Cell Signalling and Gene Expression Mediated by RET

7/29/2019 Cell Signalling and Gene Expression Mediated by RET

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MINISYMPOSIUM

Cell signalling and gene expression mediated by RET

tyrosine kinase

K . K U R O K A W A1 , K . K A W A I1 , M . H A S H I M O T O1 , Y . I T O 2 & M . T A K A H A S H I1

From the 1Department of Pathology, and the 2Equipment Center for Research and Education, Nagoya University Graduate School of Medicine,

Showa-ku, Nagoya, Japan

Abstract. Kurokawa K, Kawai K, Hashimoto M, ItoY, Takahashi M (Nagoya University Graduate

School of Medicine, Showa-ku, Nagoya, Japan).

Cell signalling and gene expression mediated by

RET tyrosine kinase (Minisymposium). J Intern Med

2003; 253: 627633.

Germline mutations of the RETproto-oncogene cause

multiple endocrine neoplasia (MEN) 2A or 2B by

different mechanisms. As is the case for other receptor

tyrosine kinases, mutant RET recruits a variety of

signalling molecules via phosphorylated tyrosine

residues present in the kinase domain and carboxy-

terminal tail. As we previously reported, the signalingvia phosphorylated tyrosine 1062 plays a crucial role

in the transforming activities of both RET-MEN2A

and RET-MEN2B mutant protein. Interestingly, this

single tyrosine residue represents a binding site for

several signalling molecules including SHC, Enigma,

SNT/FRS2, DOK and IRS1 and is responsible for ac-

tivation of the RAS/ERK, PI3-K/AKT, JNK,

p38MAPK and ERK5 signalling pathways. Amongstthese, the PI3-K/AKT and JNK pathways appeared to

be more strongly activated in the cells expressing

RET-MEN2B than in the cells expressing RET-

MEN2A, suggesting the possibility that these path-

ways may be involved in the disease phenotype. In

addition, RET is alternatively spliced to produce three

isoforms and the splicing site is present just down-

stream of tyrosine 1062. These isoforms play different

roles for the tumour development associated with

MEN 2 or the development of the kidney and the

enteric nervous system. Moreover, using differential

display analysis, we identified several genes whoseexpression is highly induced by RET-MEN2B mutant

proteins. The differential gene expression by RET-

MEN2A and RET-MEN2B may also be important for

the development of their phenotypes.

Keywords: DOK1, multiple endocrine neoplasia type

2, RET isoforms, SHC, SNT/FRS2, stanniocalcin 1.

Introduction

The RETproto-oncogene encodes a receptor tyrosine

kinase [1, 2] that is essential for the development of

the enteric nervous system and the kidney [3]. The

glial cell line-derived neurotrophic factor (GDNF)

family ligands (GFLs) including GDNF, neurturin,

persephin and artemin exert their physiological

functions via activation of RET tyrosine kinase.

However, GFLs do not bind to the extracellulardomain of RET directly but require glycosylphos-

phatidylinositol-linked cell surface proteins called

GFRas for complex formation with RET. GFRa family

members (GFRa14) show preferential binding to a

particular GFL and plays specific roles in vivo

through the preferred ligandreceptor complex

formation [4]. Gene ablation studies revealed that

the GDNF/GFRa1/RET complex formation is crucial

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for the development of the kidney and the enteric

nervous system [5].

RET mutations are responsible for the develop-

ment of several human diseases including papillary

thyroid carcinoma, multiple endocrine neoplasia

(MEN) 2A and 2B, familial medullary thyroid carci-noma (FMTC) and Hirschsprungs disease (HSCR)

[610]. More than 10 rearranged forms ofREThave

been cloned from sporadic and radiation-associated

papillary thyroid carcinomas [11, 12], whereas

germline point mutations of RET were identified in

MEN2A, MEN2B and FMTC. MEN2A or FMTC

mutations were mostly found in one of six cysteine

residues in the RET extracellular cysteine-rich do-

main [13]. These cysteine mutations induce disul-

phide-linked RET dimerization, leading to its ligand-

independent activation [14, 15]. On the contrary,

the MEN2B mutations were detected at codon 918(methionine to threonine) in most cases or at codon

883 (alanine to phenylalanine) in fewer than 4%

cases [13], which are present in the catalytic core of

the RET tyrosine kinase domain. These mutations

probably induce conformational change of the kinase

domain, resulting in activation without dimerization

[1517]. However, we do not fully understand the

mechanisms by which the different phenotypes

between MEN2A and MEN2B are caused.

Hirschsprungs disease is a congenital malforma-

tion associated with aganglionosis of the gastroin-

testinal tract. RET mutations are responsible forabout 50% of familial and 1020% of sporadic cases

of HSCR and a variety of missense, non-sense and

frame shift mutations have been identified along

with its whole coding sequence [1822]. Based on

functional analyses, at least four mechanisms of RET

dysfunction are responsible for the development of

HSCR. First, most mutations identified in the extra-

cellular domain markedly impaired the RET cell

surface expression, probably because of incorrect

folding of the RET protein [2325]. Secondly, a few

mutations in the carboxy-terminal tail impaired the

binding of adaptor proteins such as SHC to RET,resulting in the defect of RET-mediated RAS/ERK

and PI3-K/AKT signalling [26, 27]. Thirdly, the

mutations in the kinase domain, which were iden-

tified in amino acids conserved amongst members of

the tyrosine kinase family, almost completely abol-

ished the RET tyrosine kinase activity [2831].

Fourthly, some mutations in the kinase domain

severely impaired the activation of PLCc pathway

[31]. Taken together, these findings suggested that

several intracellular signalling pathways via RET

cooperatively function for the normal development

of the enteric nervous system.

Cell signalling mediated by RET

RET is alternatively spliced to produce three isoforms

(designated RET9, RET43 and RET51) that differ in

the carboxy-terminal sequence [32, 33]. There are

16 tyrosines in the intracellular domain of RET9 and

RET43 and 18 tyrosines in that of RET51. Amongst

these, tyrosines 1015 and 1062 (Y1015 and

Y1062) have been shown to be important for cell

signalling mediated by both ligand-dependent and

-independent activation of RET [12, 34]. Y1015

represents a binding site for PLCc [35], and Y1062

represents a binding site for several adaptor proteinssuch as SHC [3638], SNT/FRS2 [39, 40], IRS1

[41], DOK1 [42], DOK4 and 5 [43], and Enigma

[44] (Fig. 1). As a result, various signalling path-

ways including the RAS/ERK, PI-3K/AKT,

p38MAPK, JNK [45] and ERK5 pathways [46] are

activated mainly via phosphorylated Y1062 in RET.

Consistent with these findings, mutation of Y1062

markedly impaired the transforming activity of all

types of MEN2 mutant proteins [36].

When SHC binds to Y1062 following RET activa-

tion, it further complexes with GRB2 and GAB1/

GAB2 proteins, leading to activation of the PI3-K/AKT signalling pathway. On the contrary, asso-

ciation of SHC with the GRB2/SOS complex is

responsible for activation of the RAS/ERK pathway

[45, 47] (Fig. 1). Activation of the PI3-K/AKT and

RAS/ERK pathways resulted in the activation of

transcription factors, NFjB and CREB, respectively

[45]. Moreover, binding of SNT/FRS2 to Y1062

leads to the RAS/ERK activation because it has

GRB2 and SHP-2 binding sites in the carboxy-

teminal region [39, 40].

Using a yeast two-hybrid screen, we found that

DOK1 also binds to tyrosine 1062 in RET [42, 48].DOK1 was more strongly associated with

RET-MEN2B mutant proteins than RET-MEN2A

mutant proteins. As a result, DOK1 was highly

phosphorylated in the cells expressing RET-MEN2B.

DOK1 has six tyrosine residues that represent

binding sites for RAS-GTPase activating protein

(RAS-GAP), leading to suppression of the RAS/ERK

activation. In addition, DOK1 contains a tyrosine

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6 28 K . K UR OK AW A et al.


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residue that is a binding site for the NCK adaptor

protein. The association of NCK with DOK1appeared to be responsible for JNK and c-JUN

activation (Fig. 1). Salvatore et al. [49] reported

that the level of phosphorylation of Y1062 is

increased in PC12 cells expressing RET-MEN2B,

resulting in enhancement of activation of the ERK

and PI3-K/AKT pathways. Taken together with our

findings indicating high levels of activation of JNK

and AKT by RET-MEN2B [42, 48], it is suggested

that the enhanced signalling via Y1062 may be

involved in the MEN2B phenotype.

DOK4 and 5 also bind to Y1062 of RET and

induce ligand-dependent axonal outgrowth ofPC12 cells [43]. DOK4 is expressed broadly in

many tissues, such as brain, heart, lung and

kidney, whereas DOK5 is specifically expressed in

the brain. Because DOK4 and DOK5 associate with

neither RAS-GAP nor NCK, it is interesting to

investigate which signalling pathways are activa-

ted by them.

Regulation of cell signalling via Y1062in RET

Both SHC and SNT/FRS2 bind to pY1062 of RET viatheir phosphotyrosine-binding (PTB) domains, that

recognize the common consensus motif NKLpY.

Both PTB domains have conformational topology

but do not have sequence homology [50]. So far,

two types of the PTB domains are known. The PTB

domain of SNT/FRS2 has sequence similarity to the

PTB domain of IRS1, so these PTB domains are

called phosphotyrosine-binding domain, insulin

receptor substrate 1-like (PTBI). DOK-family pro-

teins also contain PTBI.The PTB domain of SHC binds to TrkA, that has

the canonical consensus motif for its binding

(NPXpY, X is any amino acid) in the juxtamembrane

region. The PTBI domain of SNT/FRS2 recognizes

the same consensus motif in TrkA as the PTB

domain of SHC. Consistent with this finding, the

binding mode of SNT/FRS2 PTBI to RET in vitro

resembles that of SHC PTB, depending on phos-

phorylation of Y1062 [39]. Although the PTBI

domain of SNT/FRS2 also binds to fibroblast growth

factor receptor (FGFR) [5154], the consensus

sequence for its binding in FGFR represents a uniquepeptide that does not contain phosphotyrosine. In

addition, the binding is ligand-independent. The

finding that SNT/FRS2 recognizes different receptors

with different modes suggests the interference of

signalling pathways activated by each receptor [52].

Recently, it was reported that the microdomains of

membrane, lipid rafts, play a role for regulating or

separating cell signalling via SHC and SNT/FRS2

from Y1062 of RET [55, 56]. As GFRas are GPI-

anchored at the carboxy-terminus and SNT/FRS2

has a myristylation sequence at the amino-terminus

[57], these proteins locate on rafts. RET receptortyrosine kinase locates outside rafts under unstimu-

lated conditions, and appears to be recruited to rafts

by GPI-anchored coreceptor GFRas. As a result, after

stimulation by GDNF, association of RET with SNT/

FRS2 is supposed to lead to sustained ERK activation.

On the contrary, SHC locates outside rafts, and may

be involved in the transient ERK activation as well as

the activation of the PI3-K/AKT pathway [56].

Y1062Y1015

PLC

Signal sequence Cysteine-rich

Transmembrane

Kinase domain

SHC

GAB1/2

GRB2

PI3-K

RET51

RET9RET43

AKT

SOS

SNT/FRS2 DOK1

p38MAPK

ERK5

SHP-2

NCK

JNKRAS

ERK

c-JUN

RAS-GAP

NFB CREB

GRB2++

Fig. 1 Signaling pathways medi-

ated by RET receptor tyrosine

kinase.


M I N I SY M P O SI U M : C E L L S I G N AL L I N G B Y R E T 6 2 9


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The splicing site of three RET isoforms is present

just downstream of Y1062, and their different roles

are recently demonstrated. By NIH 3T3 transfection

assay, we showed that RET51-MEN2A and RET51-

MEN2B mutant proteins have stronger transforming

activity than RET9-MEN2A and RET9-MEN2Bmutant proteins, respectively [36]. In addition, the

activity of RET43 was very low [26]. These findings

suggested that the sequences downstream of Y1062

are important for binding of adaptor proteins and

that RET51 may contribute more significantly to the

tumour development associated with MEN 2 than

RET9 and RET43. On the contrary, RET9 was

shown to be critical for the development of the

kidney and the enteric nervous system using

targeted mutagenesis mice expressing RET9 or

RET51 only [58], suggesting the possibility that

RET9 and RET51 are involved in the activation of

different signalling pathways in vivo [59].

Computational analysis of SHC PTB domain

binding to each RET isoform

To obtain supportive information about the function

of RET isoforms based on structural biology, we

simulatedthe interaction of the SHCPTB domainwith

the amino acids around Y1062 of each RET isoform

(Fig. 2). The simulations were performed based on the

model of the SHC PTB domain complexed with the

tyrosine-phosphorylated peptide of TrkA [60].

As shown in Fig. 2a, a hydrogen bond is formed

between arginine (R) 67 in the SHC PTB domain

RET51

M1064

2.37NN

H

R207 M1064

2.37 NN

H

R207

RET peptide with tyrosine 1062

SHC PTB domain

(b)

(a)

2.62

R67

pY1062

Y1062F

N

H

RET9

R207R1064

pY1062

2.47

NH

R207R1064

2.47

NH

RET43

A1064R207

A1064R207

Fig. 2 Computational analyses of SHC phosphotyrosine-binding (PTB) domain binding to RET peptide with tyrosine 1062. Simulations

of the interaction between SHC PTB domain and RET peptide with tyrosine 1062 were performed based on the model of the SHC PTB

domain complexed with the tyrosine-phosphorylated peptide of TrkA [60]. The distances between the functional residues and the potential

energy of compounds were calculated using the insight II software package. The compounds were simulated with force field parameters

based on the consistent valence force field (CVFF). The group-based cut off, 0.95 nm for the van der Waals and 0.95 nm for Coulomb

interactions, were introduced. The temperature was set at 298K. Calculations based on the simulation images were carried out using

the insight II package. (a) A hydrogen bond between phosphotyrosine (pY) 1062 in RET9 and arginine (R) 67 in SHC (2.62 A ). When

Y1062 is replaced with phenylalanine (Y1062F, light blue), the hydrogen bond cannot be formed. (b) The interaction between SHC

PTB domain and three isoforms of RET. Arginine (R) 1064 in RET9 or methionine (M) 1064 in RET51 forms a hydrogen bond with

arginine (R) 207 in the SHC PTB domain (2.47 A and 2.37 A respectively), but RET 43 does not.




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and phosphotyrosine (pY) 1062 in RET (2.62A).

When Y1062 is replaced by phenylalanine

(Y1062F), the hydrogen bond cannot be formed.

In addition, arginine (R) 1064 in RET9 or methion-

ine (M) 1064 in RET51 forms a hydrogen bond with

arginine (R) 207 in the SHC PTB domain (2.47 A

and 2.37 A respectively), but RET 43 does not (Fig.

2b). These findings are consistent with our previous

data using in vitro binding assay [26, 39]. The

current simulations suggested that the conformation

of the SHC PTB domain binding to RET9 is almost

the same as that to TrkA [60]. However, the actual

conformation of the SHC PTB domain binding to

RET51 might be changed to some degree, because

the distance between methionine (M) 1064 in

RET and arginine (R) 207 in SHC appears to be

too close to form stable interactions between these

molecules.Amongst three RET isoforms, only RET9 has the

consensus sequence for binding of the SHC SH2

domain [38, 61]. This may contribute to the stable

binding of SHC to RET9 in addition to the stable

mode of binding of the SHC PTB domain to RET9.

Gene expression and disease phenotype

We performed a differential display analysis of gene

expression using NIH3T3 cells expressing the

RET-MEN2A or RET-MEN2B mutant proteins [62].

As a result, we identified 130 known genes and 13previously unidentified sequences whose expression

was affected by each mutant protein. Of 130 known

genes, 29 genes were confirmed to be expressed

differentially by Northern blotting (Table 1). Based

on their expression patterns, they were classified

into four types: (i) 10 genes induced by both RET-

MEN2A and RET-MEN2B mutant proteins (type I),

(ii) six genes induced predominantly by RET-MEN2A

(type II), (iii) five genes induced predominantly by

RET-MEN2B (type III), (iv) eight genes repressed by

RET-MEN2A and RET-MEN2B (type IV). Type I

includes cyclin D1, cofilin, and cathepsin L and Bgenes that are known to be involved in cell growth,

tumour progression and invasion. In contrast, type

IV includes type I collagen, lysyl oxidase, annexin I,

and tissue inhibitor of matrix metalloproteinase 3

(TIMP3) genes that have been implicated in tumour

suppression. Type II and type III include various

genes with different physiological functions, and it is

unknown whether these gene expressions are really

involved in the tumour development or diseasephenotype.

To see the physiological significance of inducible

genes, we investigated whether these inducible

genes are also induced by GDNF stimulation.

Amongst 21 genes induced by RET-MEN2A and/or

RET-MEN2B, six genes including cyclin D1, cathep-

sin B, cofilin, ring finger protein 11 (RNF11),

integrin-a6, and stanniocalcin 1 (STC1) genes were

also induced in TGW human neuroblastoma cells in

response to GDNF stimulation, although the time

course of the induction was different depending on

the genes [62]. The STC1 gene was highly inducedby both MEN2B mutant protein and GDNF stimu-

lation. Because STC1 was suggestive of a role in

early skeletal development that is affected in MEN2B

patients, we stained paraffin sections of human

medullary thyroid carcinoma, arising from MEN2A,

MEN2B and sporadic cases. Interestingly, MEN2B

MTC was strongly stained with the STC1 antibody,

whereas MEN2A MTC was weakly stained and

Table 1 Gene expression induced or suppressed by RET-MEN

2A/2B mutant proteins

Type I Type II Type III Type IV

Cyclin D1 EMK2 STC1 Annexin I

RNF11 ITGA6 PheX Annexin IV

Cofilin PKA-RI EIF4G3 COLIA1Foocen TACC3 Neuropsin COLIA2

Cathepsin L PRNPA PLOD2 Lysyl oxidase

Cathepsin B MPT TIMP3

Decorin Pleiotrophin

INFb SDF1a

TB-2 like 1 protein

HSC73

Type I, the genes induced by both RET-MEN2A and MEN2B

mutant proteins.

Type II, the genes induced predominantly by RET-MEN2A mutant

protein.

Type III, the genes induced predominantly by RET-MEN2B

mutant protein.

Type IV, the genes repressed by both RET-MEN2A and MEN2Bmutant proteins.

RNF11, ring finger protein 11; INFb, interferon b; HSC73, heat

shock protein 73; EMK2, ELKL motif kinase 2; ITGA6, integrin-a6;

PKA-RI, protein kinase A regulatory subunit I; TACC3, trans-

forming acidic coiled coil-containing gene family 3; PRNPA, pri-

on-related protein A; MPT, mitochondrial phosphate transporter;

STC1, stanniocalcin 1; PheX, phosphate-regulating gene with

homology to endopeptidases on the X chromosome; EIF4G3,

eukaryotic translation initiation factor 4G3; PLOD2, procollagen-

lysine, 2-oxyglutarate, 5-dioxygenase 2; COLIA1, type I collagen

a1 chain; COLIA2, type I collagen a2 chain; TIMP3, tissue

inhibitor of metalloproteinases 3; SDF1a, stromal cell-derived

factor-1a.




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sporadic MTC without RET mutation was almost

unstained [62], suggesting a possible role for STC1

in the development of MEN2B phenotype.

Conflict of interest statement

No conflict of interest was declared.

Acknowledgements

We would like to thank members of our laboratory

for helpful scientific advice. We are grateful to

K. Imaizumi and M. Kozuka for their technical

assistance. This work was supported in part by a

grant-in-aid for COE (Center of Excellence) research

from the Ministry of Education, Culture, Science,

Sports and Technology of Japan.

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Received 12 March 2003; accepted 20 March 2003.

Correspondence: Masahide Takahashi MD, PhD, Department of

Pathology, Nagoya University Graduate School of Medicine,

65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (fax:

181 52 7442098; e-mail: [email protected]).



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Cell Signalling and Gene Expression Mediated by RET