Oncogene (1998) 17, 691 ± 697  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Nerve growth factor induced stimulation of Ras requires Trk interaction with Shc but does not involve phosphoinositide 3-OH kinase

Bengt Hallberg1, Margaret Ashcroft2, David M Loeb3, David R Kaplan2 and Julian Downward4

1Department of Cell and Molecular Biology, UmeaÊ University, S-90187 UmeaÊ, Sweden; 2Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada; 3Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA; 4Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

The TrkA receptor protein tyrosine kinase is involved in of MAP kinase and do not induce di€erentiation signalling PC12 cell di€erentiation and cessation of cell (Dikic et al., 1994; Traverse et al., 1994). division in response to nerve growth factor (NGF). To Much is known about the morphological e€ects of assess the importance of adaptor proteins and Ras in NGF on neurons; however, the precise molecular NGF control of phosphoinositide 3-OH kinase (PI 3- mechanisms leading to speci®c NGF responses from kinase), speci®c receptor mutations in Trk have been the receptor during the signal transduction process are employed. We show that phosphorylation of tyrosine not completely understood. Several proteins have been 490, but not 785, of Trk is essential for activation of described that interact with Trk, such as ERK, Shc, both Ras and PI 3-kinase in vivo, correlating with PLC-g and PI 3-kinase (Greene and Kaplan, 1995). It tyrosine phosphorylation of Shc and binding of Shc to has been shown that in response to NGF, Trk is the adaptor Grb2 and the Ras exchange factor Sos. A immediately transphosphorylated at positions Y670, mutant receptor that lacks Y490 and Y785, but contains Y674 and Y675 in the catalytic domain and on sites an introduced YxxM motif which binds the regulatory Y490 and Y785 in the non-catalytic intracellular domain of PI 3-kinase, is unable to activate Ras despite domain (Stephens et al., 1994). Tyrosine 785 in Trk causing increased PI 3-kinase activity. This indicates is known to be the docking site for PLCg (Loeb et al., clearly that activation of PI 3-kinase by itself is not 1994; Obermeier et al., 1993a); activation of PLCg sucient to cause activation of Ras, arguing against a leads to hydrolysis of PIP2, giving rise to the second model in which PI 3-kinase acts upstream of Ras. The messengers IP3 and diacylglycerol. Y490 has been Shc site of Trk is thus crucial for the activation of Ras shown to bind the adaptor protein Shc upon and PI 3-kinase. stimulation with NGF (Obermeier et al., 1993b; Stephens et al., 1994), resulting in Shc phosphoryla- Keywords: Ras; Trk; NGF; phosphatidylinositol 3- tion by Trk and interaction with the adaptor molecule kinase; apoptosis; PKB Grb2. Grb2 has been implicated in activation of Ras through interaction with the guanine nucleotide exchange factor Sos (McCormick, 1993). Activated Ras plays a major role in control of downstream Introduction signalling pathways including the Raf family of proto- oncogenic serine threonine kinases which activate the TrkA encodes the nerve growth factor (NGF) receptor MAP kinase pathway, the Ral-GDS family of guanine tyrosine kinase which is involved in transmitting nucleotide exchange factors for the Ras-related protein, signals resulting in cell di€erentiation and cessation Ral, and phosphoinositide 3-OH kinases (PI 3-kinases) of cell division in response to NGF (Greene and (Marshall, 1996). Kaplan, 1995). The NGF family of neurotrophins The PI 3-kinase family is a rapidly growing family of regulate the survival and di€erentiation of neurons in 3-OH phosphoinositide lipid kinases, many of which the peripheral and central nervous systems. Upon consist of an SH2 and SH3 domain containing treatment with NGF, the rat pheochromocytoma regulatory subunit, p85, together with a catalytic tumour cell line PC12 undergoes di€erentiation from subunit, p110 (Stephens et al., 1993). The SH2 immature chroman cells to cells which resemble containing subunit associates with an appropriate sympathetic neurons. This process is characterized by phosphotyrosine containing sequence in either an extension of neurites and development of electrical activated receptor tyrosine kinase or a tyrosine excitability. Conversely, epidermal growth factor phosphorylated cellular substrate which leads to the (EGF) stimulation of PC12 cells results in DNA activation of the catalytic p110 subunit and to synthesis and proliferation. It has been demonstrated generation of phosphatidylinositol (3,4,5) trispho- that activation of MAP kinases must be sustained for a sphate (PIP3) and phosphatidylinositol (3,4) bispho- prolonged time to induce PC12 cell di€erentiation, as sphate (PI(3,4)P2). Direct interaction of p110 with Ras seen in response to stimulation with NGF, but not has been shown to contribute to growth factor induced with EGF or insulin, which cause transient activation activation of PI 3-kinase (Klinghofer et al., 1996; Rodriguez-Viciana et al., 1994, 1996). Cell survival and neurite elongation appears to be mediated partly through increased PI 3-kinase activities in PC12 cells Correspondence: J Downward Received 5 December 1997; revised 17 March 1998; accepted 17 stimulated with NGF (Jackson et al., 1996; Kimura et March 1998 al., 1994; Yao and Cooper, 1995). Trk regulation of Ras and PI 3-kinase B Hallberg et al 692 While in some cases the direct interaction of growth nnr5 Trk wt cell line generally displays responses to factor receptors with the p85 subunit of PI 3-kinase is NGF that are similar to normal PC12 cells. The nnr5 well established, in the case of Trk it is unclear how derived cell lines Y490F (490.6), Y490/785F (490/ important this is for the activation of PI 3-kinase. In 785.21) and Y785F (785.11) express amounts of normal PC12 cells treated with NGF, PI 3-kinase mutant Trk approximately three times the level found activity cannot be found in Trk immunoprecipitates in normal PC12 cells (Stephens et al., 1994). In the (Carter and Downes, 1992; Solto€ et al., 1992). In 785.11 line, phosphorylation of Shc was also observed vitro, a phosphorylated peptide corresponding to the after stimulation with NGF, as with EGF (Figure 1). site around amino acid Y751 of Trk interacts with the Stimulation of both 490.6 and 490/785.21 with NGF p85 subunit of the PI 3-kinase, although it appears that did not result in the phosphorylation of Shc. In this site is not actually phosphorylated in vivo under contrast, after stimulation with EGF, phosphorylation physiological circumstances (Baxter et al., 1995; of Shc was observed in these two cell lines. As Obermeier et al., 1993b). However, it is known that expected, under conditions where phosphorylation of NGF is able to stimulate both the formation of Shc was observed, interaction with Grb2 was also seen Ras.GTP (Nakafuku et al., 1992; Qiu and Green, (Figure 1). As Shc phosphorylation has been suggested 1991) and 3' phosphorylated phosphoinositides (Baxter to be important in the regulation of Grb2 and Sos, Shc et al., 1995; Carter and Downes, 1992) in intact PC12 immunoprecipitates were probed with antibodies cells. In order to address the mechanism of PI 3-kinase against Sos. It was possible to observe a multi-protein activation by NGF, we have employed PC12-derived complex between Shc-Grb2-Sos in Shc immunopreci- cell lines expressing only mutant forms of Trk. In this pitates from all ®ve PC12 nnr5 cell lines stimulated study we have analysed the activation status of Ras, with EGF. However, in the nnr5-derived cell lines and correlated increased levels of GTP-bound Ras with stimulated with NGF, the Shc-Grb2-Sos complex was Shc phosphorylation. Phosphorylation of Trk Y490 is observed only in those expressing wt Trk, and not seen critical for the activation of Ras by NGF in intact in the cell lines 490.6, 490/785.21 and in 785.11 (Figure cells, and also for phosphorylation of the adaptor 1). It would be expected to observe Sos in a complex protein Shc. In addition, we have measured the levels with Shc and Grb2 in cell line 785.11, given that this of PI 3-kinase product lipids in intact PC12 cells in mutant receptor has an intact Shc binding site (Y490) response to NGF treatment, and show that PI 3-kinase and Shc phosphorylation is observed in response to activation normally correlates with Ras. To distinguish NGF stimulation of this mutant. However, in our between models in which Ras acts upstream of PI 3- hands it has not been possible to see this complex kinase (Klinghofer et al., 1996; Kodaki et al., 1994; formation: this could suggest some role for Y785 in the Rodriguez-Viciana et al., 1994, 1996), or PI 3-kinase formation of stable complexes between Shc, Grb2 and acts upstream of Ras (Hu et al., 1995), a mutant Trk Sos (see Discussion). Alternatively, the level of lacking the major autophosphorylation sites, but with expression of the Trk Y785F may have dropped in an added YxxM p85 binding motif, was analysed. This the 785.11 cell line during the course of these mutant fails to activate Ras, despite fully activating PI experiments, resulting in a reduced strength of 3-kinase, indicating that physiological activation of PI activation of Shc phosphorylation; previous work 3-kinase alone is insucient to activate Ras, and arguing against a PI 3-kinase upstream of Ras model.

Results

Complex formation of Grb2 and Sos with tyrosine phosphorylated Shc after stimulation with NGF and EGF It has been shown that NGF and EGF can stimulate the phosphorylation of Shc in PC12 cells. To study the signalling pathways involved, we have made use of a cell line named nnr5 derived from PC12 cells but lacking detectable endogenous Trk expression (Hemp- stead et al., 1992; Stephens et al., 1994). A series of cell lines have been established by transfecting nnr5 cells with mutant forms of Trk to give cells expressing only Figure 1 Tyrosine phosphorylation and association of Shc mutant Trk at levels comparable with the expression of proteins with Grb2 and Sos after stimulation in PC12 nnr5 cells Trk in wild type PC12 cells (Stephens et al., 1994). To expressing di€erent mutants of Trk. (a) Tyrosine phosphorylation assess whether Shc was phosphorylated after stimula- of Shc in non-stimulated and stimulated PC12 nnr5 derivative tion of cells expressing mutant Trk with EGF or NGF, cells. Lysates of non-stimulated (7) and stimulated PC12nnr5 6 ± lysates were immunoprecipitated with anti-Shc anti- 24, wt, 490.6, 785.11 and 490/785.21 cells with 50 ng/ml EGF for 2 min (E), and 25 ng/ml NGF for 5 min (N), were immunopre- body and the immunoprecipitated proteins probed for cipitated with anti-Shc and probed with anti-PY20. (b) Grb2 anti-phosphotyrosine (Figure 1). As has been pre- association with Shc after stimulation with EGF in PC12 nnr5 viously shown in both a PC12 cell line over-expressing cells expressing wild type and di€erent Trk mutant receptors. Trk by about 20-fold (6 ± 24) and in nnr5 cells Grb2 associates with Shc after NGF stimulation only if tyrosine 490 is phosphorylated. (c) Sos associates with Shc and Grb2 after expressing wild type Trk at about three times the stimulation with EGF in PC12nnr5 6-24, wt, 490.6, 785.11 and level in normal PC12 cells (wt), Shc was phosphory- 490/785.21. Lysates were immunoprecipitated with anti-Shc and lated after stimulation with both EGF and NGF. The probed with anti-Sos Trk regulation of Ras and PI 3-kinase BHallberget al 693

Figure 2 Growth factor induced phosphorylation of the MAP kinase Erk-2 in PC12 nnr5 6 ± 24, wt, 490.6, 785.11 and 490/ 785.21 cells. Non-stimulated lysate (7) and lysate stimulated with EGF (E) or NGF (N) (as described in Figure 1) from PC12 nnr5 derivative cells were probed with anti-Erk-2

with these cells suggested that they had wild type MAP kinase responses to NGF (Stephens et al., 1994), whereas here their response is weaker than wild type Trk PC12 nnr5 cells (see Figure 2). In Grb2 immunoprecipitates from the Trk wt and the 490/785.21 cell line it was possible to observe a Figure 3 Analysis of GTP bound to Ras in PC12 nnr5 cells complex formation between Grb2 and Sos, both in expressing wild type and di€erent Trk mutants. Cell lines were cells stimulated with EGF and NGF and in non- labelled with 32P-orthophosphate and stimulated with either EGF stimulated cells (data not shown). In addition, or NGF, as described in Materials and methods. The cells were immunoprecipitation of PLCg followed by phosphotyr- then lysed, Ras was immunoprecipitated and the amount of GTP bound as a proportion of total guanine nucleotide bound osine immunoblotting revealed similar levels of determined. Data are duplicates from at least three independent phosphorylation of PLCg in response to NGF in the experiments of each cell line Trk wt and 490.6 cell lines, while 490/785.21 and 785.11 showed minimal PLCg phosphorylation (data not shown). prior to stimulation with EGF or NGF, for 2 or 5 min, respectively. After stimulation the cells were transferred MAP kinase activation is regulated by phosphorylation to 48C, lysed, and Ras proteins immunoprecipitated of both sites Y490 and Y785 on Trk with the monoclonal antibody Y13-259. After washing, Several signalling pathways act downstream of Trk. the amount of labelled nucleotide bound to Ras was One such pathway is the MAP kinase cascade analysed by thin layer chromatography. Stimulation (Marshall, 1994). We have examined lysates from with either EGF or NGF causes activation of Ras in di€erent PC12 nnr5 derivatives and analysed the PC12 nnr5 cells expressing wt Trk. Similar levels of activation status of endogenous MAP kinase. In 6 ± Ras activation can be observed in the 6 ± 24 Trk over- 24 and wt Trk cell lines, MAP kinase is activated expressing cell line (data not shown), as has been after stimulation by EGF and NGF (Figure 2). All reported previously (Basu et al., 1994). The PC12 nnr5 PC12 nnr5 derivative cell lines activate MAP kinase derivatives 490.6, 785.11 and 490/785.21 all displayed after stimulation with EGF. However, in PC12 nnr5 similar activation of Ras after stimulation with EGF cell lines 490.6, and 490/785.11, no change of MAP (Figure 3). However, stimulation with NGF caused no kinase mobility is seen compared to non-stimulated increase in levels of Ras bound GTP in the cell lines cells, indicating that Y490 phosphorylation is essential 490.6 and 490/785.21 (Figure 3). The cell line 785.11 for MAP kinase activation. Stimulation of the PC12 shows increased levels of Ras bound to GTP when nnr5, 785.11 cell line with NGF results in a MAP stimulated with NGF. However, the elevated Ras kinase shift, but this is incomplete when compared bound GTP level reached only 50 ± 65% compared to with both the Trk wt and the 6 ± 24 cell lines after the Ras bound GTP level observed in wt Trk cells stimulation with NGF, suggesting that Y785 con- stimulated with NGF. Furthermore, the 785.11 cell line tributes to, but is not essential for, MAP kinase stimulated with NGF shows less MAP kinase activation. activation compared to the wt Trk cell line (Figure 2). These data suggest that the Shc binding site Y490 is essential for activation of the Ras/MAP kinase Shc phosphorylation correlates with Ras activation: pathway, and that the PLCg binding site Y785, stimulation of PI 3-kinase alone does not cause Ras although not essential, is required for maximal activation activation of this pathway. NGF stimulation results in an increase in the level of In order to address the e€ect of activating PI 3- GTP bound to Ras (Nakafuku et al., 1992; Qiu and kinase on Ras activation, an arti®cial Trk was Green, 1991). To evaluate the contribution of various constructed in which Y490 and Y785 were mutated tyrosine phosphorylation sites in Trk for Ras to phenylalanine and the juxtamembrane KGF activation, the PC12 nnr5 cells expressing wild type sequence required for SNT phosphorylation was and mutant Trk derivatives were examined. Figure 3 removed. To this signalling de®cient Trk was added a shows the results of such an experiment in which cells YxxM motif which, when phosphorylated, acts to bind have been labelled with 32P-orthophosphate overnight the regulatory p85 subunit of PI 3-kinase. This Trk regulation of Ras and PI 3-kinase B Hallberg et al 694 receptor is able to mediate NGF induced elevation of kinase, leading to the accumulation of elevated levels

PI(3,4)P2 and PIP3 to levels similar to the wild type of PI(3,4)P2 and PIP3 lipids in PC12 cells stimulated receptor (MA, RM Stephens, BH, JD, DRK, manu- with NGF. script submitted). However, despite this, this receptor is entirely unable to activate Ras, showing that physio- PI 3-kinase activity in anti-phosphotyrosine logical levels of PI 3-kinase activity do not contribute immunoprecipitates after NGF stimulation to Ras regulation. To study the amount of PI 3-kinase activity associated with tyrosine phosphorylated proteins after NGF Trk Y490 is required for NGF stimulated PIP and 3 stimulation of cells, lysates from the nnr5-derived cell PI(3,4)P production in vivo 2 lines were immunoprecipitated with anti-p85 a and b The addition of growth factors to PC12 cells leads to a antibodies, PY20 anti-phosphotyrosine antibodies or

rapid activation of PI 3-kinase. PI(3,4)P2 and PIP3 are with control beads, and the immunoprecipitates assayed

the products of growth factor stimulated PI 3-kinase in for PI 3-kinase activity using the substrate PI(4,5)P2.As intact cells. To address the role of Trk phosphorylation shown in Figure 5, both wt Trk and the 785.11 cell lines at Y490 and Y785 in stimulation of PI 3-kinase, the show increased PI 3-kinase activity in PY20 immuno- 32 levels of PI(3,4)P2 and PIP3 were measured in P- precipitates when compared to non-stimulated cells orthophosphate labelled cell lines. After overnight after stimulation with NGF. A twofold increased metabolic labelling, cell lines were stimulated with the activity was observed. However, this is only 4% of the appropriate growth factor and the resulting phospholi- total PI 3-kinase activity observed in anti-p85 pids extracted and deacylated. Subsequently lipid immunoprecipitates from the same cell lines. Both derivatives were analysed by HPLC. EGF stimulation 490.6 and 490/785.21 lines failed to induce any PI 3- caused substantial increases in the levels of both kinase activity in anti-phosphotyrosine immunoprecipi-

PI(3,4)P2 and PIP3 in all cell lines tested to about tates after stimulation with NGF compared to non- twenty times the levels found in unstimulated cells stimulated cells (Figure 5). The PI 3-kinase activity (Figure 4). observed in anti-phosphotyrosine immunoprecipitates Less strong e€ects were observed upon stimulation resembles the in vivo lipid results described above. with NGF. Both the wt Trk and 785.11 cell lines responded with a roughly sixfold elevation of PIP 3 PKB/Akt is regulated by Ras and PI 3-kinase whereas levels when stimulated with NGF (Figure 4). In both MAP kinase is only regulated by Ras 490.6 and the double mutant 490/785.21 cell line, only

a very small increase of PIP3 or PI(3,4)P2 occurred It has been shown that PKB/Akt is activated in following NGF treatment. This experiment implicates response to stimulation with a variety of growth Y490 of Trk as a critical site for the activation of PI 3- factors in various cell types (Marte and Downward,

Figure 5 Appearance of PI 3-kinase activity, using phosphati- dylinositol (4,5) bisphosphate as a substrate, in immunoprecipi- tates from anti-phosphotyrosine PY-20 beads from PC12nnr5 6- 24, wt, 490.6, 785.11 and 490/785.21 cells. Data are from duplicates from two experiments. Background activity in control immunoprecipitates has been subtracted amd the balance expressed as percentage of PI 3-kinase activity found in duplicate anti-p85 immunoprecipitates from the same lysates. Cells were Figure 4 E€ect of NGF stimulation for 5 min on the formation non-stimulated or stimulated for 5 min with 25 ng/ml NGF of phosphatidylinositol (3,4) bisphosphate (®lled bars) and before being lysed, divided into three equal parts, immunopreci- phosphatidylinositol (3,4,5) triphosphate (open bars) in 32P- pitated with appropriate antibody and assayed as described in orthophosphate labelled PC12nnr5 cells expressing wild type Materials and methods. Phosphatidylinositol (3,4,5) trisphosphate and mutant Trks. Lipid levels are given as percentage of the EGF levels were quanti®ed using a Phosphorimager after t.l.c. induced response (2 min treatment) for each cell line separation Trk regulation of Ras and PI 3-kinase BHallberget al 695 1997). This occurs through a PI 3-kinase dependent increased phosphorylation of this activating site (data mechanism. To assess whether PI 3-kinase and Ras are not shown). able to stimulate PKB/Akt or MAP kinase in PC12 Lysates from the above experiment were then cells, and whether this stimulation is dependent on immunoblotted for endogenous MAP kinase in order activation of Ras or PI 3-kinase, we employed the to assess its activation (Figure 6). PC12 nnr5 490/ PC12 nnr5 cell line 490/785.21 and a transient 785.21 cells transfected with V-12 Ras show activation transfection system (see Materials and methods). of MAP kinase. In contrast, transiently transfected PC12 nnr5 490/785.21 cells were transiently trans- p110-CAAX was unable to activate endogenous MAP fected with V12-Ras or p110-CAAX, both of which are kinase. These results are thus consistent with recent constitutively active, together with HA-tagged PKB/ reports in other cell types, that Ras can activate PI 3- Akt. PKB/Akt was then immunoprecipitated and kinase, which in turn is sucient to activate PKB/Akt assayed for its ability to phosphorylate histone 2B in vivo in PC12 cells. However, activation of PI 3- (Figure 6). The activated PI 3-kinase, p110-CAAX, kinase is not sucient to cause activation of MAP when transfected together with PKB/Akt resulted in a kinase in these cells, again arguing against a major role 72-fold increased PKB/Akt activity when compared to for PI 3-kinase upstream of Ras. It has been cells transiently transfected with HA-tagged PKB/Akt demonstrated previously in other cell types that the alone. Oncogenic V12-Ras stimulated PKB/Akt activ- ability of Ras to cause activation of PKB/Akt is not ity approximately 25-fold (Figure 6). The speci®city of due to the production of autocrine growth factors the activation of PKB/Akt in these kinase assays was (Marte et al., 1997). con®rmed using a phospho-speci®c antibody against the phosphorylation site at serine 473: co-expression of V12-Ras or p110-CAAX with PKB/Akt results in Discussion

The ability of NGF to cause activation of Ras in PC12-derived cells expressing physiologically relevant levels of high anity NGF receptor, Trk, is shown here to absolutely require the autophosphorylation of Trk tyrosine 490, which forms a Shc binding site. When this residue is mutated to phenylalanine, NGF fails to induce Shc phosphorylation or the formation of Shc complexes with Grb2, as has been reported previously (Stephens et al., 1994), and NGF also fails to increase the amount of GTP bound to Ras. Y490 is also absolutely required for NGF induced MAP kinase activation in cells where the level of expression of Trk is close to that in normal PC12 cells. By contrast, the PLCg binding site, Y785, is not required for the tyrosine phosphorylation of Shc or the formation of Shc/Grb2 complexes, or for the activation of Ras or MAP kinase. However, mutation of tyrosine 785 to phenylalanine does reduce the level of activation of Ras and MAP kinase by NGF, and also the extent of Shc phosphorylation. A number of possible explana- tions for this may exist. Activation of protein kinase C downstream of PLCg could contribute to activation of MAP kinase through protein kinase C phosphorylation of Raf (SoÈ zeri et al., 1992). Alternatively, a protein kinase C pathway could contribute to the activation of Ras itself: while this has been reported previously in lymphocytes (Downward et al., 1990), in most cell types PKC activation is not sucient to cause Ras activation. However, in PC12 cells activation of PKC by phorbol esters has been shown to result in elevation of GTP levels bound to Ras (Nakafuku et al., 1992). It Figure 6 Activation of PKB/Akt and Erk-2 by Ras or is therefore possible that Y785 may contribute to membrane bound p110 protein in the double mutant cell line, PC12nnr5 490/785.21. (a) Transiently transfected HA-tagged optimal Ras activation through a PKC mediated PKB/Akt activity, as ability to phosphorylate histone H2B, was pathway. Another possibility is that phosphorylation measured in the PC12 derivative cell line co-transfected with of Y785 may be needed for optimal phosphorylation of vector alone, V12-Ras or with p110-CAAX. (b) PKB/Akt activity Shc and formation of Shc/Grb2 complexes; from was determined as described in Materials and methods. Average Figure 1 this does appear to be the case, at least in activation from three transfections are shown as fold activation of PKB/Akt transfected alone. (c) Western-blot analysis of the part, although the mechanism involved is unclear. A immunoprecipitations showed equal expression levels of PKB/ PKC-dependent pathway could perhaps normally Akt. (d)Western-blot analysis of endogenous Erk-2 phosphoryla- suppress tyrosine phosphatase activities which reverse tion, seen after co-transfection of PKB/Akt with V12 Ras, but not Shc phosphorylation. Recently, a new docking protein seen with PKB/Akt vector alone or when co-transfected with P110-CAAX. (e) Western blot with anti-Ras antibodies showing has been characterized (Kouhara et al., 1997), FRS2, expression of V12 Ras in the transfected cells that is phosphorylated on tyrosine residues in response Trk regulation of Ras and PI 3-kinase B Hallberg et al 696 to FGF or NGF stimulation. FRS2 is thought to be induced by NGF stimulation of PC12 cells might be closely related, but probably not identical to, SNT able to directly in¯uence the activation state of Ras, a (Rabin et al., 1993). Upon NGF stimulation of Trk in mutant form of Trk which lacked the ability to PC12 cells, FRS2 becomes tyrosine phosphorylated autophosphorylate at Y490 or Y785 and also lacked a and binds to Grb2/Sos. It has been suggested that this juxtamembrane motif implicated in induction of could form an alternative pathway to activation of Ras neuronal di€erentiation (KGF, (Peng et al., 1995)) by NGF. However, data in Figure 3 indicates that the but contained an arti®cial p85 binding site, was presence of SNT phosphorylation in the absence of Trk expressed in nnr5 cells (Y490F/Y785F/ Y490 phosphorylation is not sucient to give Ras DKGF+YxxM at L605). This receptor is able to

activation by physiologically relevant levels of Trk. induce increases in the level of PIP3 and PI(3,4)P2 in The e€ect of mutating Y490 and Y785 on the response to NGF which are comparable to the wild induction of PI 3-kinase activity by Trk in intact cells type receptor as a consequence of the stable binding has been previously reported (Baxter et al., 1995). of p85 to the mutant Trk (MA, RM Stephens, BH, Using chimeric PDGF receptor/Trk molecules ex- JD, DRK, manuscript submitted). This suggests that pressed in PC12 cells, it was shown that Y490 was Ras activation may be most important in the

absolutely required for the elevation of PI(3,4)P2 by regulation of PI 3-kinase in cases where there is no PDGF. Our data con®rm the central role of Y490 in PI direct interaction between the growth factor receptor 3-kinase activation using whole Trk molecules rather and p85. However, this mutant of Trk, which lacks than chimeras, and also show that Y785 does not play the ability to bind or phosphorylate Shc, is completely an essential role in PI 3-kinase activation. Similarly to without e€ect on the activation state of Ras and Baxter et al. (1995) we also ®nd that in the case of MAP kinase (Figure 3). In addition, expression of an Y490F Trk, considerably reduced PI 3-kinase activity activated form of the p110a PI 3-kinase, p110-CAAX, is found in anti-phosphotyrosine immunoprecipitates fails to induce activation of MAP kinase (Figure 6) from NGF stimulated cells. It is unclear how the and fails to elevate Ras.GTP levels (S WennstroÈ mand appearance of PI 3-kinase activity in anti-phosphotyr- JD, unpublished observations). It therefore appears osine immunoprecipitates correlates with the level of PI that the levels of PI 3-kinase activity induced 3-kinase product lipids in whole cells: the maximal following stable association of p85 with a receptor amount of PI 3-kinase activity found in anti- tyrosine kinase, and by comparison also wild type phosphotyrosine immunoprecipitates is only 4% of Trk, are quite unable to induce Ras activation: it is that which can be detected in anti-p85a immunopreci- thus very unlikely that PI 3-kinase is normally able to pitates. Despite reports of an inducible association act upstream of Ras in physiological growth factor between p85 and Shc in Bcr-Abl transformed chronic mediated signalling pathways, at least in this cell type. myelogenous leukemia cells (Harrison-Findik et al., 1995) and constitutive association between p85 and Grb2 in ®broblasts (Wang et al., 1995), we were unable Materials and methods to detect PI 3-kinase activity in Shc immunoprecipi- tates (data not shown). It has been suggested that a Cells, growth factors, antibodies and plasmids 115 kDa tyrosine phosphoprotein of unknown identity can associate with p85 following NGF treatment of All PC12-derived nnr5 cell lines expressing wild type and PC12 cells (Ohmichi et al., 1994): this may account for mutant Trk have been described elsewhere (Stephens et al., 1994). Trk def (Y490F/Y785F/DKGF) and Trk PI 3-kinase activity in anti-phosphotyrosine immuno- def+YxxM (Y490F/Y785F/DKGF+YxxM at L605) nnr5 precipitates. Since Ras is able to interact directly with, cell lines are described in MA, RM Stephens, BH, JD, and stimulate, the catalytic subunit of PI 3-kinase DRK, manuscript submitted. Cells were grown in RPMI (Rodriguez-Viciana et al., 1994, 1996), the failure of 1640 containing 10% heat-inactivated horse serum (JRH Trk Y490F to stimulate PI 3-kinase activity may Biosciences) and 5% fetal bovine serum. The monoclonal simply be due to its inability to activate Ras. anti-phosphotyrosine antibody, PY-20, and antibodies However, the loss of PI 3-kinase activity in anti- against Sos, Erk and Grb2 were obtained from Transduc- phosphotyrosine immunoprecipitates from Trk Y490F tion Lab. Pan-Ras and anti-Cbl were from Oncogene expressing cells but not from PC12 cells expressing Science and Santa Cruz, respectively. Monoclonal anti-p85 dominant negative Ras (Rodriguez-Viciana et al., 1994) was from MD Water®eld. Polyclonal anti-human Shc and EGF were purchased from UBI, Lake Placid. NGF 2.5S suggests that a second pathway contributing to PI 3- was from Boehringer Mannheim. HA-PKB/Akt (from B kinase activation by NGF but not involving Ras is also Burgering and P Co€er, University of Utrecht) was controlled by phosphorylation of Y490. Activation of transiently transfected together or alone with plasmids PI 3-kinase in whole cells is therefore likely to be encoding p110-CAAX or V12 Ha-Ras (Marte et al., 1997) synergistically triggered by Ras interaction with p110 using the Lipofectamine kit from Gibco/BRL as described and tyrosine phosphoprotein interaction with p85; by manufacture. evidence for this has also emerged from other systems (Klinghofer et al., 1996). Measuring Ras.GTP levels in intact cells While several lines of evidence suggest that PI 3- kinase is a downstream e€ector of Ras (Khwaja et al., The activation state of Ras in PC12 nnr5 cell lines metabolically labelled with 32P-orthophosphate was deter- 1997; Klinghofer et al., 1996; Kodaki et al., 1994; mined as described previously (Hallberg et al., 1994). Rodriguez-Viciana et al., 1994, 1996), there have also Immunoprecipitation and immunoblotting were performed been less direct indications that PI 3-kinase might be as previously described (Basu et al., 1994), except that anti- able to act as an upstream regulator of Ras under Shc and anti-p85 antibodies were cross-linked to protein A some circumstances (Hu et al., 1995). In order to beads (BioRad) with dimethylpimelimidate (Sigma) investigate whether the levels of PI 3-kinase activity (Harlow and Lane, 1988). Trk regulation of Ras and PI 3-kinase BHallberget al 697

Measuring phosphoinositides levels in vivo (25 mM Tris, pH 7.5, 4 mM MgCl2 and 0.4 mM EGTA). 10 ml kinase bu€er 2 (250 mM PI(4,5)P (Sigma) resus- 80% con¯uent cells were incubated overnight in 500 mCi 2 pended in 3% cholate and 150 mM Tris, pH 7.5) was added [32P]orthophosphate (Amersham) in phosphate free RPMI to each reaction. The reactions were started by addition of 1640. Lipid extraction from cells and HPLC analysis was 10 ml containing 10 mCi [g-32P]ATP/reaction (Amersham) performed as described (Rodriguez-Viciana et al., 1994). and cold ATP (®nal concentration, 1.25 mM). Reactions were stopped by addition of chloroform:methanol:11.6 M Kinase assays HCl (50 : 100 : 1) after 10 min. Phosphorylated product were separated by t.l.c. and quanti®ed using a Phosphor- PKB/Akt kinase activity was assayed in vitro (Burgering imager (Molecular Dynamics). and Co€er, 1995) using Histone H2B (Boehringer Mannheim) as a substrate. Phosphorylated proteins were detected by Phosphorimager (Molecular Dynamics) and correlated against the amount of PKB/Akt protein Acknowledgements (determined by immunoblotting using enhanced chemi- Thanks to Boudewijn Burgering and Paul Co€er for the ¯uorescence and detection using a Storm 860 system). In HA-PKB contruct, and to Patricia Warne, Pablo Rodri- vitro PI 3-kinase assays were essentially performed as guez-Viciana, Stefan WennstroÈ m and Barbara Marte for described previously (Arvidsson et al., 1994) with the advice and assistance. This work was supported by the following modi®cations: the washed immobilized immune Swedish Cancer Society and by the Imperial Cancer complexes were resuspended in 20 ml kinase bu€er 1 Research Fund, UK.

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