeling as well as membrane trafficking, transcriptional regulation, and cell growth (14, 15). RhoA regulates formation of focal adhesions and subsequent assembly of stress fibers. Rac1 regulates formation of membrane lamellae, and Cdc42 triggers outgrowth of peripheral spike-like protrusions called filopodia (16–19). In the central nervous system, regulation of the actin cytoskeleton is crucial for the migration of neuron, axon guidance, development of dendrites, and morphological differentiation of oligodendrocytes that are involved in brain development (20–22). Synaptic plasticity such as long term potentiation is associated with actin cytoskeleton reorganization, indicating that the actin cytoskeleton is also important for mature brain functions (23; for review see Refs. 24 and 25).

The Rho GTPases cycle between active GTP-bound and inactive GDP-bound states that are regulated by the opposing effects of guanine nucleotide exchange factors, which catalyze the exchange of bound GDP for GTP, and GTPase-activating proteins (GAPs),3 which increase hydrolysis of bound GTP (26, 27). The activities of these regulators are modified by phosphorylation (28–31): several guanine nucleotide exchange factors and GAPs, including FRG and p190RhoGAP, are substrates for the Src family kinases (11, 32).

TCGAP (Tc10/Cdc42 GTPase-activating protein) is a recently identified RhoGAP, and it plays an important role in the regulation of insulin-induced glucose transport in adipocytes (33). In response to insulin, TCGAP translocates to the plasma membrane in adipocytes. TCGAP contains an N-terminal Phox homology domain, Src homology 3 (SH3) domain, RhoGAP domain, and several proline-rich sequences at the C-terminal region. TCGAP specifically interacts via its GAP domain with Cdc42 and TC10beta. The role of TCGAP in the brain has not been studied, although TCGAP is expressed at high levels in brain and testis (33).

In the present study, we found that the TCGAP is a novel Fyn substrate in vivo. GAP activity of TCGAP was suppressed by Fyn-mediated phosphorylation of the protein. Expression of wild-type TCGAP in rat pheochromocytoma PC12 cells suppressed NGF-induced neurite outgrowth, whereas a GAP-defective mutant of TCGAP enhanced the neurite outgrowth. NGF enhanced tyrosine phosphorylation of TCGAP through activation of Src family kinases in PC12 cells. All these data suggest that TCGAP is involved in Fyn-mediated regulation of axon and dendrite outgrowth.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Screening—A cDNA encoding the full-length of human brain-type Fyn was cloned in-frame into pGBT9 vector (Clontech) as a bait plasmid. The reporter yeast strain YJ69-2A harboring the bait plasmid was transformed with a human fetal brain cDNA library (Clontech). Transformants were selected on synthetic media lacking leucine, tryptophan, adenine, and histidine. DNA sequences of the insert in the selected transformants were analyzed by searching the BLAST data base.

DNA Constructs—A cDNA clone for human TCGAP cDNA (GenBankTM AK096338 [GenBank] ) was a kind gift from S. Sugano (University of Tokyo, Japan) and Helix Research Institute (Chiba, Japan). A GAP-inactive mutant (Arg-350 to Ile-350, R350I) and a YF mutant (Tyr-406 to Phe-406, Y406F) of TCGAP were generated by PCR. The expression plasmids for TCGAP were constructed in the pME18S vector (34). The constructs were verified by DNA sequencing. The expression plasmids pME-Fyn, pME-FynY531F, and pME-FynK299M have been described (10). The expression plasmids pME-FynR176K (an SH2-defective mutant) and pME-FynP134L (an SH3-defective mutant) were generated by the methods of Kunkel (35). The expression plasmids pEFBOS-Myc-RhoA, pEFBOS-Myc-Cdc42, and pEFBOS-Myc-Rac1 were kindly provided by Y. Takai (University of Osaka, Japan). The plasmid for GST-Cdc42/Rac-interactive binding domain of Pak (GST-CRIB) was kindly provided by C. Sasakawa (University of Tokyo, Japan). The plasmid for GST-Rho binding domain of Rhotekin (GST-RBD) was described previously (36).

Cell Culture, DNA Transfection, and Retroviral Infection—Human embryonic kidney (HEK) 293T cells and Plat-E cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2. PC12 rat pheochromocytoma cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% horse serum (Invitrogen) and 10% fetal bovine serum. HEK293T cells were transfected with plasmid DNAs by the standard calcium phosphate method. Retroviral infection of PC12 cells was performed as described (37, 38). PP2 and PP3 (Calbiochem) were added to the final concentration of 10 µM to the medium.

Antibodies—Polyclonal antibodies against TCGAP were raised by immunizing rabbits with a glutathione S-transferase (GST) protein fused with a portion of human TCGAP (antibody N: amino acid residues 1–159, antibody M: 760–926, and antibody C: 947–1126) or keyhole limpet hemocyanin conjugated with a peptide, LHSEGQTRSYC, that corresponds to amino acid residues 1116–1126 of human TCGAP (antibody P). The antibodies were affinity-purified by antigen-coupled columns. Anti-Fyn mAb was purchased from BD Transduction Laboratories. Anti-Myc mAb (9E10) and anti-phosphotyrosine (pTyr) mAb (4G10) were from Santa Cruz Biotechnology and Upstate%20Biotechnology">Upstate Biotechnology, respectively.

Preparation of Lysates, Immunoprecipitation, and Immunoblotting—Lysates of transfected cells and brain were prepared using TNE buffer (50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 5 mM EDTA, and 100 mM NaCl, 0.2 mM Na3VO4 with aprotinin at 50 units/ml). For immunoprecipitation, precleared lysates were incubated sequentially with the appropriate antibodies and Protein G-Sepharose (Amersham Biosciences) for 2 h. Immunoprecipitates were washed five times with TNE buffer. Immunoprecipitates and lysates were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were then blocked with TBST containing 3% bovine serum albumin and blotted with primary antibodies. Horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies and Renaissance Plus reagent (PerkinElmer Life Sciences) or CDP-Star (TROPIX) were used to visualize the immunoreactive proteins.

In Situ Hybridization—A partial mouse cDNA fragment (corresponding to nucleotides 2363–2560 in human TCGAP cDNA) was obtained by reverse transcription-PCR and cloned into pBluescript II SK+ (Stratagene). After verification of the sequence, the cRNA for mouse TCGAP was prepared by in vitro transcription, labeled with [35S]UTP, and used as a probe. Mouse embryo sections at embryonic days 11 (E11), E12, E13, E14, E15, and E16 were purchased from Novagen. Mice at E18 and postnatal 1–4 week of age were prepared as described (36, 39). Fresh frozen brain sections (10 µm) in the coronal, parasagittal, and sagittal plane, were mounted on glass slides (Matsunami). Sections were treated at room temperature as follows: fixation with 4% paraformaldehyde/PBS for 20 min, washing with PBS for 5 min twice, acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine (Tris)–HCl (pH 8.0), for 10 min, washing with PBS for 5 min twice, 70% ethanol for 5 min, 90% ethanol for 3 min, 100% ethanol for 3 min, chloroform for 10 min, 100% ethanol for 3 min, and then drying at room temperature for 1 h. Hybridization was performed at 55 °C for 10 h in a hybridization buffer containing 50% formamide, 10 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 17.3 mM NaH2PO4, 2.5 mM EDTA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.2% SDS, 10 mM dithiothreitol, and 10% dextran sulfate, supplemented with 1.2 x 106 cpm of 35S-labeled oligonucleotide and 40 µg of yeast tRNA. Slides were washed as follows: washing at 55 °C for 20 min in 5x SSC containing 0.1% 2-mercaptoethanol, washed at 65 °C for 30 min in 2x SSC containing 50% formamide and 1% 2-mercaptoethanol, treated with 20 µg/ml RNase in RNase buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 0.5 M NaCl), washed at room temperature for 15 min in 2x SSC, 0.1x SSC and then in 70% ethanol, 90% ethanol, and 100% ethanol each for 30 s. Sections were exposed to Hyperfilm-beta max (Amersham Biosciences) for 1 week.

Preparation of PSD Fraction—Synaptosome and PSD (one Triton X-100 extraction) of adult mouse telencephalons were prepared as described previously (36, 40).

Immunocytochemistry—Hippocampal neurons were fixed with methanol for 10 min at–20 °C, blocked with 5% normal goat serum, and then incubated with appropriate antibodies. PC12 cells were infected with mock, wild-type TCGAP, or TCGAP R350I, stimulated with 50 ng/ml NGF for 48 h, fixed with 4% paraformaldehyde/PBS, permeabilized with 0.2% Triton X-100/PBS, and blocked with 3% BSA/PBS, then incubated with appropriate antibodies. The primary antibodies were visualized using goat anti-mouse or anti-rabbit IgG conjugated to Alexa488 (Molecular Probes) or Cy3 (KPL, Jackson ImmunoResearch Laboratories).


Figure 1
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  		FIGURE 1.
Interaction between TCGAP and Fyn. A, interaction between TCGAP and Fyn in HEK293T cells. HEK293T cells were transfected with plasmids expressing TCGAP and Fyn. TCGAP-immunoprecipitates (IP) (upper panels) and cell lysates (lower panels) were subjected to immunoblotting (IB) with anti-TCGAP (M) antibody and anti-Fyn mAb. B, requirement of the SH3 domain of Fyn for interaction with TCGAP. HEK293T cells were transfected with expression plasmids for TCGAP and different mutants of Fyn (wild-type, SH3-domain mutant (SH3m), SH2-domain mutant (SH2m), and kinase-dead mutant (KM)). TCGAP-immunoprecipitates (upper panels) and cell lysates (lower panels) were subjected to immunoblotting with anti-TCGAP antibody and anti-Fyn mAb. C, phosphorylation of TCGAP by Fyn in HEK293T cells. HEK293T cells were co-transfected with plasmids expressing TCGAP and various mutants of Fyn. TCGAP-immunoprecipitates (upper panels) and cell lysates (lower panels) were subjected to immunoblotting with anti-pTyr (4G10) mAb, anti-TCGAP (C) antibody and anti-Fyn mAb. D, Fyn-mediated phosphorylation of TCGAP in the brain. Brain lysates were prepared from wild-type and fyn-knock-out mice at postnatal 1 week of age and were immunoprecipitated with anti-TCGAP (C) antibody. TCGAP immunoprecipitates and brain lysates were subjected to immunoblotting with anti-pTyr (4G10) mAb (upper panels), anti-TCGAP antibody (middle panel), and anti-Fyn mAb (lower panel)(left). Quantification of TCGAP phosphorylation in wild-type and fyn-knock-out mice (n = 3) (right). Values represent the mean ± S.E. Significance was determined by Student's t test (p < 0.001).

 
RhoGAP Assay—The RhoGAP activity of TCGAP in mammalian cells was analyzed by the Rho effector pull-down assay as described previously (36, 41). Briefly, HEK293T cells were transfected with the expression plasmids for Myc-tagged RhoA, Cdc42, or Rac1 together with or without wild-type, R350I mutant, or Y406F mutant of TCGAP in the presence or absence of different mutants of Fyn. The cells were lysed for 5 min with ice-cold lysis buffer (50 mM Tris-Cl (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 10% glycerol for Cdc42 and Rac1; 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 30 mM MgCl2, 0.1% Triton X-100, 10% glycerol for RhoA), and then the lysates were centrifuged for 5 min. The supernatants were incubated with 20 µg of GST-CRIB for 30 min for Cdc42 and Rac1, or 40 µg of GST-RBD for 60 min for RhoA. After washing the beads with lysis buffer, the bound proteins were resolved on SDS-PAGE and subjected to immunoblotting with anti-Myc mAb.

NGF Stimulation of PC12 Cells—Analysis of neurite outgrowth of PC12 cells was performed as described previously (38). PC12 cells infected with Moloney murine leukemia virus expressing mock, wild-type, Y406F mutant, and R350I mutant of TCGAP were selected with puromycin (2.0 µg/ml) for 48 h. The selected cells (5 x 104) were plated in 6-well plates and stimulated with NGF (50 ng/ml). The total neurite lengths of the cells were calculated with TI-Workbench software that was kindly provided by T. Inoue (University of Tokyo, Japan).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of TCGAP as a Novel Fyn-binding Protein—To identify proteins that interact with Fyn in the brain, we carried out yeast two-hybrid screening with full-length Fyn as bait. We obtained 80 candidate clones from a human fetal brain cDNA library comprising 8.6 x 106 individual clones. One of them encoded a partial sequence of the C-terminal portion (amino acids 894–1126) of TCGAP, a recently identified RhoGAP (33).

To determine whether TCGAP interacts with Fyn in mammalian cells, we carried out co-immunoprecipitation experiments. HEK293T cells were transfected with plasmids encoding TCGAP and full-length Fyn. As shown in Fig. 1A, Fyn was co-immunoprecipitated with TCGAP. To identify the region of Fyn that interacts with TCGAP, wild-type or mutant forms of Fyn were expressed with TCGAP in HEK293T cells. Co-immunoprecipitation experiments showed that TCGAP interacted well with kinase-dead (K299M) and SH2-defective (R176K) mutants of Fyn (Fig. 1B). In contrast, TCGAP did not interact with an SH3-defective mutant (P134K) of Fyn, suggesting that the SH3 domain is required for TCGAP binding (Fig. 1B). Moreover, Fyn was co-immunoprecipitated with TCGAP from brain lysates of wild-type mice (Fig. 1D). Therefore, we concluded that TCGAP interacts with Fyn in vivo.

Phosphorylation of TCGAP by Fyn—We next examined whether TCGAP is phosphorylated by Fyn in mammalian cells. HEK293T cells were transfected with combinations of plasmids expressing TCGAP and wild-type or mutant forms of Fyn. Co-expression of FynY531F, SH2-defective (R176K), and SH3-defective mutants (P134K) of Fyn, but not FynK299M, with TCGAP enhanced tyrosine phosphorylation of TCGAP (Fig. 1C). To confirm that TCGAP is tyrosine phosphorylated in the brain, TCGAP immunoprecipitates were prepared from brain lysates of wild-type and fyn-knock-out mice and probed with an anti-phosphotyrosine mAb (Fig. 1D). As shown in Fig. 1D, TCGAP was tyrosine-phosphorylated in both wild-type and fyn-knock-out mouse brain; the level of TCGAP phosphorylation was significantly decreased in fyn-knock-out mice. The ratio of TCGAP tyrosine phosphorylation in fyn-knock-out mice to that in wild-type mice was 0.45 ± 0.08 (p < 0.001, Student's t test). These results strongly suggest that TCGAP is a novel substrate for Fyn in the brain.


Figure 2
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  		FIGURE 2.
GAP activity of TCGAP. A, analysis of RhoGAP activity of TCGAP by Rho GTPase effector pull-down assays. HEK293T cells were transiently transfected with plasmids expressing Myc-tagged RhoA, Cdc42, or Rac1 and wild-type TCGAP (WT) or R350I mutant of TCGAP (RI). –, indicates vector control. Lysates of the cells were pulled down by GST-CRIB (for Cdc42 and Rac1) and GST-RBD (for RhoA) immobilized on glutathione-Sepharose beads. Levels of GTP-loaded RhoA, Cdc42, and Rac1 were analyzed by immunoblotting with anti-Myc mAb (upper panels). Whole cell lysates were verified by immunoblotting with anti-Myc mAb (middle panels) and anti-TCGAP (M)(lower panels). B, suppression of RhoGAP activity of TCGAP by Fyn-mediated phosphorylation. HEK293T cells were co-transfected with plasmids expressing Myc-tagged Cdc42 and wild-type TCGAP (WT) and various mutants of Fyn (constitutively active form (YF), SH3-domain mutant (SH3m), and kinase-dead mutant (KM)). Lysates were pulled down by GST-CRIB immobilized on glutathione-Sepharose beads. Levels of GTP-loaded Cdc42 were analyzed by immunoblotting with anti-Myc mAb (upper panel). Cell lysates were verified by immunoblotting with anti-Myc mAb (middle panel), anti-TCGAP (M) antibody, and anti-Fyn (lower two panels).

 
RhoGAP Activity of TCGAP in Mammalian Cells—TCGAP shows GAP activity for Cdc42 and TC10beta in vitro as described previously (33). To determine whether TCGAP exhibits RhoGAP activity in mammalian cells, we applied Rho effector pull-down methods (see "Experimental Procedures") in HEK293T cells. Wild-type TCGAP reduced the amount of GTP-loaded Cdc42 but had little effect on the GTP-load status of RhoA and Rac1 (Fig. 2A). The GAP activity of TCGAP toward Cdc42 was reduced significantly by R350I mutation of TCGAP. Arginine 350 in the GAP domain of TCGAP is conserved among the RhoGAP family proteins and is known to be required for GAP activity (42, 43). We next examined whether Fyn-mediated phosphorylation of TCGAP affects its RhoGAP activity. As shown in Fig. 2B, co-expression of active-Fyn, FynY531F, suppressed the GAP activity of TCGAP. In contrast, a kinase-dead mutant of Fyn, FynK299M, had no effect on the GAP activity of TCGAP (Fig. 2B). These results indicate that TCGAP promotes GTP hydrolysis on Cdc42 in cells, and this GAP activity is suppressed by Fyn-mediated phosphorylation of TCGAP.


Figure 3
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  		FIGURE 3.
Suppression of TCGAP by Fyn through phosphorylation at Tyr-406 of TCGAP. A, identification of Tyr-406 of TCGAP as a major phosphorylation site for Fyn. HEK293T cells were co-transfected with plasmids expressing wild-type or Y406F mutant of TCGAP and constitutive active Fyn. TCGAP-immunoprecipitates (IP)(upper panels) and cell lysates (lower panels) were subjected to immunoblotting with anti-pTyr (4G10) mAb, anti-TCGAP (C) antibody, and anti-Fyn mAb. B, Tyr-406 phosphorylation-dependent regulation of the GAP activity of TCGAP. Top, the representative Western blots of the pull-down assays are shown. HEK293T cells were co-transfected with plasmids expressing Myc-tagged Cdc42, wild-type TCGAP (WT), or Y406F mutant of TCGAP, and Fyn mutants (constitutively active (YF) and kinase-dead (KM)). Lysates were pulled down by GST-CRIB immobilized on glutathione-Sepharose beads. Levels of GTP-loaded Cdc42 were analyzed by immunoblotting with anti-Myc mAb (upper panel). Cell lysates were verified by immunoblotting with anti-Myc mAb (middle panel), anti-TCGAP (M) antibody, and anti-Fyn antibody (lower two panels). Bottom, the statistical results from five independent experiments are plotted. Data are presented as the mean ± S.E.

 
Fyn-mediated Phosphorylation Site in TCGAP—Because Fyn-mediated phosphorylation suppressed the GAP activity of TCGAP, it was likely that phosphorylation occurred in the GAP domain. Among 6 tyrosine residues present in this region, only Tyr-406 matches the consensus phosphorylation site for Fyn. To examine whether Tyr-406 is phosphorylated by Fyn, HEK293T cells were transfected with expression plasmids for wild-type TCGAP or Y406F (conversion of Tyr-406 to Phe-406) mutant of TCGAP together with active-Fyn. As shown in Fig. 3A, Y406F mutation resulted in the significant reduction of the level of TCGAP phosphorylation. This result strongly suggests that Tyr-406 is one of the major Fyn-mediated phosphorylation sites in TCGAP.


Figure 4
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  		FIGURE 4.
Pattern of TCGAP Expression. A, in situ hybridization of TCGAP mRNA in sections of developing mouse brain. Parasagittal sections of E11 (a), E12 (b), E13 (c), E14 (d), E15 (e), E16 (f), E18 (g), 1w (h), 2w (i), and 4w (j and k) were hybridized with [{alpha}-35S]UTP-labeled mouse TCGAP cRNA antisense (a–j) or sense (k) probes. B, in situ hybridization of coronal sections of 4-week-old (4w) mouse brain. Brain sections from 4-week-old mice were hybridized with an [{alpha}-35S]UTP-labeled mouse TCGAP cRNA antisense probe. C, expression of TCGAP protein in mouse brain. Mouse brain lysates from postnatal 0 day (P0) to 8 weeks (8W) were subjected to immunoblotting with anti-TCGAP (M) antibody and anti-Erk2 antibody. Erk2 was used as a loading control.

 
To examine whether Tyr-406 phosphorylation suppresses the GAP activity of TCGAP, we applied Rho effector pull-down methods. In contrast to the case with wild-type TCGAP, co-expression of active-Fyn did not suppress the GAP activity of Y406F mutant of TCGAP (Fig. 3B). In the absence of Fyn, the GAP activities of WT and Y406F TCGAP were statistically the same (Fig. 3B). These results suggest that Fyn suppresses the GAP activity of TCGAP through Tyr-406 phosphorylation.

Developmental Expression of TCGAP in Brain—To examine the distribution of TCGAP mRNA in the brain, we carried out in situ hybridization analysis (Fig. 4, A and B). Prominent expression of TCGAP mRNA was detected as early as embryonic day (E) 11 in the neural epithelia around the neural lumen. In late-stage embryonic development, TCGAP mRNA was detected in the whole brain. TCGAP mRNA was expressed at high levels in olfactory bulb, hippocampus, cerebral cortex, thalamus, and cerebellum until 1 week after birth. At postnatal weeks 2 and 4, TCGAP mRNA was expressed at high levels in olfactory bulb, cerebral cortex, corpus striatum, and hippocampus.

We next examined expression of TCGAP protein during postnatal development by Western blotting (Fig. 4C). TCGAP was expressed at high levels during the early postnatal period but gradually decreased after postnatal week 1. Taken together, these results suggest that TCGAP functions mainly in the immature brain.


Figure 5
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  		FIGURE 5.
Localization of TCGAP in neurons. A and B, localization of TCGAP in young (A) and mature (B) neurons. Hippocampal neurons were dissociated at E17.5, cultured for 2 (A) or 20 (B) days, and stained with anti-TCGAP antibody (P)(green), anti-synaptophysin mAb (red), and anti-MAP2 mAb (red). Scale bar, 25 µm. C, TCGAP in isolated PSD fraction. Samples (30 or 3 µg) of mouse total brain, synaptosomes, and PSD extracts were subjected to immunoblotting with antibodies against TCGAP (M) and PSD-95.

 
Subcellular Distribution of TCGAP—To examine the subcellular localization of endogenous TCGAP in neurons, 2- and 20-day-old cultured primary hippocampal neurons were stained with an antibody against TCGAP. In young neurons, TCGAP was localized throughout the cells (Fig. 5A). In mature neurons with mature synapses containing fully differentiated postsynaptic density (PSD), anti-TCGAP antibody labeled punctuate structures arrayed along dendrites, suggesting that TCGAP is localized to dendritic spines (Fig. 5B). Western blot analysis of subcellular fractions from mouse cerebral cortex showed that TCGAP was enriched in the isolated PSD fraction (Fig. 5C).

NGF-induced Tyrosine Phosphorylation of TCGAP in PC12 Cells—To address whether TCGAP is regulated by the NGF signaling in PC12 cells, we examined phosphorylation status of TCGAP. PC12 cells supply a useful system for examining mechanisms of neuronal differentiation and signal transduction (9, 44–46). PC12 cells were stimulated with NGF (50 ng/ml) for 20 min. TCGAP immunoprecipitates were prepared from NGF- and mock stimulated cells and probed with an anti-phosphotyrosine mAb. As shown in Fig. 6A, the level of tyrosine phosphorylation of TCGAP was significantly enhanced by NGF treatment. NGF activates Src family kinases downstream of TrkA (47). To examine that the NGF-induced phosphorylation of TCGAP is dependent on Src family kinases, we treated PC12 cells with PP2, a specific inhibitor of Src family kinases or PP3, an inactive analog of PP2 (48). As shown in Fig. 6B, PP2 significantly inhibited NGF-induced TCGAP phosphorylation. Together with our observation that the level of TCGAP phosphorylation was reduced in fyn-knock-out mice (Fig. 1D), these data suggest that TCGAP is phosphorylated by Fyn upon NGF stimulation.

Suppression of NGF-induced Neurite Outgrowth by TCGAP in PC12 Cells—To analyze the biological significance of TCGAP in neuronal cells, we examined the effect of TCGAP expression on neurite outgrowth of PC12 cells. Upon NGF stimulation, PC12 cells undergo neuronal differentiation accompanied by neurite outgrowth in a Cdc42-dependent manner (49). We used a retrovirus system to express exogenous wild-type TCGAP, the GAP-defective R350I mutant of TCGAP, or Y406F mutant of TCGAP in PC12 cells. After retroviral infection, the infected cells were selected by puromycin, and then stimulated with NGF for 2.5 days. Calculation of total neurite length revealed that exogenous expression of wild-type TCGAP as well as the Y406F mutant of TCGAP suppressed neurite outgrowth. The average lengths of neurites in WT and Y406F TCGAP-expressed cells were 77.9 ± 3.2% and 63.0 ± 5.3% that of mock infected cells, respectively. In contrast, expression of the R350I mutant of TCGAP enhanced neurite outgrowth (158.3 ± 4.2% that of mock infected cells) (Fig. 6C). These results suggest that Tyr-406-unphosphorylated TCGAP negatively regulates NGF-induced neurite outgrowth of PC12 cells through its RhoGAP activity. Overexpressed wild-type TCGAP may not be entirely phosphorylated by endogenous Fyn in response to the NGF stimulation, and the remaining Tyr-406-unphosphorylated TCGAP may attenuate the NGF-induced neurite outgrowth.


Figure 6
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  		FIGURE 6.
Regulation of NGF-stimulated neurite outgrowth in PC12 cells by TCGAP. A, enhanced tyrosine phosphorylation of TCGAP by NGF stimulation in PC12 cells. PC12 cells were stimulated with 50 ng/ml NGF for 20 min. TCGAP-immunoprecipitates (IP) were subjected to immunoblotting (IB) with anti-pTyr (4G10) mAb (upper) and anti-TCGAP (C) antibody (lower). B, suppression of NGF-induced tyrosine phosphorylation of TCGAP by inhibition of Src family kinases. PC12 cells were treated with PP2 (10 µM) or PP3 (an inactive analog of PP2) for 2 h. After stimulation with NGF (50 ng/ml) for 20 min, cells were lysed, and TCGAP-immunoprecipitates were subjected to immunoblotting with anti-pTyr mAb (upper) and anti-TCGAP antibody (lower). C, regulation of neurite outgrowth by TCGAP. PC12 cells were infected with retroviruses expressing mock vector, wild-type TCGAP, Y406F mutant of TCGAP, or R350I mutant of TCGAP. The infected cells were selected with puromycin and then stimulated with 50 ng/ml NGF for 2.5 days (upper). Scale bar, 25 µm. The total neurite length for ~50 cells was calculated for each construct. Data are from three independent experiments (lower). Values represent the mean ± S.E. Significance was determined by Student's t test.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies reveal that Src and Fyn play important roles in the regulation of axon growth, axon guidance, and neurite extension (50, 51). Src/fyn double-knock-out mice show defects in axon guidance and fasciculation (52). Cultured neurons lacking either Src or Fyn exhibit defects in neurite outgrowth in response to cell adhesion molecules (53, 54). Several Src/Fyn substrates, including p190RhoGAP, FAK, N-WASP, Cortactin, and Ephexin, have been shown to play important roles in neurite extension and axon guidance (9, 11, 12, 55, 56). However, precise roles of Src/Fyn in development of axons and neurites are still unclear. In the present study, we found that a GTPase-activating protein TCGAP is a novel Fyn substrate in vivo (Fig. 1). We also found that the RhoGAP activity of TCGAP toward Cdc42 is suppressed by Fyn-mediated phosphorylation (Figs. 2 and 3). Moreover NGF stimulation induced tyrosine phosphorylation of TCGAP through activation of Src family kinases in PC12 cells (Fig. 6). Given that Cdc42 is critical for axonal and dendritic initiation, elongation, and dynamics (57, 58), our results suggest that Fyn-mediated regulation of TCGAP is an essential step in neuronal differentiation.

Because TCGAP is expressed at high levels in developing brain (Fig. 3), we analyzed the functions of TCGAP in PC12 cells, which have been used as a model of neuronal differentiation (9, 44–46). Overexpression of wild-type TCGAP suppressed NGF-induced neurite outgrowth, whereas overexpression of a GAP-defective mutant of TCGAP enhanced the neurite outgrowth (Fig. 6), suggesting that TCGAP negatively regulates the neurite outgrowth. At the basal state, TCGAP is likely to suppress the neurite outgrowth by controlling the balance of distinct RhoGTPases. After NGF stimulation, the level of TCGAP phosphorylation was enhanced (Fig. 6A), which would result in suppression of its GAP activity of TCGAP toward Cdc42 (see below). Given that localized activation of Rac1 and Cdc42 is required for neurite extension in PC12 cells (49), the suppression of the TCGAP activity may be essential for NGF-induced neurite outgrowth in PC12 cells.

NGF-induced TCGAP phosphorylation was suppressed by PP2, an inhibitor of Src family kinases inhibitor (Fig. 6B). Together with our observation that the level of TCGAP phosphorylation was significantly reduced in fyn-knock-out mice, Fyn would be responsible for NGF-induced TCGAP phosphorylation. We have previously reported that the expression of constitutively active Fyn enhanced the NGF-dependent neurite extension in PC12 cells (9). Conversely, the expression of dominant negative mutant of Fyn diminished the NGF-dependent neurite extension (9). Furthermore, Fyn is activated by NGF stimulation in PC12 cells (59). All these results suggest that Fyn-mediated TCGAP phosphorylation is critical for NGF-induced neurite extension events in PC12 cells.

Accumulating data indicate that the functions of several GAPs are modulated by phosphorylation (60). Tyrosine phosphorylation of p190RhoGAP by Fyn causes a conformational change in p190RhoGAP, leading to enhancement of GAP activity and association with other molecules (11). Phosphorylation by Aurora B converts MgcRacGAP to a RhoGAP (61). The stability of RGS16 is enhanced by Src-mediated phosphorylation (62). We found that Tyr-406 in the GAP domain of TCGAP was one of the major Fyn-mediated phosphorylation sites, and the GAP activity of Y406F mutant of TCGAP was not suppressed by Fyn (Fig. 3). Phosphorylation of Tyr-406 of TCGAP may cause a conformational change of the GAP domain, leading to suppression of the GAP activity. Structural studies will uncover the precise mechanism of the suppression of TCGAP activity. Although, in addition to Tyr-406, TCGAP has several tyrosine residues that are phosphorylated by Fyn (data not shown), our data show that Tyr-406 phosphorylation critically contributes to the regulation of the GAP activity of TCGAP. Fyn-mediated tyrosine phosphorylation of the other sites may regulate the association of TCGAP with other proteins.

We identified the C-terminal region of TCGAP as a Fyn-binding region by yeast two-hybrid screening. The SH3 domain-defective mutant of Fyn did not interact with TCGAP (Fig. 1B), indicating that Fyn binds to the proline-rich sequence of TCGAP (63). Because the proline-rich sequence (1042VPRLPQK1048) at the C terminus of TCGAP is the unique typical sequence for binding with the SH3 domain of Fyn (64), Fyn may bind to this proline-rich sequence. Unexpectedly, although the SH3 mutant of Fyn did not interact with TCGAP, this mutant could phosphorylate TCGAP in HEK293T cells (Fig. 1C). Although, in the overexpression system used in this study, the interaction of TCGAP with Fyn was unnecessary for the Fyn-mediated TCGAP phosphorylation, in in vivo condition where the expression level of Fyn is thought to be much lower, the interaction of TCGAP with Fyn may be important for the efficient TCGAP phosphorylation. Alternatively, given that Fyn as well as Rho family GTPases is highly localized to the cell membrane, the interaction of TCGAP with Fyn may be required for membrane localization of TCGAP.

Although we detected the GAP activity of TCGAP toward Cdc42 in transfected cells (Fig. 2), Chiang et al. (33) failed to detect the TCGAP activity in cells. This discrepancy might be attributable to the differences in species (human TCGAP (1126 amino acids in length) versus mouse TCGAP (1305 amino acids in length)) and/or cells (HEK293T versus COS1). In addition, the expression levels of TCGAP and RhoGTPases in transfected cells may be also different.

Among RhoGAPs, p250GAP has the greatest similarity to TCGAP in terms of structure, expression pattern, and function (36, 65). The GAP domain of p250GAP has 70% identity with that of TCGAP. In addition, p250GAP is also implicated in the NGF-TrkA signaling (66). Within neurons, p250GAP is highly concentrated in the PSD and is thought to be involved in synaptic plasticity regulated by NMDA-type of ionotropic glutamate receptors (36, 65, 67). Because TCGAP is also localized in synapses and enriched in PSD in mature brain (Fig. 5), TCGAP may also be important for synaptic plasticity. Further studies are needed to clarify the precise roles of TCGAP in mature brain.

In summary, we found that a recently identified RhoGAP termed TCGAP is a novel Fyn substrate in the brain. We also found that NGF stimulation enhances tyrosine phosphorylation of TCGAP in PC12 cells, which would result in activation of Cdc42-mediated signaling pathways. Further studies on Fyn-mediated phosphorylation of TCGAP will unravel the molecular mechanisms underlying development of axons and dendrites in neurons.


   FOOTNOTES
 
* This work was supported by Grants-in-Aid for scientific research and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 81-3-5449-5301; Fax: 81-3-5449-5413; E-mail: tyamamot{at}ims.u-tokyo.ac.jp.

3 The abbreviations used are: GAP, GTPase-activating protein; CRIB, Cdc42/Rac-interactive binding domain; NGF, nerve growth factor; PSD, postsynaptic density; RBD, Rho-binding domain; SH2, Src homology 2; SH3, Src homology 3; GST, glutathione S-transferase; mAb, monoclonal antibody; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank S. Sugano for providing the human TCGAP cDNA clone, C. Sasakawa and Y. Takai for providing plasmids, and T. Inoue for providing TI-Workbench software.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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