1 RYK, a catalytically inactive receptor tyrosine kinase, associates ...

RYK’s interaction with EphB2, EphB3 and AF-6

1

RYK, a catalytically inactive receptor tyrosine kinase, associates with EphB2 and

EphB3, but does not interact with AF-6.

Elisabeth Trivier, Trivadi S. Ganesan

Cancer Research UK, Molecular Oncology laboratories, Weatherall Institute of

Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK

Phone : (44) 01865 222 458

Fax : (44) 01865 222 431

e-mail : [email protected]

Running title : RYK’s interaction with EphB2, EphB3 and AF-6

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 15, 2002 as Manuscript M202486200 by guest on A

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SUMMARY

RYK1 is an atypical orphan receptor tyrosine kinase, which lacks detectable kinase

activity. Nevertheless, using a chimeric receptor approach, we previously found that

RYK can signal via the MAPK pathway. Recently, it has been shown that murine Ryk

can bind to and be phosphorylated by the ephrin receptors EphB2 and EphB3. In this

study, we show that human RYK associates with EphB2 and EphB3 but is not

phosphorylated by them. This association requires both the extracellular and

cytoplasmic domains of RYK, and is not dependent on activation of the Eph receptors.

It was also previously shown that AF-6 (afadin), a PDZ domain containing protein,

associates with murine Ryk. We show here that AF-6 does not bind to human RYK in

vitro or in vivo. This suggests that there are significant functional differences between

human and murine RYK. Further studies are required to determine if RYK modulates

the signaling of EphB2 and EphB3.

1 RYK, Receptor like Tyrosine Kinase

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INTRODUCTION

RYK is an atypical orphan receptor tyrosine kinase which differs from other

members of this family at a number of conserved residues in the activation and

nucleotide binding domains, and lacks detectable catalytic activity (1-4). The extra-

cellular domain of RYK is quite short when compared to other RTKs2, being only 183

amino acids long. It appears to be largely devoid of features characteristic of other

RTKs, such as immunoglobulin-like domains (5), fibronectin type III repeats (6), or

cysteine-rich domains (7). However, it contains two leucine-rich motifs, which are

usually implicated in highly specific protein/protein interactions (8,9) as well as in cell

adhesion (10). There is also a tetrabasic protease cleavage site (KRRK) flanked by

two cysteine residues in the extracellular domain, which is predicted to give rise to two

fragments. Another interesting feature in the extracellular domain of RYK is the

presence of a WIF (Wnt inhibitory factor) module (11), suggesting the possibility that

RYK may bind to one of the members of the Wnt family of proteins.

The ability of RYK to signal downstream was investigated using a chimeric

receptor approach (2). This conclusively demonstrated that the catalytic domain of

RYK is inactive and that the amino acid alterations in the activation domain contribute

to the lack of catalytic function. However, a tyrosine-phosphorylated 75kD protein was

co-immunoprecipitated with the receptor, independently of stimulation, and activation

of the MAPK3 pathway was observed following stimulation of the receptor.

Interestingly, when the invariant lysine in sub domain II of the catalytic domain was

mutated to alanine (K334A), abolishing any existing kinase activity, the receptor failed

2 RTK, Receptor Tyrosine Kinase


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to activate the MAPK pathway and to bind and/or phosphorylate the 75 kDa protein.

Further, RYK is overexpressed in ovarian tumours, and this correlates adversely with

survival (12).

The exact function of RYK is unknown and its ligand has not been identified. A

mouse model, where Ryk was homozygously deleted by homologous recombination,

revealed an unusual, completely penetrant phenotype (13). The mice were small,

compared to wild type, and died within a week due to cleft palate. Such a phenotype

has been found in other knockout mice, including those lacking members of the

Ephrin receptor family (14). It was recently shown that, although Ryk cannot auto-

phosphorylate, it can associate and be phosphorylated by two members of the Eph

family of receptors : EphB2 and EphB3 (13). The Ephrin receptor family, which

consists of 14 members, has been shown to mediate contact-dependent cell

interactions that regulate the repulsion and adhesion mechanisms involved in the cell

guidance and assembly of multicellular structures (15,16). They are important in the

development of a range of vertebrate species, in the formation of blood vessels,

axonal guidance and metastasis of transformed cells (15,17). Yeast two-hybrid

analysis, using the cytoplasmic domain of murine Ryk as a probe, has identified an

interaction with AF-6, a PDZ domain containing protein (13). This interaction involved

the PDZ domain of AF-6 and the C-terminal region of Ryk, especially the critical C-

terminal valine residue. This valine residue is completely conserved among RYK

homologues from Drosophila to human. AF-6 is a scaffold protein found at sites of

cell-cell contact, and a target of the Ras family of proteins (18,19). Interestingly, a

3 MAPK, Mitogen Activated Protein Kinase


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subset of activated Eph receptors, including EphB2, EphB3 and EphA7, also bind to

AF-6 (20,21).

The purpose of this study was to investigate further the interaction between

RYK and the Eph receptors, and RYK and AF-6.

EXPERIMENTAL PROCEDURES

Plasmids. The following plasmids were received as gifts : Myc-AF-6, Kozo Kaibuchi

(Nara Institute of Science and Technology, Japan) (19), HA-EphB3 and HA-

EphB3K665R, Steven Stacker (Ludwig Institute for Cancer Research, Australia) (21),

EphB2, Tony Pawson (Samuel Lunenfeld Research Institute Toronto, Canada)

(22,23). The TrkA:RYK chimeric construct has been previously described (2). The

RYK.V5 plasmid was constructed by cloning RYK (acc. nº NM_002958) in pTracer

(Invitrogen) in frame with the V5 tag. A NotI site was created at the 3’ end of the cDNA

and the TGA stop codon was mutated to TGG, by PCR using the primer

TGAGCGGCCGCGAGAGGAGCCAGACGTAGG. The RYKEC construct was made by

cloning an EcoRI-HindIII fragment (nucleotides 1 to 1201) (acc. nº NM_002958) in

frame with GFP in pEGFP vector (Clontech).

Growth factor and antibodies. The recombinant human NGF4 was purchased from

R&D Systems. The N-terminal TrkA-specific monoclonal antibody (MAb) 5C3 was a

gift from U. Saragovi, the N-terminal anti-mouse RYK antibody RYK3 was a gift from

Steven Stacker (Ludwig Institute for Cancer Research, Australia) (24), the anti-human

RYK polyclonal antibody 15.2 was raised against the C-terminal region of RYK (25).

4 NGF, Nerve Growth Factor


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The following antibodies are commercially available : AF-6 (Transduction

laboratories), EphB2 (Santa Cruz Biotechnology), Trk N-terminal (Zymed), HA tag

(Roche), V5 tag (Invitrogen), His tag (Novagen), GFP mouse monoclonal (Clontech),

myc (Santa Cruz Biotechnology), antiphosphotyrosine 4G10 (Upstate biotechnology).

Transfection, immunoprecipitation, and immunoblotting. HEK293T cells were

transfected using Effectene (Qiagen) according to the manufacturer’s instructions.

For double transfections, RYK.V5 and HA.EphB3 or EphB2 were used in the ratio 4:1.

Cells were treated with 1mM sodium butyrate, a non-specific transcriptional inducer,

5 hours after transfection. Twenty four to 48 hours after transfection, cells were lysed

on ice for 15 min in lysis buffer (1% TritonX-100, 0.5% Nonidet P-40, 150 mM NaCl,

50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.5 mM EGTA) containing a protease inhibitor

cocktail (Roche) and a phosphatase inhibitor cocktail (Calbiochem). Alternatively,

RIPA buffer (1% Nonidet P-40, 1% sodium deoycholate, 0.1% SDS, 150 mM NaCl, 50

mM Tris-HCl pH 7.4, 1 mM EDTA) was used to lyse cells. Insoluble material was

removed by centrifugation for 20 min at 13,000 rpm at 4°C. Cell lysates were

incubated with antibodies and protein A or G agarose beads at 4ºC. All the

immunoprecipitations were washed twice in lysis buffer containing 1M NaCl, and

once in unsupplemented lysis buffer. For immunoblotting, the immunoprecipitates

were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) according to

standard protocols and then transferred onto nylon membranes. Antibodies were

used according to manufacturer's conditions. The 15.2 and RYK3 antibodies were

used 1/500 in 1% milk and 0.2% Tween 20 for blotting.

For the induction studies with NGF, cells were washed once with PBSA, and then


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incubated for 16h in serum-free medium, 30 hours after transfection. The quiescent

cells were stimulated with recombinant human NGF (100ng/ml) for different time

intervals (0, 5, 15, 30 or 60 min.) before being lysed.

Purification of the His-tagged catalytic domain of RYK. The catalytic domain of RYK

(nucleotide 1019 to 2138, acc. nº NM_002958) was generated by PCR using the

following primers : 5’TTAACTGCAGAGAAGTGTCACTCTTTTGGAG3’, and M13 reverse

primer from pBluescript. The cDNA was cloned as a PstI/EcoRI fragment in a

baculovirus vector (pBlueBacHis2 , Invitrogen), in frame with an His-tag on the N-

terminal end, and expressed in SF9 cells. Cells were infected with an MOI of 10,

grown at 27ºC and lysed after 4 days in : 20 mM Tris pH8, 0.5M NaCl, 1% Triton, 1%

Tween, 0.5% NP40, protease inhibitors (pepstatin, leupeptin, aprotinin, trypsin

inhibitor, PMSF). After centrifugation at 14000 rpm for 30 min., the protein was bound

on a Cobalt sepharose column (Talon, Clontech) for 1 hour at 4ºC, in lysis buffer

containing 10mM imidazole. The resin was washed thoroughly with lysis buffer

containing 10mM imidazole, and the protein was eluted in 20mM Tris pH7.5, 0.5M

NaCl, 50mM imidazole. The protein was concentrated using a Centriplus YM-10

(Amicon), dialysed against a 20mM Tris pH8 solution, and stored at -70ºC in 10%

glycerol.

In vitro kinase assay. HA.EphB3 was transiently expressed in HEK 293T cells and

immunoprecipitated using an anti-HA antibody. 1µg of purified RYK catalytic domain

was bound to protein G agarose beads using an anti-His antibody. The

immunoprecipitates were washed twice in lysis buffer containing 1M NaCl, and twice

in kinase buffer (30mM Tris pH7.5, 20mM MgCl2, 20mM MnCl2, 1mg/ml BSA). The


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proteins bound to the agarose beads were resuspended in 100 µl of kinase buffer

containing 200 µM ATP and incubated at 30ºC for 45 min. They were washed in

kinase buffer and resuspended in sample buffer. The proteins were resolved by SDS

PAGE, blotted and phosphorylation was detected using the anti-phosphotyrosine

antibody 4G10.

For the kinase assay using full length RYK as a substrate, HA.EphB3, myc.AF-6 and

RYK.V5 were transiently expressed in HEK 293T cells and immunoprecipitated using

an anti-HA tag, anti-AF-6 and anti V5 antibodies respectively. The immunoprecipitates

were washed twice in lysis buffer containing 1M NaCl, and twice in kinase buffer.

HA.EphB3 was resuspended with myc.AF-6 or RYK.V5 in 100 µl of kinase buffer

containing 200 µM ATP and incubated at 30ºC for 45 min. They were washed in

kinase buffer and resuspended in sample buffer. The proteins were resolved by SDS

PAGE, blotted and phosphorylation was detected using the anti-phosphotyrosine

antibody 4G10.

Binding Assay. A myc-tagged AF-6 construct was transfected in HEK 293T cells and

immunoprecipitated using an anti-myc antibody. Cell lysate from untransfected HEK

293T cells was also immunoprecipitated with the anti-myc antibody. The protein G

agarose beads were resuspended in PBSA and 1µg of the purified RYK catalytic

domain, RYK.cat. was added. After one hour incubation at 4ºC, the beads were

washed in lysis buffer, and resuspended in sample buffer, before being resolved by

SDS PAGE and blotted.


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RESULTS

Expression of RYK

Transfection of HEK 293T cells with a C-terminal V5-tagged RYK, followed by

immunoprecipitation by an anti-V5 antibody, and immunoblotting with the same

antibody, identified two major signals : a set of bands at around 80 kDa (comprising 3

bands of 70, 75 and 80 kDa) and another at 45 kDa. The 45 kDa band was detected

by the anti-V5 (Fig. 1A) and 15.2 antibodies (a polyclonal antibody directed against a

C-terminal epitope from RYK) (Fig. 1C). The RYK3 antibody, directed against the

extracellular domain of the receptor (24), only detected the high molecular weight

bands of 70, 75 and 80 kDa (Fig. 1B). The 45 kDa form must therefore lack the part of

the extracellular domain containing the RYK3 epitope. It is probable that the receptor

is cleaved at the tetrabasic protease cleavage site (KRRK), which would remove the

first 192 amino acids, resulting in a predicted protein of 41 kDa. Surprisingly, whereas

the anti-V5 antibody detects all the isoforms, the 15.2 antibody does not recognize the

75 and 80 kDa bands.

RYK is unable to autophosphorylate

RYK has not been shown to have catalytic activity, in vitro on a peptide

substrate, using the whole protein or the catalytic domain on its own produced in

bacteria (1,4), or in vivo using a chimeric receptor approach (2). We undertook to

study the ability of RYK to autophosphorylate using a biochemical approach. We

cloned the catalytic domain in a baculoviral vector, expressed it in SF9 insect cells and

purified it under non-denaturing conditions. An in vitro kinase assay using the purified


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protein showed that the catalytic domain of RYK is unable to autophosphorylate (Fig.

2). As a positive control, EphB3 was phosphorylated in the presence of ATP.

RYK associates with EphB2 and EphB3 but is not phosphorylated

We transfected HEK 293T cells with RYK.V5 together with HA.EphB3 or a

kinase inactive mutant of EphB3 (EphB3K665R) (21). Upon immunoprecipitation of

RYK by the anti-V5 antibody, both wild type EphB3 and EphB3K665R were detected

(Fig. 3A,B,C). This shows that EphB3 and RYK interact, and that this association is not

dependent on activation and phosphorylation of the Eph receptor. However, although

the binding with RYK was demonstrated under stringent conditions using RIPA buffer

(data not shown), there was no phosphorylation of RYK (Fig. 3D) when it is co-

expressed with EphB3. To further evaluate the ability of EphB3 to phosphorylate RYK,

we performed an in vitro kinase assay on immunoprecipitated proteins. HEK 293T

cells were transfected with HA.EphB3, RYK.V5 or myc.AF-6, and these proteins were

immunoprecipitated using the appropriate antibodies. An in vitro kinase assay was

performed using immunoprecipitated EphB3 and RYK or AF-6, and their

phosphorylation was detected with 4G10 antibody. EphB3 failed to phosphorylate full

length RYK (Fig. 4A,B,D), while the positive control AF-6 was phosphorylated (Fig.

4A,C,D). Furthermore, EphB3 was unable to phosphorylate the catalytic domain of

RYK in an in vitro kinase assay (data not shown).

A converse experiment where we co-expressed EphB3 and RYK in HEK 293T

cells, and immunoprecipitated EphB3 (Fig. 5) showed the same result. Interestingly,

only the 80 kDa form of RYK associated with EphB3, suggesting an essential role of


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the extracellular domain of RYK for this interaction.

We also investigated the interaction between RYK and EphB2 (Fig. 6). When

both receptors were expressed transiently in HEK 293T cells, and EphB2 was

immunoprecipitated, only the 80 kDa form of RYK was detected (Fig. 6A,B,C),

although it is possible that the 45 kDa band was not detected because of unspecific

bands. A phosphotyrosine blot (Fig. 6D) showed that EphB2 was activated but did not

phosphorylate RYK. This suggests that, similar to EphB3, the association between

RYK and EphB2 is dependent on the extracellular domain of RYK, and does not lead

to the phosphorylation of RYK.

In order to investigate further the association between RYK and EphB3, and

assess whether the extracellular domain of RYK is required for the interaction to take

place, we co-expressed EphB3 and a TrkA:RYK chimeric receptor, or EphB3 and TrkA

as a negative control. The TrkA:RYK chimeric receptor contains the extracellular

domain of TrkA, the NGF receptor, and the transmembrane and intracellular domains

of RYK (2). When expressed, TrkA is a 120 kDa protein, and TrkA:RYK is 140 kDa or

120 kDa depending on its state of glycosylation. The chimeric receptor, lacking the

extracellular domain of RYK, did not associate with EphB3 (Fig. 7).

We then wanted to assess if the extracellular domain of RYK on its own was

able to bind EphB3. We constructed a GFP-tagged cDNA (RYKEC) containing the

extracellular, transmembrane and juxtamembrane domains of RYK, and co-

expressed it with EphB3 in HEK 293T cells (Fig. 8). The predicted size of RYKEC

protein, together with GFP (30 kDa), is 70 kDa. On transfection, the protein migrated at

85 kDa on SDS-PAGE, probably due to glycosylation. When EphB3 was


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immunoprecipitated, RYKEC did not associate with it, suggesting that the whole

receptor is required for the interaction between RYK and EphB3.

RYK does not associate with AF-6

The C-terminal end of RYK possesses a consensus sequence for the binding

of a PDZ domain (Fig. 9). The C-terminal tyrosine and valine residues are completely

conserved. As RYK is devoid of catalytic activity and PDZ domains do not bind to

phosphorylated amino acids (26-28), it is possible that RYK may signal via a PDZ

domain containing protein. Of several PDZ domain containing proteins, only AF-6 has

been shown to bind RTKs such as EphB2 and EphB3. In the light of previous results

(13), we wished to evaluate whether AF-6 bound to RYK.

When the TrkA:RYK chimeric receptor, which contains the entire cytoplasmic

domain of RYK, and AF-6 were co-expressed in HEK 293T cells, they did not

associate, whether AF-6 (Fig. 10A,B) or the chimeric receptor (Fig. 10C,D) was

immunoprecipitated. EphB3, which was used as a positive control for the interaction,

was immunoprecipitated with AF-6 (Fig. 10E,F). The possibility that AF-6 may bind to

RYK when the latter is stimulated was excluded when no association was observed

after stimulation of TrkA:RYK with NGF (data not shown). Transfection with full length

untagged RYK could not be used in these experiments as the 15.2 antibody has low

affinity for immunoprecipitation.

To further verify if there is direct interaction between the proteins, we used the

purified His-tagged catalytic domain of RYK. AF-6 was immunoprecipitated from

transfected HEK 293T cells and mixed with RYK.cat under physiological conditions.


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Subsequently, after washing the agarose beads, proteins were resolved by SDS

PAGE gel and blotted. As shown (Fig. 11), AF-6 does not bind in the presence of

excess amount of the catalytic domain of RYK.

DISCUSSION

RYK is a receptor tyrosine kinase which belongs to a small family of individually

distinct receptors that are devoid of catalytic activity (29). The mechanism of signaling

by these receptors is best understood for erbB3 (30).

We have observed several forms of the RYK protein in transfected cells, with

different molecular weight : 45, 70, 75 and 80 kDa. The three high molecular weight

bands are full length receptors, since they are recognized by the V5 and RYK3,

specific to the extracellular domain, antibodies. The three different sizes could be due

to differences in the glycosylation status of the protein, although other post-

translational modifications could occur since the 75 and 80 kDa bands are not

detected by the 15.2 antibody. The epitope against which this antibody is directed is a

17mer peptide located at position 548-564 of the RYK protein. It contains a cysteine

that could be converted to formylglycine; this modification occurs in sulfatases at a

late stage of co-translational protein translocation into the endoplasmic reticulum

(31). There are also two glutamate residues in the peptide that could be converted to

carboxyglutamate by a carboxylase (32). If such post-translational modifications occur

in the RYK protein, the modified forms of RYK may not be recognized by the 15.2

antibody. The 45 kDa band is not detected by the RYK3 antibody directed against the

extracellular domain, and must be a truncated form of the receptor, presumably


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resulting from the cleavage of the extracellular domain. This cleavage is very likely to

take place at the tetrabasic protease cleavage site (KRRK). Ectodomain cleavage

occurs frequently among cell surface transmembrane proteins, including receptor

tyrosine kinases such as ErbB4 (33), CSF-1R5 (34), c-kit (35) (36), Met (37), or TrkA

(38). It seems to be a general mechanism to modulate receptors, by down regulating

ligand-induced signaling (39,40). Since only the full length RYK, and not the 45kDa

form, co-immunoprecipitates with EphB3 and EphB2, it seems likely that, like with

other RTKs, the ectodomain cleavage of RYK regulates the receptor, either by

preventing binding of the unknown ligand or the binding of other receptors.

The heterodimerization of RTKs belonging to two different families is unusual,

but it has been reported. The EGFR6 and PDGFR7 have been shown to

heterodimerize independently of receptor stimulation, and transactivation of EGFR

was observed after stimulation with PDGF8. EGFR transactivation by PDGF did not

depend on PDGFR kinase activity, but phosphorylation of one of the receptors, or of a

protein that links the receptors, by a Src kinase was required to maintain heterodimer

formation (41). The interaction between RYK and the Eph receptors is unusual

because RYK does not get phosphorylated and therefore may have a role different

from that of analogous interactions involving typical RTKs. RYK could act as an

inhibitor of EphB2 and EphB3, by preventing homodimerization of these receptors, but

data provided by knock out mice for these receptors seem to suggest that RYK and

these two Eph receptors may co-operate in vivo. RYK is required in normal

5 CSF, Colony Stimulating Factor6 EGFR, Epidermal Growth Factor Receptor7 PDGFR, Platelet Derived Growth Factor Receptor


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development and morphogenesis of craniofacial structures and the limbs (13).

Neonatal mice doubly deficient in EphB2 and EphB3 phenocopy RYK-deficient mice in

terms of cleft palate, but also show commissural axon defects (14), which are

reminiscent of the Drosophila drl phenotype (42-45) (Drl9 is one the RYK homologous

genes in Drosophila). RYK could cooperate with the Eph receptors in several ways.

EphB2 and EphB3 are able to bind to several ephrin B ligands, and RYK, when

heterodimerized with these receptors, could modulate their affinity to a particular

ligand. This has been observed in the EGFR family of RTKs where, for instance,

neuregulin-α can bind to erbB3 but does not activate the receptor when it is co-

expressed with EGFR (46,47). RYK could also modulate signaling downstream of the

Eph receptors by recruiting specific cytoplasmic proteins.

Our results indicate that both the intracellular and extracellular domains of RYK

are required for the interaction with EphB3. It has been observed, in the erbB family of

RTKs, that stimulation of the isolated extracellular domains of the receptors leads to

homo-oligomerization, but not hetero-oligomerization (48). This suggests that regions

outside the extracellular domain are required for heteromeric interactions, as it is the

case for RYK and the Eph receptors.

We have demonstrated in this study that the association between RYK and

EphB2 or EphB3 does not result in the phosphorylation of RYK. However, a previous

study (13) showed that, when the receptors are co-expressed, murine Ryk associates

with and is phosphorylated by EphB2 and EphB3. In both studies, co-transfections

were performed in HEK 293T cells with the same EphB3 cDNA construct, but we used

8 PDGF, Platelet Derived Growth Factor


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a human RYK cDNA, whereas the authors used the murine homologue, Ryk. The

ability of EphB3 to phosphorylate RYK could not be detected, either in vivo or in vitro.

The identity between the human and murine cDNAs is very high (93%), but our results

indicate that they do not signal in the same way. The species-dependent

phosphorylation of a substrate by a kinase has been observed in the case of p53 (49).

Murine p53 can be phosphorylated by MAPK whereas human p53 is not, suggesting

species-specific differences in the modification, and therefore possibly the regulation

of the protein.

Further, using a murine Ryk homologue, the authors of a previous study (13)

were able to show that AF-6, a PDZ domain containing protein, could interact with the

receptor via its PDZ domain, and that the C-terminal valine of Ryk was essential for

this interaction. AF-6 is a Ras effector located at sites of cell-cell junctions (18,19),

and has been shown to bind to EphB2 and EphB3 (20,21). However, we were unable

to show any interaction between the cytoplasmic domain of human RYK and AF-6.

The co-transfection experiments, performed using the TrkA:RYK chimera in order to

be able to immunoprecipitate RYK, did not correlate with the data obtained using

murine Ryk, although only the extracellular domain of RYK was missing from the

receptor. The observed differences in the data can only be explained as due to

species specificity. The C-terminal part of the human and mouse receptors, which is

the AF-6 binding peptide, are identical, but another motif, present only in the mouse

homologue, could also be required for the interaction. AF-6 binds to EphB2 and

EphB3 only if these receptors are activated and phosphorylated (20); the C-terminal

9 Drl, Derailed


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motif of the receptors is therefore not the only feature needed for the interaction with

AF-6.

In summary, our results suggest that the human homologue of RYK, although it

binds to EphB2 and EphB3, is not phosphorylated by these receptors. Further studies

exploring the mechanism by which RYK may modulate signaling by EphB2 and

EphB3 are required.

ACKNOWLEDGMENTS

We would like to thank Dr S. Stacker, Dr K. Kaibuchi, Dr T. Pawson and Dr U. Saragovi

for kindly providing us with plasmids and antibodies. This work was supported by

Cancer Research UK.

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Figure legends

Fig. 1. Transient expression of RYK. HEK 293T cells were transfected with a C-

terminal V5-tagged RYK (RYK.V5), and the receptor was immunoprecipitated with an

anti-V5 antibody. (A) immunoblotting with the anti-V5 antibody. (B) immunoblotting with

the RYK3 antibody, raised against the extracellular domain of RYK. (C)

immunoblotting with the 15.2 antibody, raised against a C-terminal peptide from RYK.

The arrows indicate the different sizes of RYK protein.

Fig. 2. In vitro kinase assay. EphB3 was immunoprecipitated from transfected HEK

293T cells using an anti-HA-tag antibody. The purified, N-terminally His-tagged

catalytic domain of RYK (RYK.cat) was bound to protein G coated agarose beads

using an anti-His antibody. An in vitro kinase assay was performed, as described in

experimental procedures. (A) immunoblotting with an anti-phosphotyrosine antibody

shows that RYK.cat is not phosphorylated, whereas EphB3 is detected. (B) the anti-

His antibody detects RYK.cat. (C) the anti-HA antibody detects EphB3.

Fig. 3. Association and phosphorylation of EphB3 and RYK. HEK 293T cells were

transfected with the indicated constructs, and RYK was immunoprecipitated using an

anti-V5 antibody. (A) the anti-HA antibody detects both the wild type and kinase dead

mutant forms of EphB3 (EphB3 and EphB3 K665R respectively) when they are co-

transfected with RYK. (B) the anti-V5 antibody detects RYK. (C) the anti-HA antibody

detects both EphB3 constructs in the cell lysate. (D) the anti-phosphotyrosine antibody


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detects phosphorylation of EphB3WT only when it is co-expressed with RYK, whereas

phosphorylation of RYK is not detected.

Fig. 4. In vitro kinase assay using full length RYK as a substrate. EphB3, RYK.V5

and AF-6 were immunoprecipitated from transfected HEK 293T cells using an anti-

HA, an anti-V5 and an anti AF-6 antibody respectively. An in vitro kinase assay was

performed using immunoprecipitated EphB3 as a kinase and RYK.V5 or AF-6 as

substrates, as described in experimental procedures. (A) the anti-phosphotyrosine

antibody blot shows phosphorylation of EphB3 and AF-6 when they are present in the

kinase assay. (B) presence of RYK detected by the anti-V5 antibody. (C) presence of

AF-6 detected by the anti-AF-6 antibody. (D) presence of EphB3 detected by the anti-

HA antibody.

Fig. 5. Association between EphB3 and RYK. HEK 293T cells were transfected with

the indicated constructs, and EphB3 was immunoprecipitated using an anti-HA

antibody. (A) the anti-V5 antibody detects RYK (shown by arrows) in the cell lysate of

transfected cells, but only detects it in the immunoprecipitates when RYK is

expressed with EphB3. (B) the anti-HA antibody shows EphB3 was expressed and

immunoprecipitated.

Fig. 6. Association and phosphorylation of EphB2 and RYK. HEK 293T cells were

transfected with the indicated constructs, and EphB2 was immunoprecipitated, using

an anti-EphB2 antibody. (A) the anti-V5 antibody detects RYK in the


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immunoprecipitates only when it is co-expressed with EphB2. (B) immunoblotting

with the anti EphB2 antibody shows that EphB2 was immunoprecipitated. (C) the cell

lysate was immunoblotted with the anti-V5 antibody to show that RYK is expressed.

(D) the anti-phosphotyrosine antibody reveals phosphorylation of EphB2 but does not

detect any phosphorylation of RYK.

Fig. 7. Absence of interaction between EphB3 and TrkA:RYK. HEK 293T cells were

transfected with the indicated constructs, and EphB3 was immunoprecipitated using

an anti-HA antibody. TrkA was used as a negative control (A) the anti-Trk antibody

detects TrkA and TrkA:RYK in the cell lysate 9shown by arrows) but not in the

immunoprecipitates. (B) the anti-HA antibody shows that EphB3 is expressed and

immunoprecipitated.

Fig. 8. Absence of interaction between EphB3 and RYKEC. HEK 293T cells were

transfected with the indicated constructs, and EphB3 was immunoprecipitated using

an anti-HA antibody. (A) the anti-GFP antibody does not detect RYKEC in the

immunoprecipitates. (B) the anti-GFP antibody shows that RYKEC was expressed.

(C) the anti-HA antibody shows that EphB3 was expressed and immunoprecipitated.

Fig. 9. Comparison of the C-terminal end of RYK across species.10 The C-terminal

residues of Drosophila, Human and Mouse RYK (Acc.nº NP_477341, S58885,

P34925, Q01887 respectively) were aligned using Clustal-X and displayed with

10 DNT, Doughnut


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MacBoxShade.

Fig. 10. Absence of interaction between AF-6 and TrkA:RYK. HEK 293T cells were

transfected with the indicated constructs; EphB3 was used as a positive control. (A)

and (B) AF-6 was immunoprecipitated with an anti-AF-6 antibody but TrkA:RYK is not

detected in the immunoprecipitates, although it is present in the cell lysate. In the

untransfected lane, the anti-AF-6 antibody detects endogenous AF-6. (C) and (D)

TrkA:RYK was immunoprecipitated but AF-6 is not detected in the

immunoprecipitates, although it is present in the cell lysate. (E) and (F) AF-6 was

immunoprecipitated using an anti-myc antibody, and EphB3 is detected in the

immunoprecipitate and the cell lysate.

Fig. 11. Absence of interaction between AF-6 and the catalytic domain of RYK. A

binding assay was carried out between AF-6 and the purified catalytic domain of RYK

(RYK.cat) as described in “experimental procedures”. In the presence of

immunoprecipitated AF-6 (myc-AF-6) from transfected HEK 293T cells and purified

RYK catalytic domain (RYK.cat) in PBSA, no binding was detected after blotting with

the anti-His antibody (A). (B) and (C) show that AF-6 and RYK.cat, respectively, were

present in the reaction.


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71

48

123

71

48

123

71

48

- + - + - + RYK.V5

IP V5IB V5

IP V5IB RYK3

IP V5IB 15.2

A B C

26

Fig.1 by guest on April 12, 2018

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173

111

80

61

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36

36

173

111

His

.RY

K.c

at

HA

.Ep

hB

3

IB PTyr

IB His

IB HA

A

B

C

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Fig. 2


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80

61

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111

173

111

80

61

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- + - + - + RYK.V5- - + + - - HA.EphB3- - - - + + HA.EphB3K665R

IP V5IB HA

IP V5IB V5

Cell LysateIB HA

IP V5IB PTyr

D

B

A

C

28

Fig.3


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121

8669

52

40

8669

52

40

184

184

121

+ - myc.AF-6 protein

- + RYK.V5 protein

+ + HA.EphB3 protein

IB PTyr

IB V5

IB AF-6

IB HA

A

B

C

D

29

Fig. 4


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121

- + + - + +- - + - - + HA.EphB3

CellLysate IP HA

IB V5

IB HA

RYK.V5

A

B

Fig. 5

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8669

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- + - + RYK.V5

- - + + EphB2

IP EphB2IB V5

IP EphB2IB EphB2

Cell LysateIB V5

IP EphB2IB PTyr

A

B

C

D

Fig. 6

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122

102

122

- + + - + + HA.EphB3

- - + - - + TrkA:RYK

- + - - + - TrkA

CellLysate IP HA

IB Trk

IB HA

A

B

Fig.7

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- + - + HA.EphB3

- - + + RYKEC.GFP

IP HAIB GFP

Cell LysateIB GFP

IP HAIB HA

A

B

C

Fig. 8

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122102

122102

204

- - + - - + - + + - + + - - + - - + myc.AF-6- + + - + + - - + - - + - - - - - - TrkA:RYK - - - - - - - - - - - - - + + - + + HA.EphB3

CellLysate IP AF-6

CellLysate IP 5C3

IB AF-6IB Trk

IB AF-6IB Trk

A

B

C

D

CellLysate IP myc

122102

204

IB HA

IB AF-6

E

F

Fig. 10

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- + myc.AF-6

+ + His.RYK.cat

IP mycIB His

IP mycIB myc

reaction mixIB His

A

B

C

Fig.11

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Elisabeth Trivier and Trivadi S. GanesanEphB3, but does not interact with AF-6

RYK, a catalytically inactive receptor tyrosine kinase, associates with EphB2 and

published online April 15, 2002J. Biol. Chem.

10.1074/jbc.M202486200Access the most updated version of this article at doi:

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