Identification of in vivo brain-derived neurotrophic factor-stimulated ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 48, Issue of December 2, pp. 3037030377, 1994 Printed in U.S.A.

Identification of in Viuo Brain-derived Neurotrophic Factor-stimulated Autophosphorylation Sites on the TrkB Receptor Tyrosine Kinase by Site-directed Mutagenesis*

(Received for publication, June 17, 1994, and in revised form, September 9, 1994)

Michelle GuitonSP, Frank J. Gunn-Moore8, Trevor N. Stittn, George D. Yancopoulosn, and Jeremy M. TavarBSIl From the rnepartment of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 lTD, United Kingdom and llRegeneron Pharmaceuticals Inc., Tarrytown, New York 10591-6707

Brain-derived neurotrophic factor (BDNF’) interacts with the TrkB receptor tyrosine kinase, the tyrosine kinase domain of which has homology with the insulin receptor subfamily of protein kinases. This includes the conservation of three regulatory tyrosines (residues 670, 674, and 675) known to play a crucial role in signal trans- mission by the insulin receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be important in phosphatidylinositol 3-kinase binding (Ober- meier, A, Lammers, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. BioZ. Chern. 268, 22963-2296611, and K540R (consensus ATP binding lysine) substitutions were transiently expressed in COS cells for analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells stably expressing wild-type TrkB. In addition, the mutants were expressed in Chinese hamster ovary cells for analysis of the ability of the receptor to mediate BDNF-stimulated transcription from a 12-0-tetradecanoylphorbol-13-acetate response element (TRE). BDNF stimulated the phosphorylation of wild-type TrkB on multiple tyrosine and serine residues. This phosphorylation occurred on tyrosines 670,674, and 675 plus two other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-dependent transcription. A Y674F/Y675F, but not Y670F, substitution dramatically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphorylation at all sites but mediated a normal BDNF- stimulated activation of a TRE. Our results demonstrate a critical role for the phosphorylation of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type TrkB.

The neurotrophic factors have received a great deal of recent interest with respect to their potential use in the treatment of a number of neurodegenerative disorders (Barinaga, 1994). At the molecular level, however, we only have a rudimentary un- derstanding of their mechanism of action. The neurotrophic factors are a family of related polypeptides and include nerve

* This work was supported by the Wellcome Trust. 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.

5 Recipient of a Medical Research Council studentship. 11 A British Diabetic Association Senior Research Fellow. To whom

correspondence should be addressed. Tel.: 44-272-288273; Fax: 44-272- 288274.

growth factor, brain-derived neurotrophic factor (BDNF),’ and neurotrophins-3, -4, and -5 (NT-3, NT-4/5, respectively) all of which promote neuronal differentiation and prevent apoptosis (Glass and Yancopoulos 1993). The receptors to which these neurotrophins bind have recently been cloned. While all neurotrophins appear to bind a common low affinity receptor (p75), the likely signal transducing component of the receptors are the transmembrane tyrosine kinases TrkA (binds nerve growth factor), TrkB (binds BDNF, NT-3, and NT-4/5), and TrkC (binds NT-3) (for reviews see Chao (1992), Meakin and Shooter (1992), and Glass and Yancopoulos (1993)). Studies with transgenic mice demonstrate a central role for the Trks in development of the nervous system (Klein et al., 1994; Smeyne et al., 1994), but similar studies with the p75 component suggest that this protein plays a more subtle and as yet largely unknown role in neurotrophin action (Lee et al., 1992).

Receptor tyrosine kinases appear to employ two mechanisms for initiating the growth factor signal. In the case of the receptors for epidermal growth factor (EGF) and platelet-derived growth factor for example, ligand-stimulated autophosphorylation of the receptor leads to the binding, via SH2 domains, of several downstream effector molecules directly to specific tyrosine phosphorylation sites within the receptor (e.g. PtdIns 3- kinase, phospholipase Cy, and Grb2) (Panayotou and Water- field, 1993). These effector molecules become activated and con- sequently further transmit the signal into the cell. The insulin and insulin-like growth factor receptors, on the other hand, are autophosphorylated on multiple tyrosines in response to ligand, but these tyrosines do not act directly as docking sites for effector molecules; this is despite the fact that one site in the insulin receptor (tyrosine 1334) has a YXXM motif, which in other receptors is capable of interacting with the p85 subunit of PtdIns 3-kinase. Instead these receptors appear to phosphorylate other substrates, the best characterized of which is insulin receptor substrate-1 (White and Kahn, 1994). Insulin receptor substrate-1 is phosphorylated on multiple tyrosines in response to insulin, and it is these tyrosines that then act as specific binding sites for the SH2 domains of PtdIns 3-kinase, Grb2, SHPTP2, and Nck (White and Kahn, 1994).

The mechanism by which the Trk family of receptors initiates its specific signal is largely unknown. PtdIns 3-kinase activity and immunoreactive p85 subunits have been found associated with TrkA (Soltoff et al., 1992) and with a chimera composed of an EGF receptor ligand binding domain fused to a Trk tyrosine kinase domain from an actin-Trk fusion oncogene (“EGFR-onc- T r k chimera (Obermeier et al., 1993a)). Others have failed to

tor; PtdIns 3-kinase, phosphatidylinositol 3-OH kinase; Ik”, mitogen- The abbreviations used are: BDNF, brain-derived neurotrophic fac-

activated protein; TRE, 12-0-tetradecanoylphorbol-13-acetate response element; SH2, src-homology 2; NT, neurotrophin; EGF, epidermal growth factor; PAGE, polyacrylamide gel electrophoresis.

30370

DkB Autophosphorylation Sites in Vivo 30371

detect any such association (Ohmichi et al., 1992). The amino acid sequence of Trk reveals a single YXXM p85 binding motif in the kinase domain at tyrosine 751. When mutated to phenylalanine in the EGFR-oncTrk chimera, the p85 subunit failed to bind (Obermeier et al., 1993a).

Other proteins that associate with the Trk family receptors are SHC (probably at tyrosine 484 of TrkB and tyrosine 490 in EGFR-oncTrk (Obermeier et al., 1993a)), phospholipase C y (probably at tyrosine 785 (Obermeier et al., 1993b)), and the MAP kinase ERKl (Loeb et al., 1992). Grb2 does not appear to associate with Trk (Suen et al., 1993).

The Trk tyrosine kinases lie in the insulin receptor subfamily of tyrosine kinases (Hanks et al., 1988). The insulin receptor is phosphorylated on tyrosines 1158,1162, and 1163, which reside in subdomain VI1 between the conserved DFG and APE found in almost all protein kinases (Hanks et al., 1988). All members of the "rk family possess the three tyrosines in homologous positions to insulin receptor tyrosines 1158, 1162, and 1163; indeed the sequence surrounding these tyrosines is almost to- tally conserved. In the insulin receptor phosphorylation of these tyrosines plays a critical role in the regulation of exogenous kinase activity (see Tavare and Siddle (1993) for a review). This suggests that one or more of these three tyrosines in the neurotrophic factor receptors may play an analogous regulatory role. A recent study by Middlemas et al. (1994) suggested that these tyrosines (residues 670,674, and 675) may be phosphorylated in TrkB; however, this conclusion was based indi- rectly on the co-migration of synthetic phosphopeptides with authentic tryptic peptides from 32P-labeled TrkB.

In the present study we sought to directly identify the sites of autophosphorylation of TrkB in intact cells by site-directed mutagenesis and explore their possible role in signaling by this receptor.

EXPERIMENTAL PROCEDURES Material~-[~~PlOrthophosphate and enhanced chemiluminescence

(ECL) reagents were fromhersham International (Amersham, United Kingdom). Synthetic phosphopeptide (IPVIENPQY(P)FGITNSQLK) was from Zinsser Analytic (Maidenhead, U. K.). Monoclonal anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology, Inc. (Lake Placid, NY), nitrocellulose from Schleicher & Schuell, Immobilon-P from Millipore Corp. (Watford, U. K.), and sequencing grade trypsin from Boehringer Mannheim (Lewes, U. K.). MG86-TrkB cells are an NIH 3T3 cell variant stably expressing the rat TrkB cDNA (Glass et al., 1991; Ip et al., 1993). Unless otherwise stated all other biochemicals were supplied by the Sigma Chemical Company (Poole, U. K,), and general purpose laboratory reagents were of analytical grade and from BDH (Poole, U. K.).

Preparation ofdntibodies-Rabbit polyclonal antibodies were raised against a keyhole limpet hemocyanin conjugated synthetic peptide corresponding to the last 14 amino acids of human TrkA (CALAQAP- PVYLDVLG; synthesized by the University of Bristol Molecular Recog- nition Center). This antibody (pan-Trk) is cross-reactive with TrkA, TrkB, and TrkC.' A second rabbit polyclonal antibody (crTrkBeetD) was raised to the extracellular domain of rat TrkB generated by amplifylng the region corresponding to amino acids 1-396 of rat TrkB by polymerase chain reaction (using primers 5'-T"TTAGGCCCCCTTGCCCCAT- GTCCTGC-3' and 5'-TT'NTCTAGACTACCGATTGGTTTGGTC-3'). This was subcloned into the pMAL' bacterial expression vector (New England Biolabs, Inc.), thereby fusing the extracellular portion of TrkB to the Escherichia coli maltose binding protein. This fusion protein was overexpressed and purified, and rabbits were immunized by subcuta- neous injection. Western blot analysis demonstrated that this antiserum was highly specific for TrkB with no detectable reactivity toward T r k A . 3

plasmid pBSK--trkB (rat TrkB sequence cloned into Bluescript SK- Site-directed Mutagenesis-In vitro mutagenesis was performed on

(Stratagene)) as outlined in "Muta-Gene Phagemid In Vitro Mutagen- esis Version 2" (Bio-Rad) with the following primers: 5"CGGGATGTAT-

F. J. Gunn-Moore, M. Guiton, and J. M. Tavare, unpublished data, F. J. Gunn-Moore and J. M. Tavare, unpublished observations.

TCAGCACCGAC-3' for introducing a Y670F mutation; 5"AGCAC- CGACTTCTTCCGGGTTGGT-3' for a Y674FN675F mutation; 5'-CAG- GAGGTGTTCGAGCTGATG-3' for a Y751F mutation; and 5"CTGGC- CGTGAGGACGCTGAAG-3' for a K540R mutation. Wild-type and mutant receptor cDNAs were then subcloned into the mammalian expression vectors pECE (Ellis et al., 1986) and pcDNAIneo (Invitrogen), and the entire region containing the mutated residues was resequenced.

Expression of DkB Receptors in COS Cells by Dansient lhnsfection and Western Blot Analysis-COS cells were transfected with 5 pg of TrkB DNA (in the pECE vector) using DEAE-dextran as described (Gluzman, 1981). 48 h after transfection the cells were serum-starved for 2 h and extracted, and TrkB was immunoprecipitated using 2.5 mg of protein A-Sepharose and 1:lOO dilution of anti-TrkB antibodies a t 4 "C for 2 h as previously described for the insulin receptor (Tavare et al., 1991). Samples of crude cell lysates or immunoprecipitates were run on 6% SDS-PAGE gels, and the proteins were electrophoretically transferred to Immobilon-P. Western blotting was performed using the ECL system (Amersham International) as described (Tavare et al., 1991), using 1:500 dilution of pan-TrkB serum or 1 pg/ml 4G10 as primary antibodies.

Analysis of DkB Receptor Phosphorylation Sites in Intact Cells-This was a modification of the method described by Tavare et al. (1991). COS cells transfected with TrkB constructs and CHO.T, which overexpress the insulin receptor, or MG86-TrkB cells (-80% confluent) were meta- bolically labeled with l mCi of [32P]orthophosphate/dish as described (Tavare et al., 1991). Cells were incubated with or without ligands for 5 min prior to extraction and precipitation of 32P-labeled TrkB or insulin receptors with pan-Trk ( 1 : l O O dilution) or anti-insulin receptor antibody 83.14 ( 1 : l O O dilution (Soos et al., 198611, respectively, as described (Tavare et al., 1991).

Two-dimensional Phosphopeptide M~pping-~~P-Labeled receptors were separated by SDS-PAGE using a Miniprotean I1 system (Bio-Rad). Proteins were transferred onto nitrocellulose, and the location of labeled receptors was detected using a PhosphorImager (Molecular Dynamics). Membrane squares containing the receptors were excised and treated with 0.5% (w/v) PVP-40 in 100 mM acetic acid for 1 h a t 37 "C. Membranes were then washed extensively using distilled water. The protein was digested and eluted from the membrane in 100 mM NH,HCO,, pH 8.2, containing 5% (v/v) acetonitrile and 5 pg of trypsin a t 37 "C for 16 h. A further 5 pg of trypsin was added for an additional 24 h.

Phosphopeptides were separated on thin layer chromatography plates as described by Tavare and Denton (1988) except that electrophoresis was allowed to proceed for 2.5 h. The phosphopeptides were detected using a PhosphorImager. Phosphoamino acid analysis was performed as described (Cooper et al., 1983) with the constituent phosphoamino acids separated at pH 3.5 on cellulose chromatography plates.

Zhznscription Assays-The plasmid pCol.CAT, containing the 580- base pair 5' region (-517 to +63) of the human collagenase (Col) promoter upstream of chloramphenicol acetyltransferase cDNA, was gen- erously provided by Dr. R. Medema (University of Utrecht, The Netherlands). The collagenase promoter was amplified by polymerase chain reaction using pCol.CAT as the template via standard techniques, and the resulting 602-base pair fragment was subcloned into the luciferase vector, pGL2-basic (Promega, Madison, WI). This gave the plasmid pCol.Luc, which possesses the firefly luciferase gene under the control of the growth factor-responsive collagenase promoter. pSV2.CAT, containing the SV40 promoter upstream of the chloramphenicol acetyltransferase gene, was provided by Dr. A. Beckett (Department of Pathology, University of Bristol).

1 x lo6 CH0.T cells were seeded into 100-mm diameter Petri dishes in Ham's F-12 medium (Gibco, Paisley, U. K.) supplemented with 5% (v/v) bovine fetal calf serum, 10 rn Hepes, 200 unitdm1 benzylpenicil- lin, 100 pg/ml streptomycin, and 250 pg/ml G418. After 24 h, cells were transfected for 90 min with 3 pg of TrkB cDNA (and mutants in pcDNAIneo), 4 pg of pCol.Luc, 4 pg of pSV.CAT, and 250 pg/ml DEAE-dextran in 3 ml of serum-free Ham's F-12 medium. Cells were then incubated for 4.5 h in the same medium (minus DEAE- dextran and DNA) supplemented with 5% (v/v) fetal calf serum plus 100 p~ chloroquine and subsequently incubated for 16-20 h in this medium a t 0.5% (v/v) serum without chloroquine. After preincubation for 1 h in serum-free medium, insulin (100 nM) or BDNF (100 ng/ml) was added, and the culture was continued for a further 24 h. The cells were har- vested and assayed for chloramphenicol acetyltransferase (Nielsen et al., 1989) and luciferase activity (using a commercially available kit; Promega). The ratio of luciferase activity (photons/pg of protein) to chloramphenicol acetyltransferase activity ( d p d p g of protein) provided a specific measure of collagenase promoter activity corrected for varia- tions in transfection efficiency.

30372 D k B Autophosphorylation Sites in Vivo IPVIENPOYFGITNSQLK TCPOEWELMLGCWOR

Y4E4 K M O Y F ~ O W ~ W ~ y m ~ 7 8 5

FIG. 1. Schematic representation of TrkB and the position of the mutations employed in this study. The figure shows a linear representation of the TrkB polypeptide. Indicated are the potential phosphorylation sites (O), transmembrane sequence (solid box), and tyrosine kinase homology region ( h e a d y shaded).

RESULTS

A rat TrkB cDNA was used throughout these studies (for sequence see Middlemas et al. (1991)) and was subcloned into either pECE (for expression in COS cells) or pcDNAIneo (for expression in CH0.T cells). We introduced into this cDNA several point mutations (Fig. 1). These included a K540R substitution (the residue corresponding to the consensus lysine found at the active site of all protein kinases (Hanks et al., 198811, single Y670F and Y751F substitutions, and a Y674FN675F double substitution. The entire fragment used as template for mutagenesis was completely resequenced to confirm the absence of spurious mutations.

Expression of n-kB and Its Mutants in COS Cells-The wild- type TrkB and various mutants were transiently transfected into COS cells. 48 h post-transfection the cells were extracted, and the crude cell lysates were analyzed by Western blotting with pan-Trk and anti-phosphotyrosine antibodies. As shown in Fig. 2a the expression level of wild-type and mutant TrkBs was similar in all cell transfections (TrkB runs with an apparent M, of - 120,000 in COS cells). Anti-phosphotyrosine antibody blotting revealed that wild-type TrkB was highly phosphorylated in the absence of added BDNF (Fig. 2b, lane 2). Addition of BDNF or NT-3 (both a t 50 ng/ml) to the cells for 5 min had no apparent effect on the extent of reactivity of the anti-phosphotyrosine antibody with TrkB (data not shown). This is in contrast to insulin receptors expressed in COS cells, which exhibited a marked insulin stimulation of tyrosine phosphorylation of the 0-subunit as judged using this technique (data not shown). This suggests that TrkB is constitutively phosphorylated in COS cells in the absence of added ligand (see also below).

A number of other tyrosine-phosphorylated proteins were observed in the anti-phosphotyrosine Western blots, of apparent M, -100,000, -80,000, -75,000, and -42,000 (Fig. 2b). The level of tyrosine phosphorylation of these bands roughly paralleled the level of TrkB tyrosine phosphorylation. We cannot exclude the possibility that these represent proteolytic fragments of TrkB; however, this is unlikely as we observed no low molecular weight 32P-labeled fragments in pan-Trk precipitates from 32P-labeled cells (see below). I t is likely, therefore, that some of these bands may represent intracellular substrates for TrkB (e.g. PtdIns 3-kinase p85 subunit). The M, 42,000 phos- phoprotein may be the MAP kinase isoform ERK2.

Anti-phosphotyrosine blotting of extracts from mock transfected cells revealed a small band just above wild-type TrkB (Fig. 2b, lane 1; see further discussion below). Introduction ofthe Y670F mutation had little apparent effect on the level of tyrosine phosphorylation (Fig. 2b, lane 3 ) . However, the Y674F/ Y675F double substitution and the single K540R mutation dramatically reduced tyrosine phosphorylation of TrkB (Fig. 2b, lanes 4 and 6; note that the “nonspecific” band just above TrkB was unaffected). The Y751F mutation caused a dramatic -90% reduction in tyrosine phosphorylation of TrkB (Fig. 2b, lane 5; this result was confirmed using a separate clone of this plasmid).

no-dimensional Phosphopeptide Mapping and Phos- phoamino Acid Analysis of DkB Expressed in COS Cells-The extent of 32P labeling of the receptor mutants almost exactly

a aTrk

”

1 2 3 4 5 6

205- 1 116-1

80-

49-

-TRKB

“TRKB

1 -1

1 2 3 4 5 6 FIG. 2. Expression and phosphorylation of TrkB and its mu-

tants in COS cells. COS cells were mock transfected (lane 1 ) or transfected with wild-type (lane 2), Y670F (lane 3 ) , Y674FR675F (lane 4 ) , Y751F (lane 5 ) , and K540R (lane 6) mutants. After 48 h, the cells were extracted and solubilized in sample buffer prior to separation by SDS- PAGE, Western blotting with pan-Trk antibody (panel a, aZM) or anti- phosphotyrosine antibody (panel h, CrPY), and detection by ECL. The migration of the molecular weight markers (~10.~ kDa) is indicated.

paralleled their reactivity with anti-phosphotyrosine antiserum (compare Fig. 3a with Fig. 2b). Phosphoamino acid analysis clearly demonstrated that wild-type TrkB was phosphorylated on tyrosine and serine residues with a small amount of phosphothreonine (Fig. 3b, lane 1). The phosphoamino acid content was unaffected by the Y670F substitution (lane 2 ) but was dramatically reduced by the Y674FN675F, Y751F, and K540R substitutions (lanes 3 ,4 , and 5, respectively). Note that a residual amount of phosphotyrosine (and phosphoserine) was still apparent in these mutants; this is probably due to con- tamination with the nonspecific band detected in anti-phosphotyrosine Western blots (Fig. 2b, lane 1) . It was not possible to eliminate this band when excising the TrkB band from the gels.

Fig. 4 (panel a ) shows a typical phosphopeptide map of insulin-stimulated insulin receptors isolated from 32P-labeled CH0.T cells. Insulin stimulated the phosphorylation of at least 13 phosphopeptides. These include peptides corresponding to the three major tyrosine autophosphorylation sites found in the regulatory domain (residues 1158,1162, and 1163) recovered as five phosphopeptides (of related sequence DIYETDYYRK), which are mono- (Il), di- (I2 and 12’), or tri- (I3 and 13’) phosphorylated and cleaved by trypsin at arginine 1155 and either arginine 1164 (11, 12, and 13) or lysine 1165 (12’ and 13’) (see Tavar6 and Denton (1988) for further discussion).

The tryptic peptide predicted to be generated from TrkB containing tyrosines 670, 674, and 675 has the sequence DVYSTDYYR(VGGHTMLP1R). This differs by two internal residues from the equivalent insulin receptor peptide DIYET- DYYR(K), i.e. a conserved valine for isoleucine (no charge difference) and a serine for glutamate (a small if any charge difference at pH 3.5; see Boyle et al. (1991)). In TrkB, trypsin would cleave this peptide at the arginine (residue 676) following the vicinal tyrosines (i.e. as in the insulin receptor) and at the next available carboxyl terminal arginine (residue 6861, which would introduce an extra two positive charges into the

D k B Autophosphorylation Sites in Vivo 30373

205-

116-

80 -

49-

Pi .

PS. PT.

PY.

Origin.

a

1 2 3 4 5

b

-TRKB

1 2 3 4 5 FIG. 3. Phosphoamino acid analysis of ”3 isolated from ”P-

labeled COS cells. COS cells were transfected with wild-type (lane 1 ), Y670F (lane 21, Y674FN675F (lane 3 ), Y751F (lane 4) , and K540R (lane 5) TrkB mutants. After 48 h the cells were labeled with [“P]P,, and receptors were immunoprecipitated with CyTrk antiserum as described under “Experimental Procedures.” The isolated proteins were separated by SDS-PAGE and visualized using a PhosphorImager (panel a). Bands corresponding to TrkB were subjected to phosphoamino acid analysis (panel b ) as described under “Experimental Procedures.” The migration of the molecular weight markers ( X I O - ~ kDa) is indicated inpanel a, and inorganic phosphate (Pi), standard phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) are indicated in panel b.

peptide. If tyrosines 670,674, and 675 were phosphorylated in TrkB, then we would predict that tryptic digestion of 32P- labeled TrkB would generate three peptides a t positions equivalent to peptides 11, 12, and I3 of the insulin receptor (i.e. cleaved at arginine 676) and peptides running in equivalent positions but shifted toward the anode by a distance equivalent to two positive charges if cleaved a t arginine 686.

Indeed this was the case. The peptide map of wild-type TrkB exhibited approximately 12 distinct phosphopeptides (Fig. 4, panel d ). Peptides T4, T5, and T6 appeared to co-migrate with insulin receptor peptides I1,12, and I3 (compare panels a and d of Fig. 4 and a mixture of peptides from TrkB and insulin receptors in panel c) . Peptides cleaved at arginine 686 were not easily identified but probably reside in the mixture of peptides running above the origin.

Phosphoamino acid analysis confirmed the existence of phosphotyrosine in peptides T4, T5, and T6 along with very significant amounts of phosphoserine (Fig. 5). This suggests, therefore, that peptides T4, T5, and T6 correspond to mono-, di-, and triphosphorylated peptides containing tyrosines 670, 674, and 675 and that serine 671 may also be phosphorylated.

Consistent with our initial assignment of peptides T4, T5, and T6, the TrkB Y670F mutant (Fig. 4, panel f ) lacked peptide T6 (the triphosphopeptide; however, overexposure of the maps did reveal small amounts of peptide T6 in this mutant (data not shown)). The Y674FN675F mutant on the other hand exhibited a dramatically lower phosphorylation of all phosphopeptides;

the only peptides apparent were T1, T2, and a peptide running significantly above T5 (Fig. 4, panel g).

In agreement with Western blotting with anti-phosphotyrosine antibodies, neither BDNF nor NT-3 had any apparent effect on the phosphorylation of any of the phosphopeptides (data not shown). This is despite the fact that two-dimensional phosphopeptide mapping is a greatly more sensitive technique than Western blotting, i.e. we could not even detect an increase in the ratio of peptides T5 (diphospho) to T6 (triphospho). This again suggests that TrkB is constitutively phosphorylated in COS cells in the absence of ligand.

Consistent with the anti-phosphotyrosine blotting, a K540R substitution caused a dramatic reduction in the phosphorylation of all phosphopeptides (Fig. 4, panel j ) , but surprisingly this was also the case with a Y751F substitution (Fig. 4, panel h).

Two other phosphotyrosine-containing peptides were consis- tently identified, peptides T1 and T3; the latter is more apparent in MG86-TrkB cells (see below). The identity of these peptides was not unequivocally determined in the current study. However, peptide T3 appeared to co-migrate with a synthetic peptide based on the tryptic peptide predicted to be produced from tyrosine 484 (sequence IPVIENPQY(P)FGITNSQLK), the site of which has been proposed to bind SHC (Obermeier et al., 1993a). Peptide T1 remains to be identified but has the appro- priate hydrophobicity and charge a t pH 3.5 to correspond to a tryptic peptide containing tyrosine 785, which is reported to be involved in phospholipase Cy binding (Obermeier et al., 1993b; Middlemas et al., 1994). This remains to be established more directly using site-directed mutagenesis. Several serine phosphopeptides were also identified migrating as peptides T2 and T7-10 (Figs. 4 and 5). Finally, identical phosphopeptide maps were obtained if TrkB was precipitated with anti-TrkB““” antibodies, which recognize the ligand binding domain of TrkB (data not shown), suggesting that the pan-Trk serum is unaffected by the phosphorylation state of the receptor.

Analysis of BDNF-stimulated Phosphorylation Sites in MG86-DkB Cells-We turned to the stably transfected MG86 cell line, which expresses the rat TrkB cDNA, to determine the pattern of phosphopeptides obtained from TrkB a t a lower level of expression (approximately 100-fold lower per MG86-TrkB cell than in a single transiently transfected COS cell). The addition of BDNF to these cells produced a marked increase in tyrosine autophosphorylation as assessed by Western blot analysis with anti-phosphotyrosine antibodies (Fig. 6a) and in pan- Trk precipitates isolated from 32P-labeled cells (Fig. 6b). Phos- phoamino acid analysis (Fig. 6c) demonstrated that BDNF promoted a large increase in both tyrosine and serine phosphorylation of TrkB in these cells.

Two-dimensional phosphopeptide mapping of 32P-labeled TrkB revealed maps that were very similar to those we obtained from transiently transfected COS cells. BDNF stimulated phosphorylation of almost all peptides. The maps clearly demonstrate the phosphorylation of tyrosines 670,674, and 675 (peptides T4, T5, and T6) in addition to peptides T1 and T3 plus the major serine phosphopeptides T2 and one or more of T7-9. Expression of the receptor in these cells was not high enough to allow us to determine whether peptides T4, T5, and T6 were also phosphorylated on serine.

Interestingly the ratio of diphospho (peptide T5) to triphospho (peptide T6) peptides from the regulatory domain is approximately the same in BDNF-stimulated MG86-TrkB cells as it is in transiently transfected COS cells. This again suggests that TrkB is constitutively phosphorylated in the absence of ligand in COS cells at high levels of expression.

30374 DkB Autophosphorylation Sites in Vivo

b IR

f Y670F --.

h Y751F

K540R ,”. . - .’

FIG. 4. Two-dimensional phosphopeptide mapping of TrkB isolated from S2P-labeled COS cells. CH0.T cells stimulated with insulin or transiently transfected COS cells were incubated with [32P]Pi, and insulin receptors or TrkB was immunoprecipitated as described under “Experimental Procedures.” The isolated proteins were digested with trypsin and separated by two-dimensional thin layer chromatography. The maps shown are: human insulin receptor (panel a) , a mixture of phosphopeptides from human insulin receptors and wild-type TrkB (panel.c), wild-type TrkB (panel d), TrkB-Y670F (panel f ) , TrkB-Y674F/Y675F (panelg) , TrkB-Y751F (panel h) , and TrkB-K540R (panel j).Akey identlfylng the major human insulin receptor phosphopeptides is shown in panel b and to the major phosphopeptides obtained from TrkB in panel e. Insulln receptor peptides I1,12, I2’, 13, and 13’ were formerly C1, B3, B2,A2, and A1 and have been redesignated for simplicity. Refer to TavarB et al. (1991) for a more complete discussion of the identity of the other insulin receptor phosphopeptides. DNP, dinitrophenol.

Pi - 1 PT ps 4

Origin pyI T I T2 T3 T4 T5 T6 T7 T8 T9 TI0

FIG. 5. Phosphoamino acid analysis of individual TrkB tryptic phosphopeptides. Peptides were scraped from a peptide map of wild- type TrkB (e.g. seepanel d of Fig. 4) and analyzed by phosphoamino acid analysis as described under “Experimental Procedures.” The migration of inorganic phosphate (Pi) and standard phosphoserine (PS) , phosphothreonine (PT), and phosphotyrosine (PY) are indicated.

Effects of DkB Autophosphorylation Site Mutations on BDNF-stimulated Gene Expression in Chinese Hamster Ovary Cells-COS cells are an inappropriate setting in which to study signaling by TrkB because of the extremely high levels of protein expression obtained and because TrkB appeared to be constitutively phosphorylated in the absence of added ligand. We attempted to introduce the TrkB cDNAs into PC12 cells; however, we found these cells to be particularly refractory to trans-

fection. Thus we examined the effect of the various point mutations on the ability of TrkB to signal to the nucleus in CH0.T cells (which overexpress human insulin receptors (Ellis et al., 1986)). The addition of insulin to these cells caused an approx- imate 25-fold increase in transcription from a collagenase promoter (see Fig. 8). We have shown this effect to be mediated through the TRE of this promoter4, which binds the AP-1 com- plex (i.e. Fos.Jun heterodimers). CH0.T cells possess very low basal AP-1 activity and are thus particularly useful for the study of this signaling pathway.

The expression of wild-type TrkB in these cells caused a substantial increase in basal transcription from the collagenase promoter (equivalent to that seen with insulin alone), which was stimulated a further -%fold when BDNF was included in the incubation medium (see Fig. 8). Neither the Y670F or Y751F substitutions had any significant effect on the ability of TrkB to signal this response. This is in stark contrast to the Y674FN675F and K540R substitutions, which caused a dramatic reduction in both basal and BDNF-stimulated transcriptional activity. Transfection of the cells with the latter two mutants did not alter the viability of the cells, as promoter activity from the constitutive SV40 promoter was unaffected (data not shown).

DISCUSSION

Phosphorylation of DkB on &rosines 670, 674, and 675 in the “Regulatory Domain”-The phosphorylation of TrkB was

G. A. Rutter and J. M. TavarB, unpublished observations.

a b

lFkB Autophosphorylation Sites in Vivo

a

30375

2 0 5 - p l - T R K B 116- I

luIl - + a PY

C

Pi -

PS - PT - PY -

Origin- L -

- + 32 P

+

TRKB

FIG. 6. Expression and phosphorylation of TrkB in MG86 cells. In panel a , MG86-TrkB cells were serum starved and treated in the absence (-) or presence (+) of 50 ng/ml BDNF for 5 min. The cells were extracted and solubilized in sample buffer, and TrkB was immunoprecipitated with crTrk antiserum prior to separation by SDS-PAGE followed by Western blotting with anti-phosphotyrosine antibody (09Y) and detection by ECL. In panel b, MG86-TrkB cells were labeled with V2PlPi prior to incubation in the absence (-1 or presence (+) of 100 ng/ml BDNF for 5 min. Cells were extracted, and TrkB was immunoprecipitated with aTrk antiserum and separated by SDS-PAGE. The figure shows an image of the gel using a PhosphorImager. In panel c, the band containing TrkB was excised and subjected to phosphoamino acid analysis. The migration of the molecular weight markers ( x ~ O - ~ kDa) is shown in panels a and b. Inorganic phosphate (Pi), standard phosphoserine (PS), phosphothreonine (PT), and phosphotyrosine (PY) are indicated in panel c.

studied in both COS cells and MG86"hkB cells. In COS cells the receptor appeared to be constitutively phosphorylated in the absence of added ligand. However, the pattern of phosphorylation of TrkB in non-ligand-stimulated COS cells was essentially identical to that observed in BDNF-stimulated MG86- TrkB cells (compare Fig. 4d with Fig. 7b and c; note the low basal TrkB phosphorylation in MG86-TrkB cells). In addition, the pattern of phosphorylation of the human insulin receptor in COS cells is virtually indistinguishable from that obtained in CH0.T cells (Tavar6 and Dickens, 1991; Tavar6 et al., 1991, 1992). Thus COS cells represent a suitable setting for perform- ing phosphorylation site analysis despite the level of overexpression achieved.

Wild-type TrkB expressed in COS cells exhibits the constitutive presence of three peptides (T4, T5, and T6), which we identified as mono- (T4), di- (T5), and tri- (T6) phosphorylated peptides derived from the regulatory domain of TrkB (containing tyrosines 670,674, and 675). This conclusion was based on: (i) co-migration of peptides T4, T5, and T6 with insulin receptor peptides 11, 12, and 13, respectively (compare Fig. 4a with 4d and the peptide mixture in 4c), which we have previously identified as mono- (111, di- (I2), and tri- (13) phosphopeptides from the regulatory domain tyrosines 1158, 1162, and 1163 of the insulin receptor (Tavar6 and Denton, 1988); and (ii) mutagenesis of tyrosine 670 to phenylalanine (Y670F mutant) in which

C

lated from "P-labeled MG86 cells. MG86-TrkB cells were labeled FIG. 7. 'ho-dimensional phosphopeptide mapping of "rkB iso-

with I"yPIP, prior to incubation in the absence (panel a ) or presence (panels b and c ) of 100 ng/ml BDNF for 5 min. Cells were extracted, and TrkB was immunoprecipitated with aTrk antiserum followed by digestion with trypsin and separation of peptides by two-dimensional thin layer chromatography. Panels a and b are one experiment, and panel c is from a separate experiment. Each map contains receptor isolated from 5 ( a and b ) or 10 (c) dishes of cells. The subsequent application of more protein to the plate caused peptides to appear more diffuse and peptides T7-9 to co-migrate.

the triphosphopeptide T6 was virtually eliminated (Fig. 4f). In COS cells the K540R mutation almost completely elimi-

nated constitutive phosphorylation of TrkB, consistent with its central role in phosphoryl transfer. The fact that this mutation also reduced serine phosphorylation suggests that the serine kinase(s) involved would only phosphorylate TrkB either (i) after their activation by an enzymatically active TrkB tyrosine kinase or (ii) after prior autophosphorylation of TrkB on tyrosine. That the pattern of phosphorylation of TrkB tyrosine phosphopeptides in COS cells and in MG86-TrkB cells was essentially the same (including the ratio of T5 (di-):T6 (tri-) phosphopeptides) suggests that whatever mechanism caused constitutive phosphorylation in COS cells is essentially equivalent to that induced by ligand in MG86-TrkB cells. This is likely to involve receptor dimerization (Schlessinger and Ullrich, 1992) induced either by ligand in MG86-TrkB cells or by overexpression in COS cells.

A significant amount of phosphoserine was present in peptides T4, T5, and T6 and may have been due to the autophosphorylation of serine 671 by the TrkB tyrosine kinase or by other cellular serinehhreonine kinases. That this site is phosphorylated needs to be established unequivocally by site- directed mutagenesis, as we cannot rule out the possibility that three nonrelated serine phosphopeptides co-migrated with peptides T4, T5, and T6. Interestingly, however, upon overexposure of the maps, a very small amount of a phosphopeptide could be detected to the left of peptide T6 and in a position where a tetraphosphopeptide would be predicted to migrate (data not shown). It is possible that all three tyrosines and serine 671

?FkB Autophosphorylation Sites in Vivo 30376

140

120

u) .- - 5 100

$

I

r

80 5 .- b

0 60 > ._ I

m 0

2 40

c

- 1

20

0 BDNF INSULIN

MUTANT Wildtype Y670F Y6741675F Y751F K540R Empty vector

FIG. 8. Effects of autophosphorylation site mutations in TrkB on BDW-stimulated transcriptional activation in CH0.T cells. CH0.T cells were co-transfected with pCol.Luc, pSVB.CAT, and empty pcDNAIneo or pcDNAIneo with the indicated TrkB cDNA inserts. The cells were treated for 24 h with the indicated ligands and extracted, and the luciferase and chloromphenicol acetyltransferase activities were measured as described under "Experimental Procedures." Data are nor- malized to the luciferase/chloromphenicol acetyltransferase ratio found in extracts from cells co-transfected with wild-type TrkB DNA and treated with BDNF (i.e. lane 2) and are means 2 S.D. of data from at least two separate experiments.

could be phosphorylated in this peptide. The Y670F mutant, which essentially lacks the triphos-

phopeptide T6, behaved in a similar fashion to the equivalent human insulin receptor mutant (i.e. Y1158F) where the mutation has no effect on autophosphorylation of tyrosines 1162/ 1163 in in vitro phosphorylated receptor preparations (Zhanget al., 1991). However, in intact cells the Y1158F mutation of the insulin receptor promotes a large decrease in autophosphorylation (Wilden et al., 1992a).5 The Y670F mutation in TrkB appeared to have little or no effect on the ability of the receptor to stimulate the tyrosine phosphorylation of a number of cellular proteins in COS cells (Fig. 2b) or to mediate BDNF-stimulated gene transcription from a TRE (Fig. 8). The equivalent mutation in the insulin receptor appears to partially block signaling by this receptor (Wilden et al., 1990, 1992b). This suggests that tyrosine 670 of TrkB and tyrosine 1158 of the insulin receptor play subtly different functions in regulating the activity of their respective receptors.

Mutagenesis of TrkB at tyrosines 674/675 caused a dramatic inhibition of total phosphate incorporation into TrkB much in the same way as an equivalent Y1162/1163F mutation in the human insulin receptor affects its phosphorylation in intact cells (Tavare and Dickens, 1991; Wilden et al., 1992a). The Y674FN675F double substitution also appeared to block tyrosine phosphorylation of intracellular proteins in COS cells (Fig. 2b) and basal or BDNF-stimulated gene transcription in CH0.T cells (Fig. 8). An equivalent Y1162/3F mutation in the insulin receptor almost completely blocks signaling (Ellis et al., 1986; Tavare and Dickens, 1991; Wilden et al., 1992b). Hence, these residues in TrkB and the insulin receptor appear to play a very similar role.

Mutagenesis of the vicinal tyrosines in the TrkB related

E. A. Hammond and J. M. Tavare, unpublished observations.

p70trk oncoprotein reduces the transforming activity of this protein in NIH 3T3 cells (Mitra, 1991). Tyrosines in subdomain VII, and equivalent to tyrosine 674 of TrkB, have also been shown to be critical for the regulation of signaling by other tyrosine protein kinases, e.g. pp60""" (Kmiecik et al., 1988) and the hepatocyte growth factor receptor, c-Met (Ferracini et al., 1991). Furthermore, phosphorylation of MAP kinase on ThrlE3 and Tyrla5, also in subdomain VII, by MAP kinase kinase acti- vates MAP kinase kinase activity (Anderson et al., 1990; Ahn et al., 1991; Crews et al., 1992).

In the insulin receptor, phosphorylation of the regulatory domain serves to activate the kinase such that it will then phosphorylate other exogenous substrates (White et al., 1988; Flores-Riveros et al., 1989; Dickens and Tavare, 1992). Further- more, phosphorylation of these residues precedes phosphorylation of two tyrosines (1328 and 1334) at the carboxyl terminus of the P-subunit (Tavare and Denton, 1988). It is tempting to speculate that the tyrosines in the regulatory domain serve a similar function in TrkB; for example, phosphorylation of tyrosines 674 and 675 may be required to activate the kinase such that other tyrosines within TrkB (e.g. Tyr484 and Tyr'a5) or exogenous substrates (e.g. phospholipase Cy) are phosphorylated. In support of this idea, the exogenous tyrosine kinase activity of the p70Lrk oncogene is dramatically reduced by mutagenesis of the vicinal tyrosines (Mitra, 1991).

Clearly, therefore, phosphorylation of residues in this subdomain of protein kinases plays a critical role in regulating their biological activity. The crystal structure of ERK2 places tyrosine 185 of the regulatory loop of subdomain VI1 in the active site of the enzyme (Zhang et al., 1994) blocking substrate binding. Tyrosines 674 and 675 may play a similar role in TrkB; only when they are phosphorylated does a local conformational change occur allowing phosphorylation of other peptides and proteins and thus signaling.

Phosphorylation of D k B on Other !Pyrosines--Two other major tyrosine phosphopeptides were observed, T1 and T3 (Fig. 4d). The latter appeared to co-migrate with a synthetic tryptic phosphopeptide containing tyrosine 484, the putative SHC binding site (Obermeier et al., 1993a). However, the recovery of this peptide was inconsistent (in Fig. 7b it is quite prominent; on most occasions, however, it was hardly detectable), and its assignment remains to be determined by site-directed mutagenesis. Peptide T1 was also not assigned; it has the correct charge at pH 3.5 (approximately -1.0) and extreme hydrophobicity, however, to be derived from the tryptic peptide containing tyrosine 785 (ASPVYLDILG) a putative phospholipase Cy binding site (Obermeier et al., 199313; Middlemas et al., 1994).

We could obtain no direct evidence for the phosphorylation of tyrosine 751, a putative PtdIns 3-kinase binding motif (Ober- meier et al., 1993a). Mutagenesis of this residue for phenylalanine caused a dramatic reduction in tyrosine and serine phosphorylation of the receptor in COS cells (Fig. 2b and Figs. 3 and 4). The tryptic peptide that would possess this tyrosine has the sequence TCPQEVYELMLGCWQR, and thus, with an approximately neutral charge, we cannot exclude the possibility that it contributes to one of the tyrosine phosphopeptides observed in our maps. However, it should be noted that methionine 754 at Tyr-3, which makes up the proposed p85 binding motif (YXXM), is conserved in almost all members of the tyrosine kinase family (Hanks et al., 1988). Being within subdomain XI of the tyrosine kinase homology region, this methionine is important either structurally or in catalysis making it unlikely, perhaps, that this amino acid is involved in binding the p85 subunit of PtdIns 3-kinase. Furthermore, all receptor tyrosine phosphorylation sites identified to date, which act as docking sites for proteins with SH2 domains, lie outside the tyrosine kinase homology region,

DkB Autophosphorylation Sites in Vivo 30377

either in kinase inserts or within the amino- and carboxyl- terminal extremities flanking the tyrosine kinase homology region (Carpenter, 1992; Pawson and Gish, 1992; Schlessinger and Ullrich, 1992; van der Geer and Hunter, 1993).

Perhaps mutagenesis of tyrosine 751 causes a small struc- tural perturbation that leads to the dramatic reduction in tyrosine phosphorylation of TrkB. Surprisingly, we found that this mutant was still capable of apparently normal BDNF-dependent signaling in CH0.T cells (Fig. 8). This agrees with Obemeier et al. (1994), who found that the mutation had no effect on the ability of the receptor to mediate neurite outgrowth in PC12 cells. We cannot exclude the possibility that the Y751F TrkB mutant is more extensively phosphorylated in CH0.T cells than in COS cells due to a potential difference in kinase:phosphatase ratio. Due to the low transfection efficiency we experienced in CH0.T cells (approximately 2%)6, we were unable to examine the phosphorylation state of TrkB in CH0.T cells.

Phosphorylation of lFkB on Serine Residues-We also no- ticed a considerable phosphorylation of TrkB on serine residues in response to BDNF (peptides T2 and T7-TlO). Peptide T8 may also contain a small amount of phosphothreonine (Fig. 5). The role of these phosphorylation events is not known. How- ever, they may play a role in regulation of signaling as has been shown for the EGF receptor where phosphorylation at threonine 654 by protein kinase C (Cochet et al., 1984; Friedman et al., 1984; Downward et al., 1985; Davis, 1989) and at serine 1046/7 by calciundcalmodulin-dependent kinase (Countaway et al., 1992) inhibits EGF-dependent tyrosine kinase activity Our preliminary experiments (not shown) suggest that phorbol esters enhance the phosphorylation of peptides T7-T9 in COS cells under conditions where these residues are already highly phosphorylated in the basal state. It will be interesting to determine the effect of phorbol esters on the phosphorylation state of these residues and tyrosine kinase activity of TrkB in MG86-TrkB cells.

Concluding Remarks-We have directly demonstrated that tyrosines 670, 674, and 675 are phosphorylated in wild-type TrkB and that tyrosines 674 andor 675 are critical for signaling. The phosphorylation of all tyrosine and serine residues was stimulated either (i) by BDNF in MG86-TrkB cells, which express relatively low levels of TrkB or (ii) by overexpression of TrkB in COS cells. In the latter case, constitutive phosphorylation is presumably a result of ligand-independent receptor dimerization induced by receptor overexpression. It will now be interesting to determine the extent to which tyrosines 674 and 675 are important in signaling by TrkB in neuronal cells, a more physiologically relevant setting. As these tyrosines are completely conserved in TrkA and TrkC, it will also be of interest to establish whether they are phosphorylated and play a similar role in these related neurotrophic factor receptors.

barn (Department of Medicine, University of Bristol) for help and useful Acknowledgments-We are particularly grateful to Dr. David Daw-

discussions. We are also indebted to Dr. Rene Medema (University of Utrecht) for the pCol.CAT plasmid and Dr. Guy Rutter (University of Bristol) for helpful discussions and cloning the pCol.Luc plasmid.

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