Gtk, a Src-related tyrosine kinase, induces NGF-independent neurite outgrowth in PC12 cells through activation of t h e Rap1 pathway:

Transcription

1 JBC Papers in Press. Published on June 30, 2000 as Manuscript M Gtk, a Src-related tyrosine kinase, induces NGF-independent neurite outgrowth in PC12 cells through activation of t h e Rap1 pathway: RELATIONSHIP TO SHB TYROSINE PHOSPHORYLATION AND ELEVATED LEVELS OF FOCAL ADHESION KINASE. Cecilia Annerén 1, Kris A. Reedquist 2, Johannes L. Bos 2 and Michael Welsh 1 1.) Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. 2.) Laboratory of Physiological Chemistry and Center of Biomedical Genetics, Utrecht University, Utrecht, the Netherlands Corresponding author: Michael Welsh Department of Medical Cell Biology Box 571, Biomedicum S Uppsala, Sweden Phone: Fax: Michael.Welsh@medcellbiol.uu.se Running title: Neurite outgrowth of PC12 cells expressing mouse Gtk Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

2 SUMMARY The rat pheochromocytoma cell line PC12 is extensively used as a model for studies of neuronal cell differentiation. These cells develop a sympathetic neuron-like phenotype when cultured in the presence of nerve growth factor, NGF. T h e present study was performed in order to assess the role o f mouse Gtk (previously named Bsk/Iyk), a cytoplasmic tyrosine kinase belonging to the Src-family, for neurite outgrowth in PC12 cells. We report that PC12 cells stably overexpressing Gtk exhibit a larger fraction of cells with neurites as compared to control cells, and this response i s not accompanied by an increased ERK activity. Treatment o f the cells with the MEK-inhibitor PD98059 did not reduce t h e Gtk-dependent increased in neurite outgrowth. Gtk expression induces a NGF-independent Rap1 activation, probably through altered CrkII signaling. We observe increased CrkII complex formation with p130cas, focal adhesion kinase (FAK) and Shb in PC12-Gtk cells. The expression of Gtk also correlates with a markedly increased content of FAK, phosphorylation of the adaptor protein Shb an association between these two proteins. Transient transfection of Gtk overexpressing cells with RalGDS-RBD o r Rap1GAP, inhibitors of the Rap1 pathway, reduces the Gtk dependent neurite outgrowth. These data suggest that Gtk participates in a signaling pathway, perhaps involving Shb, FAK and Rap1, that induces neurite outgrowth in PC12 cells.

3 The rat pheochromocytoma tumor cell line PC12 is commonly used to study the signaling pathways involved in neuronal cell differentiation. These cells mature into sympathetic-like neurons upon the addition of nerve growth factor (NGF) 1 (1,2). In serum-free conditions and in the absence of NGF, PC12 cells undergo programmed cell death (3). NGF induces cell cycle arrest and differentiation by binding and activating the TrkA receptor tyrosine kinase. This causes tyrosine phosphorylation of Shc (4) and fibroblast growth factor receptor substrate-2 (FRS-2) which stimulate the extracellular-signal regulated kinase (ERK) pathway; phospholipase C-γ (PLC-γ) (4), which stimulates protein kinase C and yields an increase in intracellular calcium and; phosphatidylinositol 3-kinase (PI3-kinase) which activates Akt, to mediate neuronal survival (for a recent review see (5)). The Ras/ERK cascade has been demonstrated to be both necessary and sufficient for NGF-induced differentiation of PC12 cells (6). Activation of ERK by growth factors can trigger either cell growth o r differentiation, and a transient activation of ERK is thought to stimulate proliferation whereas a sustained activation induces differentiation (7). Although the Ras pathway is considered to be of major importance for NGF-induced ERK activation, a recent study suggested that Ras is responsible for the initial activation of ERK, whereas the sustained activation is mediated by Rap1 (8). Rap1 (9) is a Ras-family member which shares with Ras many downstream effectors, including Raf1 a n d RalGEFs. The involvement of Rap1 in NGF-induced ERK activation is controversial since it was recently shown that NGF fails to activate Rap1 in PC12 cells (10). Mouse Gtk (previously named Bsk/Iyk) (11,12) is a non-receptor protein-tyrosine kinase (PTK) belonging to the Src family of PTKs.

4 Mouse Gtk is highly homologous to human Frk/Rak (13,14) and rat Gtk (15). Little is known about the function of these tyrosine kinases and they have been suggested to be members of a subgroup within the Srcfamily with certain specific characteristics compared to the other Src family members. We have previously investigated the importance of two tyrosine residues within the tail of Gtk, namely Y497 and Y504, for NIH3T3 (16) and RINm5F cell proliferation (17). It was shown that expression of a kinase-active mutant that could enter the nucleus (Gtk Y497/504F in NIH3T3 cells, Gtk Y504F and Gtk Y497/504F in RINm5F cells) reduced the cell proliferation rate. Furthermore we observed that the Y504F- a n d Y497/504F-Gtk mutants increase the mrna levels of glucagon in the RINm5F cells. These findings raise the possibility that Gtk might b e involved in differentiation and maturation of cells. To investigate this hypothesis we have transfected PC12 cells with Gtk and studied the effects on neurite outgrowth in the absence and presence of NGF. We observe that Gtk overexpression induces NGF-independent neurite outgrowth and Rap1 activation, probably through activation of the CrkII-C3G pathway. This could be the consequence of increased levels of focal adhesion kinase (FAK) and phosphorylation of the Shb adaptor protein. EXPERIMENTAL PROCEDURES Materials- Fetal Calf Serum, Trypsin-EDTA and penicillin/streptomycin solutions were purchased from HyClone Europe Ltd. (Cramlington, UK). Dulbecco's Modified Eagle's Medium (DMEM) and Lipofectamin were from Gibco BRL (Gaithersburg, MD, USA), Geneticin, sodium orthovanadate, phenylmethylsulfonylflouride (PMSF), leupeptin, Triton

5 X-100, dithiothreitol (DTT), trypsin, ribonuclease A, Nonidet P40, horse serum and phosphotyrosine were from Sigma Chemical Co. (St. Louis, MO, USA). Immobilon-P was from Millipore Corporation (Bedford, MA, USA), horse radish peroxidase linked donkey anti-rabbit or anti-mouse IgG, enhanced chemiluminescence (ECL) detection system, Rainbow Molecular Weight Standard, Protein A/G Sepharose, glutathione Sepharose and Hyperfilm were from Amersham Pharmacia Biotech (Uppsala, Sweden). Recombinant human NGF-β was from Boehringer- Mannheim Biochemica (Mannheim, Germany). pires-egfp was from Clontech (Palo Alto, CA, USA), PD98059 was from Calbiochem (La Jolla, CA, USA), Gtk antiserum was made according to (16) and Shb antibody according to (18), anti-trka (19) was a kind gift from Louis F. Reichardt from UCSF (San Fransisco, USA), the monoclonal antiphosphotyrosine antibody (4G10) was from Upstate Biotechnology (Lake Placid, NY, USA), the anti-phospho p44/p42 MAPK antibody recognising phosphorylated Thr202/Tyr204 ERK and the anti- phospho Tyr490 TrkA were from New England Biolabs (Beverly, MA, USA), t h e anti-c3g, polyclonal anti-crkii and anti-p44/p42 MAPK antibody were from Santa Cruz (Santa Cruz, CA, USA). All other antibodies were from Transduction Laboratories (Lexington, KY, USA). Stable Transfection and Cell Culture- Wild-type PC12 cells (10 7 ) were electroporated (330 V, 1000 F) with 15 µg of Gtk cdna inserted into the expression vector pcdna3.1(-)/myc-his B or empty vector. 24 h after electroporation, 0.75 mg/ml Geneticin (G418) was added for clonal selection. Surviving clones were picked and expanded before Western blot analysis for Gtk expression. Cells were maintained in Dulbecco's Modified Eagle's Medium, DMEM supplemented with 10 % fetal calf serum (FCS), 5 % horse serum (HS) at 37 C in 5 % CO 2.

6 Neurite Outgrowth- PC12 cells were cultured for 72 h in medium with a full serum supplement with or without the presence of 20 ng/ml NGF and/or PD98059 (a final concentration of 20 µm, added 10 min prior to NGF). The percentage of cells with neurites extending two diameters of the cell body was counted every 24 hours. Western Blot Analysis on Lysates- Subconfluent cells were grown overnight in medium containing 2 % FCS and 1 % HS and stimulated with 100 ng/ml NGF for the indicated time-points. Cells were then washed with cold phosphate-buffered saline (PBS), briefly sonicated in SDS-sample buffer (containing β-mercaptoethanol and 2 mm PMSF), and subjected to Western blot analysis. The membranes were incubated with the indicated antibodies and the immunoreactivity was subsequently detected by ECL. In Vitro Kinase Assay- PC12 cells cultured to subconfluence in 10 c m dishes and maintained in 2 % FCS, 1 % HS overnight, were stimulated for 10 min. with 100 ng/ml NGF and washed with cold PBS containing 100 µm orthovanadate. Cells were lysed in 150 mm NaCl, 30mM Tris ph 7.5, 10 mm EDTA, 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 0.1 % SDS, 100 µm orthovanadate, 2 mm PMSF, 100 U/ml Trasylol (aprotinin) and 0.05 mm Leupeptin, and the nuclei removed by centrifugation. Gtk was precipitated with Gtk antibody and protein A Sepharose and the beads were thoroughly washed and subsequently subjected to an in vitro kinase reaction by incubation in the presence of 40 mm Hepes ph 7.5, 10 mm MgCl 2, 3 mm MnCl 2, 10 % glycerol, 1 mm dithiothreitol and 7µCi [γ32p]atp for 15 min. The reaction was stopped by addition of SDS-sample buffer. All solutions were supplemented with 100 µm sodium orthovanadate. The proteins were separated by SDS-PAGE and Western transfer was performed. The filters

7 were either directly exposed or blotted with phosphotyrosine (4G10) antibody. Immunoprecipitation and Immunoblotting- PC12 cells cultured t o subconfluence in 10 cm dishes were maintained in low serum as indicated above and stimulated with NGF (100 ng/ml) for the indicated time-points, thoroughly washed with cold PBS containing 100 µm orthovanadate and lysed in 300 µl lysis buffer (20 mm Hepes ph 7.5, 150 mm NaCl, 1 % Triton X-100, 10 % glycerol, 1 mm EDTA, 300 µm orthovanadate, 100 units/ml aprotinin, 2 mm PMSF, 1 mm DTT, 0.05 mm Leupeptin, 20 mm calpain inhibitor, N-acetyl-Leu-Leu-norleucinal) for CrkII, FAK, C3G, Cas and (150 mm NaCl, 0.5 % Triton X-100, 50 m M Tris ph 7.5, 1 mm EDTA, 300 µm orthovanadate, 100 units/ml aprotinin, 2 mm PMSF, 1 mm DTT, 0.05 mm Leupeptin, 20 mm calpain inhibitor N-acetyl-Leu-Leu-norleucinal) for Shb and TrkA. Lysates were clarified at xg for 10 min. at 4 C and the supernatants were incubated on ice with the indicated antibodies for 1 hour followed by incubation with protein A or G Sepharose beads for 1 hour. The beads were washed with PBS + 1 % Triton X µl SDS-sample buffer was added and the proteins were separated by SDS-PAGE followed by Western blotting with the indicated antibodies. Crk-SH2 Binding Assay- The construction, expression and purification of GST-CrkSH2 fusion protein has been described previously (20,21). PC12 cells cultured to subconfluence in 10 cm dishes were serumstarved over night and stimulated with NGF (100 ng/ml) for 10 min a n d thoroughly washed with cold PBS containing 100 µm orthovanadate and lysed in 300 µl lysis buffer (150 mm NaCl, 0.5 % Triton X-100, 50 m M Tris ph 7.5, 1 mm EDTA, 300 µm orthovanadate, 100 units/ml aprotinin, 2 mm PMSF, 1 mm DTT, 0.05 mm Leupeptin, 20 mm calpain inhibitor N-acetyl-Leu-Leu-norleucinal). Lysates were clarified at 13000

8 xg for 10 min. at 4 C and the supernatants were incubated on ice with GST-CrkSH2 fusion protein immobilized to glutathione Sepharose beads for 1 hour. The beads were washed with PBS + 1 % Triton X-100 and 5 0 µl SDS-sample buffer was added. The proteins were separated by SDS- PAGE followed by Western blot analysis for Shb and FAK. Rap1/Ras Activity Assay- PC12 cells cultured to subconfluence in 10 c m dishes were serum-starved over night and stimulated with NGF (100 ng/ml) for 10 min and thoroughly washed with cold PBS containing µm orthovanadate and lysed in 300 µl lysis buffer (1 % NP-40, 50 m M Tris ph 7.5, 20 mm MgCl 2, 200 mm NaCl, 10 % glycerol, 100 µm orthovanadate, 100 units/ml aprotinin, 2 mm PMSF, 0.05 m M Leupeptin). Lysates were clarified at xg for 10 min. at 4 C a n d the supernatants were incubated on ice with GST-RalGDS-RBD fusion protein (see (22) for details) immobilized to glutathione sepharose beads for 1 hour. The beads were washed with PBS + 1 % Triton X-100 and 50 µl SDS-sample buffer was added. The proteins were separated by SDS-PAGE followed by Western blot analysis for Rap1 or Ras. Transient Transfection- The pmt2ha-ralgds-rbd construct was generated by PCR amplification of RalGDS encoding amino acid as a Sal1/Not1 fragment and subsequently subcloned into Sal1/Not1- digested pmt2ha expression vector. The pmt2ha-rap1gap was generated by subcloning full-length Rap1GAP into the pmt2ha expression vector (according to (23)). Parental PC12 and PC12-Gtk cells cultured in 2 cm dishes were transfected with 0.3 µg pires-egfp and 2 µg RalGDS-RBD or Rap1GAP construct using 1.8 % Lipofectamin for 3 hours in 37 C in serum free medium. As control, cells were transfected with 0.3 µg pires-egfp-vector and 2 µg of carrier DNA. After 24 hours, cells were left unstimulated or treated with 50 ng/ml NGF for 48 or 2 4

9 hours and the GFP positive cells with neurites extending two diameters of the cell body were counted in a Zeiss fluorescence microscope. RESULTS Neurite outgrowth of PC12 cells expressing Gtk- To elucidate if Gtk affects the differentiation of PC12 cells we established PC12 cell lines stably overexpressing the wild-type Gtk cdna. Two clones (PC12 Gtk-7 and Gtk-10) were found to have increased intracellular levels of a 5 5 kda Gtk protein, compared with the control cells as assessed by Western blot analysis (Fig. 1A). A significant portion of the Gtkexpressing PC12 cells displayed a flattened phenotype and extended neurites when cultured in the absence of NGF. As seen in Fig. 1B, t h e Gtk-10 cells which express higher levels of Gtk than the Gtk-7 cells also show the more altered morphology, with a higher fraction of cells with neurites after 72 hours in culture as well as more somal flattening compared with the Gtk-7 cells. To investigate if NGF could induce further differentiation of these cells, a time-course experiment in response to NGF was performed. The cells were cultured for 24 h before treatment with 20 ng/ml NGF f o r another 72 hours. The Gtk-expressing cells responded to NGF, and t h e fraction of cells with neurites was significantly higher at all time-points compared with the control cells (PC12 or PC12-neo, Fig. 2). The NGFinduced increment in neurite outgrowth compared with the outgrowth before NGF-treatment was similar in the Gtk and control cells. Gtk kinase activity is not affected by NGF- To assess the activity of Gtk in PC12 cells we performed an in vitro kinase assay using PC12-Gtk10 and parental PC12 cells which were treated with NGF (100 ng/ml) for 10 min or left unstimulated. The amount of Gtk present in the lysates

10 and the total amount of tyrosine phosphorylation were determined by Western blot analysis using Gtk or phosphotyrosine antibody. [ 32 P]- incorporation into a 55 kda band was specifically detected in the immunoprecipitates from the Gtk overexpressing cells and NGF h a d little impact on Gtk autophosphorylation (Fig. 3). This 55 kda band was tyrosine phosphorylated as demonstrated by Western blot analysis with the specific phosphotyrosine antibody, 4G10. Western blot analysis o f PC12-Gtk10 lysate for phosphotyrosine also showed a strong 57 kda band just above the position of Gtk. Another protein of 125 kda was also tyrosine phosphorylated to a larger extent in the cell lysates from PC12-Gtk cells compared with the control cells. Due to the proximity o f Gtk to IgG it was not possible to assess the Gtk-levels in the immunoprecipitates but the amount Gtk present in the lysate was similar in stimulated and unstimulated PC12-Gtk cells. Wild-type PC12 cells express low but detectable levels of Gtk. NGF-independent neurite outgrowth in Gtk-expressing cells is n o t caused by the MAPK-pathway- It is well established that the MAPK (mitogen-activated protein kinase) pathway is important for differentiation of PC12 cells, and therefore we wanted to assess the activation of p42 and p44 ERK in the Gtk expressing cells. Phosphorylation of p42/p44 ERK was assessed after stimulation with 100 ng/ml NGF for 0, 2, 10 and 60 min and related to their total amounts by Western blot analysis and densitometric scannings. Addition of NGF stimulates ERK phosphorylation in both Gtk overexpressing and control cells (Fig. 4A), without an elevation of the basal phosphorylation of ERK in PC12-Gtk cells. Moreover, the NGFinduced activation is even somewhat delayed in these cells, with significantly decreased phosphorylation of both p42 and p44 ERK at 2

11 min., suggesting that the MAPK pathway is not responsible for the basal neurite outgrowth induced by Gtk. To exclude the possibility of a minor increase in ERK activation in PC12-Gtk cells we investigated if the MEK-inhibitor PD98059 could influence neurite outgrowth of PC12-Gtk. Cells were cultured as described above but in the presence or absence of PD98059 (20 µm), which was added 10 min prior to the addition of NGF (20 ng/ml), and cells with neurites were counted (Fig. 4B). PD98059 inhibited the effects on neurite outgrowth induced by NGF but could not lower the fraction of PC12-Gtk cells with neurites below that of the basal state. Thus, no correlation between ERK activation and neurite outgrowth in response to Gtk overexpression can be found, suggesting that Gtk transmits a MAPK-independent differentiation signal in PC12 cells. Gtk expressing cells exhibit elevated TrkA phosphorylation- NGFinduced differentiation of PC12 cells is mediated by the TrkA receptor which becomes phosphorylated after NGF binding. We thus wanted t o see if TrkA phosphorylation was elevated in the PC12-Gtk cells. Cells were left unstimulated or treated with NGF (100 ng/ml) for 10 min followed by TrkA immunoprecipitation using a specific TrkA antibody. Western blot analysis for phosphotyrosine was performed and t h e amount of phosphorylated TrkA was compared with the total amount present in lysate and immunoprecipitate. We observe a TrkA specific band in both lysates and immunoprecipitates corresponding to kda. In addition we detect two bands of kda displaying TrkA immunoreactivity which showed no signs of tyrosine phosphorylation. NGF stimulated TrkA phosphorylation in the control cells. The tyrosine phosphorylation of TrkA following NGF-treatment was similar in the control and Gtk overexpresssing cells, whereas the unstimulated cells from both Gtk-expressing clones exhibited more

12 tyrosine phosphorylation of TrkA compared with the control cells (Fig. 5A). Furthermore, the total amount of TrkA was lower in the PC12-Gtk cells compared with the control, suggesting an attempt to downregulate this receptor as a consequence of its constitutive activation. When TrkA binds NGF, the tyrosine at position 490 becomes phosphorylated and this generates a binding site for the adaptor protein Shc. We therefore performed Western blot analysis of equal amounts of crude lysate from cells stimulated with NGF for 10 or 6 0 min. using an antiserum specifically recognizing the tyr-490 phosphorylated form of TrkA. There is a sustained increase of tyr-490 phosphorylation after NGF stimulation in the control cells. The Gtk expressing cells, however, show an elevated basal phosphorylation of tyr-490 which cannot be increased further with NGF indicating that tyr- 490 is a phosphorylation site involved in Gtk-mediated activation. The adaptor protein Shb is phosphorylated in Gtk-expressing cell- We have previously shown that PC12 cells overexpressing the adaptor protein Shb exhibit enhanced NGF-induced differentiation, assessed as neurite outgrowth (24). It was found that NGF induced phosphorylation of p57 Shb in the overexpressing cells and therefore it was suggested that Shb could be involved in the transmission of NGF-dependent differentiation signals in PC12 cells. To determine if Gtk could influence Shb phosphorylation we immunoprecipitated Shb from extracts of cells incubated in the absence or presence of NGF and examined its degree of tyrosine phosphorylation. High amounts of phosphorylated Shb were observed in the Gtk-expressing cells (Fig. 6) whereas control cells exhibited no detectable phosphorylation despite the presence of similar amounts of Shb in all these immunoprecipitions. NGF did not exert any apparent effect on Shb phosphorylation in this experiment, which is in line with previous experiments studying Shb phosphorylation in

13 parental PC12 cells (24). When exposing the phosphotyrosine blot for a long time, a 125 kda band appeared in the immunoprecipitates (result not shown). To assess if this band could be focal adhesion kinase, FAK, the blot was stripped and reprobed with antibody against FAK, demonstrating the appearance of a 125 kda band in the Gtk-10 cells but not in the control cells (Fig. 6). PC12 Gtk- cells express elevated levels of FAK- FAK has been postulated t o play a central role in the cellular response to the extracellular matrix and for cell morphology and motility (for review, see (25)). Due to t h e Gtk-dependent FAK-Shb association we decided to study FAK tyrosine phosphorylation and expression in PC12-Gtk cells. The FAK protein levels in cell extracts were much increased in both Gtk-7 and Gtk-10 PC12 cells (Fig. 7A and B) compared with parental PC12 cells. Mock transfected PC12-neo cells express similar levels of FAK as parental cells indicating that the elevated FAK levels in PC12-Gtk cells are not caused by clonal selection (result not shown). Immunoprecipitation with anti- FAK antibody and Western blot analysis for phosphotyrosine was performed and the results show that the degree of FAK phosphorylation is increased to the same extent as the overall FAK content (Fig. 7A). The effect of FAK on the cytoskeleton may involve the binding t o p130cas. CrkII and C3G have been shown to associate with Cas (reviewed in (26)) and C3G has been identified as a guanine nucleotide exchange factor for Rap1 (for review see (27)). To determine if Gtk is involved in regulating this pathway, cell lysates were immunoprecipitated using anti-crkii, anti-cas or anti-c3g antibody and the phosphorylation and protein complex formation were analyzed (Fig. 7B-D). The expression of CrkII, Cas, and C3G was similar in Gtk expressing cells and control cells. Immunoprecipitation of CrkII from cell extracts and subsequent Western blot analysis for phosphotyrosine,

14 CrkII, Cas, FAK and C3G revealed an NGF-independent phosphorylation of CrkII and an association of FAK and Cas to CrkII to a higher extent in the Gtk expressing cells compared with the control cells. The Gtk cells exhibited augmented Cas tyrosine phosphorylation (Fig. 7C), and this might explain the increase in the association between Cas and CrkII. C3G-phosphorylation was similar in the Gtk cells compared with t h e control cells (Fig. 7D). To examine if the SH2 domain of CrkII mediates an association with Shb, we performed a pull-down experiment using a GST-Crk-SH2 fusion protein. The Crk-SH2 protein was incubated with lysates of PC12-Gtk and control cells and precipitated proteins were analyzed by immunoblotting (Fig. 7E). The phosphotyrosine blot revealed stronger bands of kda and 57 kda in t h e unstimulated Gtk overexpressing cells compared with the control cells. One possible explanation for the observed decrease of the 135 kda phosphotyrosine band after NGF stimulation could be that its binding sites for the CrkII-SH2 fusion protein are blocked by a NGF-dependent association of endogenous proteins that occurs in intact cells. Shb a n d FAK were found to bind the SH2-domain of CrkII in a Gtk-dependent manner as determined by immunoblotting with specific antibodies for Shb and FAK and the Shb band was detected at the same position as the 57 kda phophotyrosine band. Since it is known that FAK associates with p130cas directly, and since we show that Shb can bind both CrkII a n d FAK it is likely that FAK associates with CrkII via Cas or Shb. Rap1 activation is increased in Gtk expressing cells. GTP-bound Rap1 associates with high selectivity and specificity to RalGDS in vitro(28), therefore we performed a Rap1 activity assay using a GST-RalGDS-RBD fusion protein. Immunoblotting of precipitated Rap1 provides a qualitative representation of Rap1-GTP binding, corresponding t o quantitative changes detected using classical GDP/GTP-binding ratio

15 techniques (22). The relative Rap1 activation, as determined by the amount Rap1 bound to RalGDS relative the total amount of Rap1 in t h e lysate, was assessed. We also measured the amount of activated Ras bound to RalGDS-RBD by blotting for Ras. As shown in Fig. 8, Ras is activated by NGF in both PC12-Gtk and control cells to a similar degree. However, there is a significant NGF-independent increase in the activation of Rap1 in Gtk expressing cells compared with parental PC12 cells. We conclude that Gtk overexpression induces a constitutive increase in the amount of GTP-bound endogenous Rap1. Transient expression of RalGDS-RBD and Rap1GAP reduces neurite outgrowth in PC12-Gtk cells- To assess to what extent NGF-dependent activation of Rap1 in Gtk overexpressing PC12 cells is responsible for the increase in neurite outgrowth in these cells, transient transfections of RalGDS were performed. When expressed, this construct will yield a product, which contains the 97 amino-acid long Rap1 binding domain (RBD), thus associating with Rap1 and blocking its interaction with physiological downstream effectors (23). Control PC12 cells and Gtk overexpressing PC12 cells were transiently transfected with the pires- EGFP-vector alone or together with a RalGDS-RBD-construct and cultured in the absence or presence of NGF (50 ng/ml) and fluorescent cells with neurites were counted. RalGDS-RBD expression significantly decreased neurite outgrowth of PC12-Gtk cells (Fig. 9A), which h a d been cultured for two days in the absence and presence of 50 ng/ml NGF, by 53 % and 39 %, respectively, but did not affect NGF-dependent neurite outgrowth of parental PC12 cells, indicating that Rap1 activation is at least partially responsible for the differentiation caused by Gtk overexpression. The differences in neurite outgrowth between NGF-treated and untreated control- or RalGDS-RBD-transfected PC12- Gtk cells was similar, 20 % and 18 %, respectively, suggesting that NGF-

16 induced differentiation is not dependent on Rap1 activation in the Gtk cells. This is in line with a previous study showing that Rap1 is n o t activated by NGF (10). Although RalGDS-RBD can also interact with Ras, it has been reported that the RalGDS-RBD is rather specific for Rap1, with a n affinity in vitro for active Rap1 about 100-fold greater than that for active Ras (28). However, to exclude the possibility that the effects o f RalGDS-RBD were due to interactions with Ras, we also performed transient transfection of the Rap1-specific GTPase activating protein, Rap1GAP, together with EGFP and counted GFP expressing cells with neurites as described above (Fig. 9B). Expression of Rap1GAP significantly decreased neurite outgrowth of PC12-Gtk cells in t h e absence and presence of NGF, but did not affect NGF-dependent neurite outgrowth of parental PC12 cells. Thus, using two different strategies for inhibiting Rap1 signaling, these experiments demonstrate a critical role for Rap1 in mediating Gtk-induced neurite outgrowth. DISCUSSION We have previously shown that Gtk plays a role in inhibiting cell proliferation in NIH3T3 (16) and RINm5F cells (17). Furthermore Gtk also regulates hormone production in the insulin producing cell line RINm5F (17), raising the possibility that this kinase plays a role for cell differentiation. The PC12 cells are commonly employed for studies o n neuronal cell differentiation, and neurite outgrowth in response to v- Src expression sets a precedent for the possibility that other Src family members may operate in a similar fashion (29). In this study we show that wild-type Gtk is kinase active and has a great impact on NGFindependent neurite outgrowth when overexpressed in PC12 cells. This could either reflect the aberrant activation of new routes promoting

17 neurite extension or the amplification of the physiological signaling pathway of endogenous Gtk, which is expressed at very low levels. Gtk could be one member of a family of kinases which all serve a similar role. Due to the possibility of negative regulation of Gtk activity through phosphorylation of C-terminal tyrosines in PC12 cells, we also attempted to express Gtk Y504F and Gtk Y497/504F mutants, but failed t o obtain clones that would survive and divide. We presently cannot assess to what extent there is negative regulation of Gtk kinase activity in PC12 cells. However, the transfection of PC12 cells with Gtk Y497/504F resulted in single cells or clusters of cells with large flattened cell bodies and long branched neurites which ceased to grow and eventually died (result not shown). Several studies have reported that overexpression or constitutive activation of proteins that activate the Ras-MAPK pathway induce spontaneous differentiation of PC12 cells or enhance the response t o differentiation factors (i.e. TrkA (30), MEK1 (6), Ras (31), Raf (32), PKCε (33), and Crk (34)). It has been argued that NGF-induced differentiation of PC12 cells is associated with prolonged phosphorylation and activation of ERK. We did not observe any increase in ERK activation by Gtk neither in the presence or absence of NGF. Moreover, the MEK1 inhibitor PD98059, a known suppressor of NGFinduced differentiation (35), did not reduce the basal neurite outgrowth of PC12 cells overexpressing Gtk. These results suggest that the Gtk-mediated differentiation of PC12 cells is ERK-independent, although it cannot be excluded that Gtk overexpression induces a weak but prolonged ERK activation, undetectable by Western blot analysis, induced by some other factor than MEK1. Other reports of ERKindependent signals promoting neurite outgrowth in PC12 cells have been presented, i.e. the signaling through SAPK/JNK (36,37), p38 (38),

18 Shb (24) and βpdgf-r (39), suggesting other pathways that control neurite outgrowth. Rap1, a small GTPase of the Ras family (9,40), has been suggested to play a role in the sustained activation of ERK by acting through B-Raf (8,41,42) but a recent study reports that NGF fails to activate Rap1 (10) making this issue controversial. The amino acid sequences of Rap1 and Ras show about 50 % identity to each other. Due to this high sequence similarity, Rap1 can bind to Ras effector molecules, however, not always activating them. Thus, it has been suggested that Rap1 functions as a Ras antagonist, suppressing Ras-dependent signaling, including the ERK pathway (43). Several distinct pathways may transduce signals towards Rap1 activation: An increase in intracellular calcium, release of diacylglycerol (DAG), camp synthesis and activation of C3G or some other Rap1 guanine exchange factor. We investigated Rap1 activity in PC12-Gtk cells and found a significant increase in GTPbound Rap1. We also observed a partial suppression of neurite outgrowth in Gtk overexpressing cells after transient transfection with RalGDS-RBD and Rap1GAP constructs clearly indicating that Rap1 activation is required for a significant proportion of the neurite outgrowth in PC12-Gtk cells. Several pieces of evidence have been presented arguing for the CrkII-signaling pathway as responsible for this effect. Firstly, overexpression of Crk in PC12 cells has previously been reported to induce neurite formation (34). Secondly, we show a n increased association of Cas and FAK with CrkII in PC12-Gtk cells. C3G, a specific guanine exchange factor for Rap1 (44), was also present in this complex. C3G is known to bind the N-terminal SH3-domain of CrkII and as a consequence the Crk-C3G complex is translocated to the plasma membrane via binding of the Crk SH2 domain upon stimulation (reviewed in (27)). The observed binding of Cas to CrkII could serve

19 this purpose of retargeting C3G. Thirdly, we observe a markedly increased phosphorylation of Shb and an association of Shb with t h e SH2 domain of CrkII. The preferred binding site for the CrkII SH2 domain contains a proline in position three downstream of the phosphorylated tyrosine (45). There are three candidate tyrosines in Shb with a proline in this position, namely Y333, Y355 and Y384. If o n e or several of these are phosphorylated they could serve as binding sites for CrkII. We have shown that PC12 cells overexpressing Shb (24) exhibit an increased neurite outgrowth in response to NGF, and this is at least partially dependent on Rap1 activation 2. NGF induces phosphorylation of overexpressed Shb and an increased association between CrkII and an unknown phosphotyrosine protein of kda. Tyrosine phosphorylation of Shb appears to coincide with neurite outgrowth in both the Gtk and Shb overexpressing PC12 cells, being NGF-independent in the former and NGF-dependent in the latter case. The final argument in favor for a role of CrkII in Gtk-dependent Rap1 activation is the observation of an increased FAK content and its association with Shb and CrkII. Upon activation and phosphorylation in cell adhesions, FAK associates with a tyrosine kinase, i.e. Src, and this promotes the direct binding of downstream signaling proteins such a s p130cas. Phosphorylation of p130cas then allows the association of CrkII to the complex via its SH2 domain (for reviews see (27,46)). The interaction between FAK and CrkII, perhaps via Cas, observed in PC12- Gtk might be a consequence of the increased level of phosphorylated FAK and this in turn may contribute, via C3G, to the increase in Rap1 activation. Shb could possibly serve a similar role as p130cas for CrkII- C3G retargeting to the proximity of FAK since Shb interacts with both FAK and CrkII. A recent report by Altun-Gultekin and coworkers showing that v-crk expressing PC12 cells exhibit a flattened phenotype

20 with broad lammelipodia and an upregulation in the expression of FAK (47), similarly to what we observe in Gtk overexpressing cells, supports the hypothesis that FAK is involved in neurite outgrowth of PC12 cells. However, since FAK controls cellular responses to the extracellular matrix, including adhesion, spreading and migration, the phenotype observed in the v-crk and Gtk overexpressing cells may be a combination of differentiation and induced spreading. The elevated FAK levels observed in PC12-Gtk cells could be due to increased gene transcription or reduced FAK degradation by proteolytic proteins as a consequence of its activation and association with other proteins. The SAPK/JNK pathway has been suggested to play a role in transducing NGF- and ERK-independent neurite outgrowth (36,37). We therefore studied SAPK/JNK phosphorylation in PC12-Gtk and parental PC12 cells before and after NGF treatment by Western blot analysis using specific antisera against phosphorylated SAPK/JNK. We could n o t detect any increase in SAPK/JNK phosphorylation in the Gtk expressing cells making it unlikely that this pathway is involved in Gtk-mediated neurite outgrowth (results not shown). The observed increased phosphorylation of TrkA, Cas and Shb in PC12-Gtk cells could either result from the direct phosphorylation by Gtk or as a consequence of the activation of another kinase. The strong increase in Shb phosphorylation raises the possibility that Shb is a substrate for Gtk o r at least an early effector for Gtk signaling. To summarize the results in the present study we suggest the following model for Gtk induced neurite outgrowth (Fig. 10). Overexpression of Gtk causes augmented TrkA and Shb phosphorylation and FAK overexpression. Shb and p130cas associate with FAK, thus generating binding sites for the CrkII-C3G complex via the CrkII SH2-domain. This will activate Rap1 and some downstream

21 signaling pathway, such as AF6, Nore1, Krit or Ral (48-51) or perhaps some unknown pathway that induces neurite outgrowth. Acknowledgements We are grateful to Dr Dan Lindholm for introducing us to the Zeiss fluorescence microscope. The work was supported by the Juvenile Diabetes Foundation International, the Swedish Medical Research Council (31X-10822), the Swedish Diabetes Association, the Novo- Nordisk Foundation and the Family Ernfors Fund. REFERENCES 1. Tischler, A. S., and Greene, L. A. (1975) Nature 258, Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, Greene, L. A. (1978) J. Cell. Biol. 78, Obermeier, A., Bradshaw, R. A., Seedorf, K., Choidas, A., Schlessinger, J., and Ullrich, A. (1994) EMBO J. 13, Klesse, L. J., and Parada, L. F. (1999) Microsc. Res. Tech. 45, Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994) Cell 77, Marshall, C. J. (1995) Cell 80, York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. (1998) Nature 392, Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, Zwartkruis, F. J., Wolthuis, R. M., Nabben, N. M., Franke, B., a n d Bos, J. L. (1998) EMBO J. 17,

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25 Footnotes 1 The abbreviations used are: NGF, nerve growth factor; MAPK, mitogenactivated protein kinase; ERK, extracellular-signal regulated kinase; MEK, MAP kinase kinase; FAK, focal adhesion kinase; Shb, Src homology 2-domain protein of beta-cells; Cas, Crk-associated substrate; C3G, Crk SH3 binding GEF; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; RBD, Rap1-binding domain; GST, glutathione-s-transferase; EGFP, enhanced green florescence protein. 2 Lu, L., Annerén, C., Reedquist, K. A., Bos, J. L., Welsh, M., (2000) Nerve growth factor-dependent neurite outgrowth in pheochromocytoma PC12 cells overexpressing the Src homology 2-domain protein Shb requires activation of the Rap1 pathway. Submitted.

26 Fig. 1. PC12-Gtk cells extend neurites in the absence of NGF. A, Protein expression levels in PC12 cells stably transfected with Gtk cdna. B, Neurite outgrowth of PC12-Gtk7 and PC12-Gtk10 cells. The cells were cultured on glass slides for 48 h in 10% FCS and 5% HS, gently fixed in cold acetone and stained with hematoxylin and eosin (HE). Original magnification; 100 X Fig. 2. PC12 cells expressing Gtk can be further stimulated b y NGF. Parental PC12, neo-transfected control cells, Gtk-7 and Gtk-10 cells were cultured in parallel in the presence or absence of NGF ( 2 0 ng/ml) for 72 h, cells with neurites extending two diameters of the cell body were counted every 24 h. Means± SEM were calculated in each case for at least 5 independent experiments. *, P<0.05, **, P<0.01, ***, P<0.001 (vs. parental and PC12-neo cells, each tested independently). All comparisons were performed by ANOVA and Student-Newman Keuls-test. Fig. 3. Gtk kinase activity is not affected by NGF. Control PC12 cells and Gtk-10 cells cultured to subconfluence and maintained in 2% FCS, 1% HS overnight, were stimulated for 10 minutes with 100 ng/ml NGF, immunoprecipitated with Gtk antibody and subsequently subjected to an in vitro kinase reaction by incubation in the presence of 7µCi [γ32p]atp for 15 min. The reaction was stopped by addition of SDS-sample buffer. The proteins were separated by SDS-PAGE a n d Western transfer was performed. The filters were either directly exposed (top panel) or blotted with phosphotyrosine antibody (4G10). Crude lysate was analyzed for the presence of Gtk and tyrosine phosphorylated proteins.

27 Fig. 4. Basal neurite outgrowth in Gtk expressing cells is n o t dependent on ERK activity. A, Subconfluent cells were grown overnight in medium containing 2 % FCS and 1 % HS and stimulated with 100 ng/ml NGF for the indicated time-points. Cells were then washed with cold PBS, briefly sonicated in SDS-sample buffer and subjected to Western blot analysis. The membranes were first incubated with anti-phospho p44/p42 ERK antibody, then stripped and reprobed for total ERK. The p42 and p44 immunoreactive components have been indicated (right). ERK activation was measured as tyrosine phosphorylation of p44 and p42 versus total amount of ERK in t h e lysate. Columns; Means± SEM from densitometric scannings of six separate experiments. *, P<0.05, (Gtk expressing cells vs. control cells). All comparisons were performed by ANOVA and Student-Newman Keuls-test. B, Parental PC12 and PC12Gtk-10 cells were cultured in parallel with 20 ng/ml NGF only or together with 20 µm PD98059 for 7 2 hours. The inhibitor was added 10 minutes prior to NGF. Cells with neurites extending two diameters of the cell body were counted every 24 hours. Means± SEM were calculated in each case for four independent experiments. Fig. 5. The TrkA receptor is phosphorylated in PC12-Gtk cells. A, Subconfluent cells (parental PC12 cells, Gtk-7 and Gtk-10 cells) were cultured overnight in medium containing 2% FCS and 1% HS a n d stimulated with 100 ng/ml NGF for 10 min. Cells were immunoprecipitated with anti-trka antibody and subjected to Western blot analysis for phosphotyrosine (4G10) and TrkA. Total amount of TrkA in cell extracts was also assessed. B, Cells were stimulated as above with 100 ng/ml NGF for the indicated time-points. Cells were

28 lysed in SDS-sample buffer, briefly sonicated and subjected to Western blot analysis for tyr-490 phosphorylated TrkA. Fig. 6. The adaptor protein Shb is phosphorylated and associates with FAK in Gtk-expressing cells. Subconfluent cells (parental PC12 cells and Gtk-10 cells) were cultured overnight in medium containing 2% FCS and 1% HS and stimulated with 100 ng/ml NGF for 10 min. Cells were immunoprecipitated with anti-shb antibody and subjected to Western blot analysis for phosphotyrosine (4G10). The membrane was then stripped and reprobed for FAK and Shb. Fig. 7. Activation of the CrkII signaling pathway and elevated FAK levels in PC12-Gtk cells. A, Subconfluent cells (parental PC12 cells, Gtk-7 and Gtk-10 cells) were cultured overnight in medium containing 2% FCS and 1% HS and stimulated with 100 ng/ml NGF f o r 10 min. Cells were immunoprecipitated with anti-fak antibody and subjected to Western blot analysis with phosphotyrosine antibody. The membrane was then stripped and reprobed for FAK. 10% of the cell extract was subjected to Western blot analysis for total FAK. B, Cells were cultured as above and stimulated with 100 ng/ml NGF for 3 min. Cells were immunoprecipitated with anti-crkii antibody and subjected to Western blot analysis with phosphotyrosine antibody. The membrane was then stripped and reprobed for CrkII, p130cas, FAK and C3G (bottom). 10% of the cell extract was subjected to Western blot analysis for CrkII, p130cas, FAK and C3G (top). C, Cells were cultured a s in A and immunoprecipitated with p130cas antibody. The immunoprecipitates were subjected to Western blot analysis for phosphotyrosine (4G10) and p130cas. Total amount of p130cas in lysate was assessed as above. D, Cells were cultured as in A and

29 immunoprecipitated with C3G antibody. The immunoprecipitates were subjected to Western blot analysis for phosphotyrosine and C3G. Total amount of C3G in crude lysate was assessed as above. E, Control and PC12-Gtk cells were cultured as above, lysed and incubated with GST- CrkSH2 fusion protein immobilized to glutathione sepharose. The beads were washed and SDS-sample buffer was added. The proteins were subjected to Western blot analysis for phosphotyrosine (4G10), Shb and FAK. Fig. 8. Higher levels of Rap1 activation in Gtk- cell extract versus control PC12 extract. Subconfluent Gtk-10 cells and control PC12 cells cultured overnight in 1% HS and 2% FCS were stimulated with NGF (100 ng/ml) for 10 min, thoroughly washed and lysed. Lysates were clarified and incubated on ice with GST-RalGDS-RBD fusion protein immobilized to glutathione sepharose beads for one hour. The beads were washed and SDS-sample buffer was added. The proteins were subjected to Western blot analysis for Ras or Rap1 (top). The relative Rap1 activation was assessed as the amount of Rap1 bound t o the fusion protein versus total amount of Rap1 in lysate. Columns; Means± SEM from densitometric scannings were calculated from four to five separate experiments. ***, P<0.001 (Gtk-10 vs. control cells) using Student's unpaired t - test Fig. 9. Inhibition of the Rap1 pathway decreases Gtk-dependent neurite outgrowth. A, Parental PC12 and PC12-Gtk cells, cultured in 2 cm dishes were transfected with 0.3 µg pires-egfp and 2 µg RalGDS- RBD. After a 24 hour culture period, cells were left unstimulated o r treated with 50 ng/ml NGF for 48 hours. The GFP positive cells with neurites extending two diameters of the cell body were counted and

30 means± SEM were calculated from four separate experiments. *, P<0.05 (GFP vs GFP+RalGDS-RBD-transfected cells) using Student's paired t - test. B, Parental PC12 and PC12-Gtk cells, cultured in 2 cm dishes were transfected with 0.3 µg pires-egfp and 2 µg Rap1GAP. After a 24 hour culture period, PC12-Gtk/PC12 cells were left unstimulated or treated with 50 ng/ml NGF for 24/48 hours. The GFP positive cells with neurites extending two diameters of the cell body were counted and means± SEM were calculated from three separate experiments. **, P<0.01 (GFP vs GFP+Rap1GAP-transfected cells) using Student's paired t - test. n.d. indicates not determined. Fig. 10. Hypothesised model for Gtk-induced neurite outgrowth. Overexpression of Gtk in PC12 cells causes augmented TrkA and Shb phosphorylation which results in the association between FAK and Shb. FAK further binds p130cas which forms a complex with CrkII by binding via its SH2 domain. The SH3 domain of CrkII binds C3G, a guanine exchange factor for Rap1, which activates some unknown downstream effector of Rap1 that stimulates neurite outgrowth.

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