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Vol.
10,
No.
8
MOLECULAR
AND
CELLULAR
BIOLOGY,
Aug.
1990,
p.
4202-4210
0270-7306/90/084202-09$02.00/0
Human
trk
Oncogenes
Activated
by
Point
Mutation,
In-Frame
Deletion,
and
Duplication
of
the
Tyrosine
Kinase
Domain
FRANQOIS
COULIER,"2
RAMESH
KUMAR,"3
MARY
ERNST,'
RUDIGER
KLEIN,3
DIONISIO
MARTIN-ZANCA,l
AND
MARIANO
BARBACIDl
3*
BRI-Basic
Research
Program,
National
Cancer
Institute-Frederick
Cancer
Research
Facility,
Frederick,
Maryland
217011;
INSERM
Unite
119,
13009
Marseille,
France2;
and
Squibb
Institute
for
Medical
Research,
P.O.
Box
4000,
Princeton,
New
Jersey
0854340003
Received
19
March
1990/Accepted
16
May
1990
Malignant
activation
of
the
human
trk
proto-oncogene,
a
member
of
the
tyrosine
protein
kinase
receptor
family,
has
been
implicated
in
the
development
of
certain
human
cancers,
including
colon
and
thyroid
papillary
carcinomas.
trk
oncogenes
have
also
been
identified
in
cultured
cells
transfected
with
various
DNAs.
In
this
study,
we
report
the
characterization
of
three
in
vitro-generated
trk
oncogenes,
trk2,
trk4,
and
trk5
(R.
Oskam,
F.
Coulier,
M.
Ernst,
D.
Martin-Zanca,
and
M.
Barbacid,
Proc.
Natl.
Acad.
Sci.
USA
85:2964-2968,
1988),
in
an
effort
to
understand
the
spectrum
of
mutational
events
that
can
activate
the
human
trk
gene.
Nucleotide
sequence
analysis
of
cDNA
clones
of
trk2
and
trk4
revealed
that
these
oncogenes
were
generated
by
a
head-to-tail
arrangement
of
two
trk
tyrosine
protein
kinase
domains
connected
by
a
purine-rich
region.
These
oncogenes
code
for
cytoplasmic
molecules
of
67,000
(p67tk2)
and
69,000
(p69tT4)
daltons.
In
contrast,
the
product
of
the
trk5
oncogene,
gp95kkS,
is
a
cell
surface
glycoprotein
of
95,000
daltons.
This
oncogene
was
generated
by
a
153-base-pair
in-frame
deletion
within
sequences
coding
for
the
extracellular
domain
of
the
trk
receptor.
This
activating
deletion
encompasses
a
triplet
coding
for
one
of
the
nine
cysteine
residues
that
the
trk
receptor
shares
with
the
product
of
the
highly
related
trkB
tyrosine
protein
kinase
gene.
Introduction
of
a
single
point
mutation
(TGT--AGT)
in
this
codon
resulted
in
a
novel
trk
oncogene
whose
product,
gp140s-45,
differs
from
the
nontransforming
trk
proto-oncogene
receptor
in
a
single
amino
acid
residue,
Ser-345
instead
of
Cys-
345.
These
results
illustrate
that
multiple
molecular
mechanisms,
including
point
mutation,
internal
deletion,
and
kinase
domain
duplication,
can
result
in
the
malignant
activation
of
the
human
trk
proto-oncogene.
Tyrosine
protein
kinases
are
emerging
as
key
regulatory
elements
in
signal
transduction
(17,
21).
Unfortunately,
their
pivotal
position
within
the
cellular
machinery
may
have
a
negative
side
effect.
Mutations
within
many
tyrosine
protein
kinase
loci
lead
to
malignant
transformation,
presumably
as
a
result
of
imbalances
within
those
signal
transduction
processes
that
they
are
meant
to
control
(17,
21).
Oncogenic
tyrosine
protein
kinases
transduced
by
retroviruses
exhibit
multiple
and
diverse
mutations,
a
reflection
of
the
strong
selection
process
that
occurs
during
viral
replication
(19,
20).
In
contrast,
oncogenic
tyrosine
protein
kinases
of
nonviral
tumors,
including
those
of
humans,
exhibit
single
mutations,
perhaps
a
consequence
of
the
limited
number
of
mutagenic
events
allowed
within the
cellular
genome.
For
instance,
neu
oncogenes
activated
in
carcinogen-induced
tumors
exhibit
a
single
point
mutation
within
their
transmembrane
coding
domain
(3,
18).
Similarly,
the
c-abl
oncogenes
of
human
leukemias
owe
their
transforming
properties
to
reproducible
rearrangements
with
specific
sequences
within
the
bcr
locus
(14).
Malignant
activation
of
trk
oncogenes
in
human
tumors
results
from
genetic
rearrangements
in
which
its
catalytic
tyrosine
kinase
domain
is
fused
to
sequences
derived
from
various
unrelated
loci
(4,
10,
13).
These
rearrangements
lead
to
the
generation
of
chimeric
molecules
and
allow
the
ectopic
expression
of
the
trk
kinase
in
different
tissues,
depending
upon
the
nature
of
the
locus
involved
in
this
mutational
event.
To
date,
trk
oncogenes
have been
detected
in
a
tumor
of
the
ascending
colon
and
in
a
significant
fraction
of
thyroid
papillary
carcinomas
(4,
10).
In
addition,
trk
*
Corresponding
author.
oncogenes
have
been
generated
during
the
course
of
gene
transfer
(8,
13).
Transfection
of
NIH
3T3
cells
with
DNA
isolated
from
a
mammary
carcinoma
cell
line
resulted
in
the
fusion
of
the
trk
kinase
to
sequences
derived
from
the
L7a
ribosomal
protein
(8,
22).
We
have
previously
reported
the
frequent
generation
of
trk
oncogenes
as
a
result
of
transfection
of
NIH
3T3
cells
with
nontransforming
trk
proto-oncogene
cDNA
sequences
(13).
Some
of
these
trk
oncogenes
code
for
cytoplasmic
kinases
reminiscent
of
the
products
of
trk
oncogenes
identi-
fied
in
human
tumors.
Other
oncogenes
code
for
cell
mem-
brane
glycoproteins.
These
results
indicate
that
the
trk
locus
can
participate
in
the
generation
of
distinct
classes
of
trans-
forming
proteins
(13).
We
undertook
the
present
studies
to
analyze
the
molecular
structure
of
these
trk
oncogenes
and
their
respective
gene
products
in
an
effort
to
understand
the
mechanisms
underlying
the
oncogenic
activation
of
the
human
trk
proto-oncogene.
MATERIALS
AND
METHODS
Isolation
of
cDNA
clones.
Total
cellular
RNA
was
prepared
by
the
guanidinium
isothiocyanate-cesium
chloride
method,
and
the
poly(A)-containing
fraction
was
isolated
by
affinity
chromatography
on
oligo(dT)-cellulose
columns
(9).
cDNA
was
synthesized
by
oligo(dT)
priming
on
poly(A)-containing
RNAs
(cDNA
Synthesis
System;
Amersham
International)
isolated
from
third
cycle
NIH
3T3
transformants
derived
from
the
trk2
(E29-913
cells)
and
trk4
(E18-93
cells)
onco-
genes.
cDNA
libraries
were
prepared
in
lambda
ZAP
vectors
(Stratagene
Cloning
Systems),
and
106
bacteriophages
were
plated
on
a
lawn
of
Escherichia
coli
BB4
cells.
Phages
were
absorbed
onto
nitrocellulose
filters
and
lysed,
and
their
4202
ACTIVATION
OF
HUMAN
trk
ONCOGENES
4203
DNAs
were
hybridized
under
stringent
conditions
(65
h
at
420C
in
5x
SSC
[lx
SSC
is
0.1
M
NaCl,
0.015
M
sodium
citrate],
50%
formamide,
lx
Denhardt
solution)
with
a
nick-translated
1.2-kilobase-pair
(kbp)
BalI-EcoRI
DNA
fragment
of
the
trk
oncogene
that
encompasses
its
entire
tyrosine
protein
kinase
catalytic
domain
(10).
Positive
lambda
clones
were
plaque
purified
as
described previously
(9)
and
subsequently
converted
to
plasmid
clones
by
the
automatic
excision
process
(Stratagene
Cloning
System).
Construction
of
pRK25.
Poly(A)-containing
RNA
was
iso-
lated
from
E67-52
cells,
a
second
cycle
NIH
3T3
transfor-
mant
derived
from
the
trk5
oncogene
by
the
guanidinium
isothiocyanate-CsCl2
procedure
(9)
followed
by
oligo(dT)
enrichment.
A
1-p.g
portion
of
this
poly(A)-containing
RNA
was
converted
into
double-stranded
cDNA
by
the
Gubler
and
Hoffman
(6)
procedure
by
using
a
reagent
kit
(Invitro-
gen).
One-tenth
of
this
cDNA
(5
p.l)
was
amplified
by
the
polymerase
chain
reaction
(PCR)
technique
(16).
The
5'
primer
(no.
1709),
5'-GGCTGGATCCTCACAGAGCTGGA-
3',
overlaps
a
BamHI
cleavage
site
(underlined)
located
at
positions
764
to
769
in
the
trk
cDNA
(11).
The
3'
primer
(no.
1712),
5'-TCGGGTCCATCGGATCGGAGG-3',
overlaps
an
NcoI
cleavage
site
(underlined)
at
positions
1875
to
1880.
A
control
PCR
amplification
was
set
up
by
using
pDM38
DNA,
a
plasmid
containing
our
longest
trk
proto-oncogene
cDNA
clone
(2,673
base
pairs
[bp])
as
a
template
(11).
Thirty
cycles
of
amplification
were
performed
with
denaturation
at
94°C
for
1
min,
annealing
at
50°C
for
2
min,
and
polymerization
at
72°C
for
3
min.
The
polymerization
time
was
extended
by
5
s
after
each
cycle.
A
10-pI
portion
of
the
total
reaction
(200
,ul)
was
tested
by
agarose
gel
electrophoresis.
The
remainder
was
subjected
to
proteinase
K
digestion,
organic
extrac-
tions,
and
ethanol
precipitation.
DNA
was
resuspended
in
water
and
digested
with
BamHI
and
NcoI.
After
phenol
extraction,
the
restricted
DNA
was
resuspended
in
water
and
subcloned
into
a
pDM38
vector
prepared
by
partial
BamHI
and
total
NcoI
digestion.
The
resulting
plasmid,
pRK25,
was
verified
by
restriction
endonuclease
digestions
and
nucleotide
sequencing
(both
strands)
of
the
insert.
Construction
of
pRK26.
A
single
point
mutation
(T-*A)
was
introduced
in
nucleotide
1117
of
the
trk
proto-oncogene
cDNA
clone
by
PCR-aided
mutagenesis.
This
change
con-
verts
a
TGT
sequence
coding
for
a
cysteine
residue
(Cys-
345)
into
a
serine-coding
triplet,
AGT.
Two
segments
of
the
trk
proto-oncogene
cDNA
clone
present
in
pDM38
were
amplified
by
PCR.
Primers
no.
1709
(see
above)
and
no.
2039
(5'-GAGGCGCAGACTCCCGTGCCGCAC-3')
amplified
a
342-bp
fragment.
Primers
no.
1712
(see
above)
and
no.
2040
(5'-GTGCGGCACGGGAGTCTGCGCCTC-3')
amplified
a
760-bp
segment.
Primers
no.
2039
and
2040
(nucleotides
1105
to
1128)
overlap
an
Hinfl
cleavage
site
(underlined)
created
by
designing
an
A/i
mispriming
over
the
first
base
of
the
wild-type
TGT
codon.
This
created
Hinfl
site
is
unique
in
the
amplified
1.
1-kbp
DNA
segment
located
between
the
BamHI
and
NcoI
cleavage
sites.
Amplified
DNAs
were
processed
as
described
above,
separately
subjected
to
Hinfl
digestion,
and
ligated
to
each
other
by
T4
DNA
ligase
(Bethesda
Research
Laboratories,
Inc.).
The
ligated
products
were
digested
with
NcoI
and
BamHI
and
electrophoresed
in
a
1.0%
agarose
gel.
A
1.1-kbp
DNA
fragment
was
purified
by
electroelution
and
subcloned
into
pDM38
previously
di-
gested
with
BamHI
(partial)
and
NcoI.
The
resulting
plas-
mid,
pRK26,
was
verified
by
Hinfl
digestion
and
nucleotide
sequence
analysis.
Cell
labeling
and
immunoprecipitation.
Subconfluent
cul-
tures
(10-cm
dishes)
were
preincubated
for
30
min
and
labeled
with
[35S]methionine
(50
,Ci/ml,
1,200
Ci/mmol;
ICN
Radiochemicals)
for
3
h
in
methionine-free
Dulbecco
modified
Eagle
medium
containing
10%
dialyzed
calf
serum.
Cells
were
washed
with
phosphate-buffered
saline,
lysed
in
radioimmunoprecipitation
buffer,
and
immunoprecipitated
with
polyclonal
rabbit
antibodies
raised
against
either
a
bacterially
made
p7otrk
protein
(12)
or
a
synthetic
peptide
corresponding
to
the
14
carboxy-terminal
residues
of
the
trk
proto-oncogene
product
(11).
The
resulting
immunocom-
plexes
were
precipitated
with
protein
A-Sepharose
beads
(Pharmacia,
Inc.)
and
resolved
by
sodium
dodecyl
sulfate
(SDS)-polyacrylamide
gel
electrophoresis
on
8%
polyacryl-
amide
slab
gels.
Protein
kinase
assays.
Subconfluent
cultures
(10-cm
dishes)
were
washed
twice
with
phosphate-buffered
saline
and
lysed
in
radioimmunoprecipitation
buffer
containing
100
,uM
sodium
vanadate,
5
mM
phenylmethylsulfonyl
fluoride,
and
0.2
U
of
aprotinin
per
ml.
Clarified
lysates
were
incu-
bated
with
the
polyclonal
antisera
indicated
above.
The
resulting
immunocomplexes
were
precipitated
with
pro-
tein
A-Sepharose
beads
and
resuspended
in
50
,ul
of
50
mM
HEPES
(N-2-hydroethylpiperazine-N'-2-ethanesulfonic
acid)-HCl
buffer
containing
20
mM
MnCl2,
5
mM
MgCl2,
1
mM
dithiothreitol,
50
p.M
ATP,
and
200
,uCi
of
[-y-32P]ATP
(6,000
Ci/mmol)
per
ml.
After
incubating
for
10
min
at
30°C,
the
immunoprecipitates
were
washed
with
radioimmunopre-
cipitation
buffer
and
the
32P-labeled
proteins
were
analyzed
by
SDS-polyacrylamide
gel
electrophoresis
on
8%
slab
gels.
RESULTS
Generation
of
novel
trk
oncogenes
during
gene
transfer.
Transfection
of
NIH
3T3
cells
with
nontransforming
plas-
mids
carrying
either
the
tyrosine
protein
kinase
domain
or
the
entire
trk
proto-oncogene
coding
sequences
results
in
the
frequent
generation
of
trk
oncogenes
(13).
As
summarized
in
Table
1,
we
have
characterized
the
products
of
42
indepen-
dently
generated
trk
oncogenes
by
using
two
antisera
raised
against
bacterially
synthesized
p7o'rk
(12)
and
against
a
synthetic
peptide
corresponding
to
the
14
carboxy-terminal
residues
of
the
normal
trk
protein
(11).
Twelve
of
these
in
vitro-generated
oncogenes
have
been
described
in
a
previous
study
(13).
Each
of
the
trk
oncogenes
derived
from
plasmids
containing
the
catalytic
domain
of
the
trk
kinase
(pDM17
and
pDM22)
codes
for
cytoplasmic
proteins
of
sizes
ranging
between
60,000
and
79,000
daltons
(Table
1).
In
contrast,
most
of
the
oncogenes
generated
during
transfection
of
cDNA
sequences
encoding
the
entire
trk
proto-oncogene
product
(pDM38)
code
for
glycoproteins
of
sizes
ranging
between
83,000
to
178,000
daltons
(Table
1).
The
peptidic
backbones
of
these
glycoproteins
were
found
to
be
in
the
range
of
69,000
to
100,000
daltons.
Since
the
molecular
mass
of
the
nonglycosylated
form
of
the
trk
proto-oncogene
prod-
uct
is
80,000
daltons
(11),
it
is
likely
that
loss
of
trk
sequences
as
well
as
gain
of
additional
genetic
information
may
have
played
a
role
in
the
generation
of
these
pDM38-
derived
trk
oncogenes.
Molecular
cloning
of
trk2
and
trk4
oncogenes.
In
order
to
characterize
these
in
vitro-generated
trk
oncogenes,
we
isolated
cDNA
clones
from
two
representative
pDM22-
derived
oncogenes,
trk2
and
trk4.
cDNA
libraries
were
prepared
in
X
ZAP
vectors
by
reverse
transcription
of
poly(A)-containing
RNAs
isolated
from
third
cycle
NIH
3T3
transformants
derived
from
the
trk2
and
trk4
oncogenes.
Third
cycle
transformants
were
used
to
eliminate
the
pres-
ence
of
additional
trk-related
transcripts
unrelated
to
the
VOL.
10,
1990
4204
COULIER
ET
AL.
TABLE
1.
Generation
of
novel
trk
oncogenes
during
gene
transfer
Donor
DNA'
trk
oncogenes
Frequency
(oncogenes
per
trk
oncogene
products
10n
transfected
cells)
(size
in
daltons)
pDM22
+
carrier
DNA
17
oncogenes
(trkl,
trk2,
trk3,
trk4, trk7,
trk8,
trklO,
3.7
60,000-79,000
trkll,
trkl2,
trkl3,
trkl7, trkl8, trkl9,
trk20,
trk2l,
trk22,
trk23)
pDM22
11
oncogenes
(trk24,
trk25,
trk26,
trk27,
trk28,
trk29,
3.7
63,000-79,000
trk3O,
trk3l,
trk32,
trk33,
trk34)
pDM17
+
carrier
DNA
3
oncogenes
(trk39,
trk40,
trk4l)
1.1
70,000-130,000
pDM17
1
oncogene
(trk42)
0.6
70,000
pDM38
+
carrier
DNA
7
oncogenes
(trk5,
trk6,
trk9,
trk35,
trk36,
trk37,
trk38)
1.9
62,000-178,000b
pDM38
2
oncogenes
(trkl4,
trkl5)
0.7
89,000-175,000b
a
pDM22
directs
the
synthesis
of
a
nontransforming
36,000-dalton
polypeptide
which
corresponds
to
the
kinase
catalytic
domain
of
the
human
trk
oncogene
(10,
12),
under
the
control
of
a
murine
sarcoma
virus
long
terminal
repeat.
pDM17
is
identical
to
pDM22,
except
it
does
not
contain
any
promotor
sequences.
pDM38
codes
for
the
entire
human
trk
proto-oncogene
product,
under
the
control
of
a
murine
sarcoma
virus
long
terminal
repeat
(11,
13).
b
The
products
of
these
oncogenes
are
glycoproteins,
except
for
p62`/6
(13).
transformation
process.
About
106
recombinant
phages
from
each
cDNA
library
were
screened
with
a
trk
tyrosine
kinase-
specific
probe
and
were
plaque
purified,
and
those
exhibiting
the
longest
cDNA
insert
were
converted
into
plasmid
clones
as
described
in
Materials
and
Methods
and
subsequently
subcloned
in
the
mammalian
expression
vector
pMEX
(11).
Two
expression
plasmids-pFC38,
which
carries
a
3.0-
kbp
cDNA
insert
of
the
trk2
oncogene,
and
pFC36,
which
contains
a
2.9-kbp
cDNA
insert
of
the
trk4
oncogene-were
next
assayed
in
gene
transfer
assays
to
determine
whether
they
possessed
transforming
activity.
Both
plasmids
effi-
ciently
induced
morphologic
transformation
of
NIH
3T3
cells
with
a
specific
activity
(>2
x
105
focus-forming
units
per
,ug)
comparable
to
that
of
pDM16,
an
expression
vector
carrying
a
cDNA
clone
of
the
original
trk
oncogene
isolated
from
a
human
colon
carcinoma
(10).
Representative
foci
of
transformed
cells
were
isolated,
subcloned
in
agar,
and
submitted
to
immunoprecipitation
analysis
by
using
a
poly-
clonal
rabbit
antiserum
raised
against
a
peptide
correspond-
ing
to
the
carboxy
terminus
of
the
trk
proto-oncogene
product
(11).
As
shown
in
Fig.
1,
NIH
3T3
cells
transformed
by
pFC38
(trk2
oncogene)
expressed
a
trk-related
protein
of
67,000
daltons,
indistinguishable
from
p67trk2,
the
product
of
the
trk2
oncogene
present
in
the
NIH
3T3
transformant
used
to
prepare
the
trk2
cDNA
library
(13).
Similarly,
NIH
3T3
cells
transformed
by
the
expression
vector
pFC36
carrying
the
trk4
oncogene
cDNA
clone
expressed
a
trk-related
protein
of
69,000
daltons,
indistinguishable
in
size
from
p69'rk4,
the
product
of
the
trk4
oncogene
(Fig.
1)
(13).
These
results
demonstrate
that
the
cDNA
inserts
present
in
pFC38
and
pFC36
expression
vectors
represent
biologically
active
clones
of
the
in
vitro-generated
trk2
and
trk4
oncogenes.
Structural
analysis
of
cDNA
clones
of
trk2
and
trk4
onco-
genes.
We
next
submitted
these
trk2
and
trk4
oncogene
cDNA
clones
to
structural
analysis.
Both
clones
exhibited
almost
identical
restriction
endonuclease
maps
(Fig.
2).
Many
of
the
mapped
restriction
sites
were
repeated
in
both
halves
of
these
cDNA
clones,
suggesting
a
tandem
arrange-
ment.
Partial
nucleotide
sequence
analysis
confirmed
this
structure.
As
shown
in
Fig.
2,
the
trk2
oncogene
consists
of
a
head-to-tail
tandem
of
the
trk
tyrosine
kinase
catalytic
domain
present
in
pDM22,
the
expression
plasmid
that
originated
the
trk2
and
trk4
oncogenes
(13).
Translation
of
the
trk2
oncogene
product,
p67trk2,
is
likely
to
initiate
at
the
same
methionine
used
to
express
the
pDM22
product,
p36trk.
This
methionine
is
followed
by
four
amino
acid
residues
(Ala-Gly-Ile-Ser)
derived
from
a
linker
used
to
generate
pDM22
and
by
a
321-amino-acid-long
kinase
catalytic
region
derived
from
the
trk
proto-oncogene
(residues
458
to
778)
(13).
The
trk
proto-oncogene
sequences
end
at
position
779,
just
12
residues
from
its
carboxy
terminus
(Fig.
2).
These
5'
trk
sequences
are
followed
by
a
purine-rich
87-bp-long
DNA
segment
derived
from
the
5'
noncoding
region
of
pDM22.
These
sequences
have
their
origin
in
the
5'
noncoding
domain
of
the
tropomyosin
gene
involved
in
the
generation
of
the
original
human
trk
oncogene
isolate
(11).
Since
this
G+A-rich
region
lacks
in-frame
terminators,
it
serves
to
A
B
C
D
MW
p
II
P
IP
1P
I
92.5K
-
69K
-
.
,
-p69trk4
4~
p67trk2
46K-
25K
-
FIG.
1.
Immunoprecipitation
analysis
of
the
trk2
and
trk4
onco-
gene
products.
[35S]methionine-labeled
cell
extracts
of
NIH
3T3
cells
transformed
by
the
trk2
oncogene
(E29-913
cells)
(A);
pFC38
DNA,
a
plasmid
carrying
a
cDNA
clone
of
the
trk2
oncogene
(E98-1711
cells)
(B);
the
trk4
oncogene
(E18-93
cells)
(C);
and
pFC36
DNA,
a
plasmid
carrying
a
cDNA
clone
of
the
trk4
oncogene,
(E98-1111
cells)
(D)
were
incubated
with
either
preimmune
rabbit
serum
(P)
or
a
polyclonal
rabbit
antiserum
raised
against
a
synthetic
peptide
corresponding
to
the
14
carboxy-terminal
amino
acid
resi-
dues
of
the
human
trk
proto-oncogene
product
(11)
(I).
The
resulting
immunoprecipitates
were
analyzed
by
SDS-polyacrylamide
gel
elec-
trophoresis
as
described
in
Materials
and
Methods.
Gels
were
exposed
to
Kodak
XAR
film
for
24
h
at
-70°C
with
the
help
of
an
intensifier
screen.
The
migration
of
the
products
of
the
trk2
onco-
gene
(p67trk2)
and
the
trk4
oncogene
(p691rk4)
is
indicated
by
arrows.
Coelectrophoresed
molecular
size
markers
included
phosphorylase
B
(92,500
daltons
[Da]),
albumin
(69,000
Da),
ovalbumin
(46,000
Da),
and
ox-chymotrypsinogen
(25,000
Da).
MOL.
CELL.
BIOL.
ACTIVATION
OF
HUMAN
tr*
ONCOGENES
4205
*
_i
CC
6
(9
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VOL.
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c,
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rb
at
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ct
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Cl)
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Cl
w
30
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c
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c,
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cl
e,^
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oc
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>
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el
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z
m
w
4206
COULIER
ET
AL.
A
B
1
2
3
TM
*]l
at
SP,
IE7
ATG
BamHl
T1
0
NcoI
trk
proto-oncogeneL
trk
5
oncogene
E
I
TK
y
T
AG
-
1353
-
1078
-
872
-
603
1125
bp
972
bp
FIG.
3.
(A)
Schematic
representation
of
the
strategy
used
to
generate
a
cDNA
clone
of
the
trk5
oncogene.
Poly(A)-containing
RNA
isolated
from
a
second
cycle
NIH
3T3
transformant
(E67-52
cells)
was
reverse
transcribed.
Sequences
contained
between
a
BamHI
cleavage
site
present
in
the
extracellular
domain
and
an
NcoI
cleavage
site
located
in
the
kinase
region
were
amplified
by
PCR
as
described
in
Materials
and
Methods.
The
thick
bar
represents
trk
proto-oncogene
coding
sequences,
including
the
signal
peptide
(crosshatched
bar),
transmembrane
domain
(solid
bar),
and
kinase
domain
(hatched
bar).
The
cysteine
residues
(small
dots)
and
potential
sites
for
N-glycosylation
(inverted
triangles)
are
also
indicated.
The
amplified
mutant
972-bp
BamHI-NcoI
fragment
was
used
to
replace
the
wild-type
1,125-bp
BamHI-NcoI
DNA
fragment
present
in
the
trk
proto-oncogene
cDNA
clone,
as
described
in
Materials
and
Methods.
(B)
Agarose
gel
electrophoresis
analysis
of
BamHI-NcoI
DNA
fragments
amplified
from
the
trk
proto-oncogene
cDNA
clone
present
in
the
expression
vector
pDM38
(lane
1)
and
DNA
complimentary
to
trk5
oncogene
transcripts
present
in
E67-52
cells
(lane
2).
Lane
3
contains
HaeIII-cleaved
d1X174
DNA
used
as
molecular
size
markers.
connect
the
5'
and
3'
kinase
domains
of
the
trk2
oncogene
(Fig.
2).
The
carboxy-terminal
catalytic
domain,
unlike
the
one
located
at
the
amino
terminus,
contains
an
intact
car-
boxy
terminus,
as
deduced
from
direct
nucleotide
sequenc-
ing
and
by
the
ability
of
the
antiserum
elicited
against
the
trk
carboxy-terminal
peptide
to
immunoprecipitate
p67'rk2
(Fig.
1).
The
structure
of
the
trk4
oncogene
was
found
to
be
very
similar
to
that
of
trk2.
The
upstream
tyrosine
kinase
catalytic
domain
was
terminated
just
five
nucleotides
3'
from
the
breakpoint
in
the
trk2
oncogene
(Fig.
2).
In
addition,
the
trk4
oncogene
product
contains
a
slightly
longer
stretch
of
the
G+A-rich,
tropomyosin-derived
sequences
(22
nucleotides
longer
than
the
corresponding
segment
in
the
trk2
onco-
gene).
The
remaining
3'
sequences
in
trk4
are
the
same
as
in
trk2,
including
a
complete
tyrosine
kinase
catalytic
domain
and
an
intact
carboxy-terminal
tail
(residues
458
to
790
of
the
trk
proto-oncogene)
(11).
The
additional
nine
amino
acid
residues
present
in
p69'rk4
(two
residues
derived
from
trk
sequences
and
seven
encoded
for
by
the
additional
purine-
rich
connecting
sequences)
are
likely
to
account
for
its
slightly
larger
molecular
weight
(Fig.
1).
Molecular
characterization
of
the
trk5
oncogene.
Transfec-
tion
of
NIH
3T3
cells
with
an
expression
plasmid
(pDM38)
containing
a
cDNA
clone
of
the
trk
proto-oncogene
also
results
in
the
frequent
generation
of
transforming
genes
(Table
1).
Unlike
the
pDM22
DNA-derived
oncogenes,
those
generated
during
transfection
of
pDM38
DNA
code
for
glycoproteins
associated
with
cellular
membranes.
In
this
study,
we
have
characterized
one
of
these
oncogenes,
trk5.
The
trk5
oncogene
codes
for
a
120,000-dalton
cell
surface
glycoprotein
slightly
smaller
than
the
mature
product
of
the
trk
proto-oncogene,
gpl40rk
(13).
These
results
suggest
that
a
small
deletion
may
account
for
the
malignant
activation
of
the
trk5
oncogene.
To
test
this
hypothesis,
we
isolated
poly(A)-containing
RNA
from
E67-52
cells,
a
second
cycle
NIH
3T3
transfor-
mant
containing
the
trk5
oncogene.
This
RNA
was
submitted
to
Si
nuclease
analysis
by
using
probes
derived
from
the
transmembrane
and
extracellular
domain
of
the
trk
proto-
oncogene.
This
strategy
was
based
on
the
assumption
that
deletions
within
the
tyrosine
kinase
catalytic
domain
would
likely
result
in
inactive
nontransforming
molecules.
Poly(A)-
containing
RNA
isolated
from
E67-52
cells
fully
protected
an
end-labeled
antisense
probe
derived
from
the
5'
end
of
the
extracellular
domain
of
the
trk
proto-oncogene
(nucleotides
1
to
757).
In
contrast,
two
similar
probes
encompassing
the
3'
half
of
the
extracellular
domain
and
the
transmembrane
region
(nucleotides
771
to
1420
and
nucleotides
957
to
1420)
were
cleaved
by
the
S1
enzyme,
yielding
identical
fragments
of
220
bp
(data
not
shown).
These
results
mapped
the
putative
deletion
in
the
trk5
oncogene
within
these
extracel-
lular
domain
sequences
somewhere
between
nucleotide
757
(3'
end
of
the
fully
protected
probe)
and
nucleotide
1200
of
the
trk
proto-oncogene
cDNA
clone.
On
the
basis
of
this
information,
we
utilized
a
PCR-aided
cloning
strategy
to
isolate
a
partial
cDNA
clone
of
the
trk5
oncogene
to
precisely
define
its
structural
alteration(s).
For
this
purpose,
we
synthesized
a
23-mer
5'
sense
amplimer
corresponding
to
nucleotides
760
to
782
of
the
trk
proto-
oncogene
which
encompassed
a
BamHI
cleavage
site
and
a
21-mer
3'
antisense
amplimer
complementary
to
nucleotides
1866
to
1886
of
the
trk
proto-oncogene
which
included
an
Ncol
cleavage
site.
As
shown
in
Fig.
3,
these
primers
MOL.
CELL.
BIOL.
1-1
ACTIVATION
OF
HUMAN
trk
ONCOGENES
4207
A
TUNICAMYCIN
I
-
MW
P
I
200K
B
+
-
-
-
+
P
I
P
I
P
I
Flanking
Direct
Repeats
eTCC
(373rd
codon)
---153
bp
Deletion
*GTG
(321st
codon)
FIG.
4.
Nucleotide
sequence
analysis
of
the
region
surrounding
the
153-bp
deletion
in
the
trk5
oncogene
reveals
flanking
direct
repeats
5'ATGGCTCC3'
and
5'ATGGCTGCC3'
(vertical
arrows).
Codon
numbers
correspond
to
those
of
the
trk
proto-oncogene.
The
position
of
the
endpoints
of
the
153-bp
in-frame
deletion
in
the
trk5
oncogene
is
indicated
by
a
horizontal
arrow.
amplified
the
expected
1.1-kbp
DNA
fragment
from
cDNA
prepared
from
NIH
3T3
cells
overexpressing
the
trk
proto-
oncogene
(E25-427
cells).
Instead,
the
same
set
of
primers
amplified
a
970-bp
DNA
fragment
from
the
corresponding
trk5-transformed
E67-52
cells
(Fig.
3).
Nucleotide
sequence
analysis
of
this
amplified
DNA
fragment
revealed
that
the
trk5
oncogene
contains
a
153-bp-long
in-frame
deletion
cor-
responding
to
nucleotides
1048
to
1200
of
the
trk
proto-
oncogene.
Interestingly,
these
sequences
are
flanked
by
an
8-bp
direct
repeat,
ATGGCT(G)CC,
that
may
have
facili-
tated
their
deletion
during
the
course
of
gene
transfer
(Fig.
4).
No
other
differences
with
the
previously
published
se-
quence
of
the
trk
proto-oncogene
were
found
in
this
ampli-
fied
DNA
fragment.
These
results
predict
that
the
trkS
oncogene
will
code
for
a
glycoprotein
identical
in
sequence
to
that
encoded
by
the
trk
proto-oncogene,
except
for
the
deletion
of
residues
322
to
372
(11).
To
demonstrate
that
this
51-amino-acid
deletion
was
di-
rectly
responsible
for
the
transforming
properties
of
the
trk5
oncogene,
we
replaced
the
wild-type
1.1-kbp
BamHI-NcoI
segment
of
the
trk
proto-oncogene
cDNA
clone
pDM38
by
the
amplified
970-bp
BamHI-NcoI
DNA
fragment.
The
re-
sulting
plasmid,
pRK25,
was
capable
of
transforming
NIH
3T3
cells
with
an
efficiency
of
at
least
105
focus-forming
units
per
pLg
of
DNA,
a
transforming
activity
comparable
to
that
of
the
original
human
trk
oncogene
(10).
NIH
3T3
cells
trans-
formed
by
pRK25
DNA
were
isolated,
subcloned
in
agar,
and
submitted
to
immunoprecipitation
analysis
by
using
the
rabbit
antiserum
elicited
against
the
trk
carboxy-terminal
sequences.
As
shown
in
Fig.
5,
pRK25-derived
transfor-
mants
expressed
two
glycoproteins
of
120,000
and
95,000
daltons,
indistinguishable
in
size
from
those
expressed
in
trk5-transformed
E28-381
cells.
More
importantly,
immuno-
0-
92.5K
! _
69K-
gpl
2Otrk5
gpg5trk5
-
p71
trk5
46K-
FIG.
5.
Immunoprecipitation
analysis
of
the
trk5
oncogene
prod-
ucts.
[35S]methionine-labeled
cell
extracts
of
NIH
3T3
cells
trans-
formed
by
the
trk5
oncogene
(E67-52
cells)
(A)
and
pRK25
DNA,
a
plasmid
carrying
a
trk
proto-oncogene
cDNA
clone
containing
the
153-bp
deletion
present
in
the
trk5
oncogene,
(B38-91
cells)
(B)
were
incubated
with
either
preimmune
rabbit
serum
(P)
or
a
polyclonal
rabbit
antiserum
raised
against
a
synthetic
peptide
corresponding
to
the
14
carboxy-terminal
amino
acid
residues
of
the
human
trk
proto-oncogene
product
(11)
(I).
The
resulting
immunoprecipitates
were
analyzed
by
SDS-polyacrylamide
gel
electrophoresis
as
de-
scribed
in
Materials
and
Methods.
Parallel
cultures
were
metaboli-
cally
labeled
either
in
the
absence
(-)
or
in
the
presence
(+)
of
10
p.g
of
tunicamycin
per
ml.
Gels
were
exposed
to
Kodak
XAR
film
for
24
h
at
-700C
with
the
help
of
an
intensifier
screen.
The
migration
of
the
unglycosylated
(p71l'5),
partially
glycosylated
(gp95'),
and
mature
(gp120'k5)
forms
of
the
trk5
oncogene
product
is
indicated
by
arrows.
Coelectrophoresed
molecular
size
markers
included
myosin
(200,000
Da),
phosphorylase
B
(92,500
Da),
albumin
(69,000
Da),
and
ovalbumin
(46,000
Da).
precipitation
of
tunicamycin-treated
cells
revealed
that
the
polypeptidic
backbones
of
the
products
of
the
recombinant
pRK25
DNA
and
the
trk5
oncogene
also
had
identical
electrophoretical
mobilities
(Fig.
5).
These
results
demon-
strate
that
pRK25
codes
for
a
representative
cDNA
clone
of
the
in
vitro-generated
trk5
oncogene.
A
single
amino
acid
substitution
can
generate
a
trk
onco-
gene.
The
51-amino-acid-long
deletion
responsible
for
the
malignant
activation
of
the
trk5
oncogene
encompasses
a
domain
highly
conserved
between
the
two
members
of
the
trk
subfamily
of
cell
surface
receptors,
trk
and
trkB
(7,
11).
This
suggests
that
this
region
may
play
an
important
role
in
determining
the
structure
of
these
receptors.
One
of
the
conserved
residues
is
Cys-345,
one
of
nine
cysteines
shared
by
the
extracellular
domains
of
the
trk
and
trkB
gene
products
and
the
only
cysteine
residue
present
in
this
deleted
domain
(7).
Since
cysteine
residues
play
an
important
role
in
determining
the
secondary
and
tertiary
structure
of
growth
factor
receptors,
we
decided
to
investigate
whether
mutation
of
this
particular
residue
might
result
in
the
malignant
activation
of
the
trk
proto-oncogene.
For
this
purpose,
we
replaced
the
TGT
triplet
coding
for
Cys-345
(nucleotides
1117
to
1119)
by
the
serine-coding
AGT
sequence.
To
engineer
this
mutation,
we
amplified
two
DNA
fragments
from
the
trk
proto-oncogene
cDNA
clone.
One
of
these
fragments
extended
from
the
BamHI
cleavage
site
described
above
(nucleotides
764
to
769)
to
sequences
over-
lapping
the
TGT
triplet.
The
second
DNA
fragment
ex-
tended
from
sequences
overlapping
this
TGT
codon
to
the
A
C
G
T
VOL.
10,
1990
4208
COULIER
ET
AL.
A
B
C
MW
F
~p
1
P
1r
P
200K
-
A
B
C
D
i
I1
-
.r
I
MW
+
-
+
-
+
-
+
-
200K
-
gpl
40trk/gpl
4OS345--,.
gpl
I
otrkIgpl
1
OS345
--
92.5K
-
69K
-
-
J
gp1
20trk5
em
,gp95trk5
gpl
40trklgp1
4OS345
.
gpi
1
otrklgpi
1
0S345
,
92.5K
-
|_-
gpl
20trk5
..,gp95trk5
69K
-
S:
46K
-
46K
-
FIG.
6.
Immunoprecipitation
analysis
of
the
product
of
the
trkS345
oncogene.
[35S]methionine-labeled
cell
extracts
of
NIH
3T3
cells
expressing
the
trk
proto-oncogene
(E25-427
cells)
(A);
NIH
3T3
cells
transformed
by
pRK26
DNA,
a
plasmid
carrying
the
trkS345
oncogene,
(B38-42
cells)
(B);
and
NIH
3T3
cells
transformed
by
the
reconstructed
trkS
oncogene
(B38-91
cells)
(C)
were
incubated
with
either
preimmune
rabbit
serum
(P)
or
a
polyclonal
rabbit
antiserum
raised
against
a
synthetic
peptide
corresponding
to
the
14
carboxy-
terminal
amino
acid
residues
of
the
human
trk
proto-oncogene
product
(11)
(I).
The
resulting
immunoprecipitates
were
analyzed
by
SDS-polyacrylamide
gel
electrophoresis
as
described
in
Materials
and
Methods.
Gels
were
exposed
to
Kodak
XAR
film
for
24
h
at
-70'C
with
an
intensifier
screen.
The
migration
of
the
products
of
the
trk
proto-oncogene
(gp14otrk
and
gpllO`rk),
the
trkS345
oncogene
(gp140S345
and
gpllOS345),
and
the
trkS
oncogene
(gp12o0rks
and
gp9tk5)
is
indicated
by
arrows.
Coelectrophoresed
molecular
size
markers
were
those
described
in
the
legend
to
Fig.
5.
NcoI
cleavage
site
used
to
generate
the
trkS
oncogene
(nucleotides
1875
to
1880).
The
primers
utilized
in
these
amplifications
carried
a
T-*A
mismatch
at
position
1117
of
the
trk
proto-oncogene
cDNA
clone
that
resulted
in
the
creation
of
a
unique
Hinfl
cleavage
site
in
the
amplified
DNA
fragrnents
(see
Materials
and
Methods).
Ligation
of
these
amplified
DNAs
after
digestion
with
Hinfl
generated
a
1.1-kbp
BamHI-NcoI
DNA
fragment
identical
to
that
present
in
the
trk
proto-oncogene,
except
that
it
contained
a
serine-
coding
AGT
sequence
instead
of
the
wild-type
TGT
codon.
Replacement
of
the
wild-type
1.
1-kbp
BamHI-NcoI
DNA
fragment
of
pDM38
by
this
PCR-amplified
fragment
yielded
pRK26,
a
plasmid
capable
of
directing
the
synthesis
of
a
trk
protein
carrying
a
single
amino
acid
substitution
(Cys-*Ser)
in
residue
345.
Transfection
of
NIH
3T3
cells
with
pRK26
DNA
resulted
in
their
malignant
transformation
with
an
efficiency
of
102
to
103
focus-forming
units
per
,ug
of
DNA.
This
transforming
activity
is
100-
to
1,000-fold
lower
than
that
of
the
trkS
Qncogene.
Whether
mutations
other
than
Ser-345
may
confer
higher
levels
of
transformation
to
this
oncogene
remains
to
be
tested.
Representative
foci
of
trans-
formed
NIH
3T3
cells
were
isolated,
cloned
in
semisolid
agar,
and
submitted
to
immunoprecipitation
analysis.
As
shown
in
Fig.
6,
the
products
of
pRK26
were
indistinguish-
able
from
those
encoded
by
its
nontransforming
allele,
the
trk
proto-oncogene.
These
results
indicate
that
replacement
of
a
single
amino
acid
residue
is
sufficient
to
confer
trans-
forming
activity
to
the
trk
proto-oncogene
and
suggest
that
the
conserved
Cys-345
residue
may
play
an
important
role
in
defining
the
appropriate
tertiary
structure
of
the
normal
trk
cell
surface
receptor.
FIG.
7.
Comparison
of
the
protein
kinase
activities
of
the
trk
proto-oncogene
protein
and
the
products
of
the
trk5
and
trks345
oncogenes.
Cell
extracts
derived
from
NIH
3T3
cells
(A),
NIH
3T3
cells
expressing
the
trk
proto-oncogene
(E25-427
cells)
(B),
NIH
3T3
cells
transformed
by
the
trkS345
oncogene
(B38-42
cells)
(C),
and
NIH
3T3
cells
transformed
by
the
reconstructed
trk5
oncogene
(B38-91
cells)
(D)
were
immunoprecipitated
with
a
polyclonal
rabbit
antiserum
raised
against
a
synthetic
peptide
corresponding
to
the
14
carboxy-terminal
amino
acid
residues
of
the
human
trk
proto-
oncogene
product
either
in
the
presence
(+)
or
in
the
absence
(-)
of
10
1Lg
of
competing
peptide
and
analyzed
for
protein
kinase
activity
as
described
in
Materials
and
Methods.
32P-labeled
samples
were
analyzed
by
SDS-polyacrylamide
gel
electrophoresis.
Gels
were
exposed
to
Kodak
XAR
film
for
12
h
at
-70°C
with
an
intensifier
screen.
The
migration
of
the
products
of
the
trk
proto-oncogene
(gp140'rk
and
gpllOtrk),
the
trkV345
oncogene
(gp140S345
and
gpllOS345),
and
the
trkS
oncogene
(gpl20rks
and
gp95WrI)
is
indicated
by
arrows.
Coelectrophoresed
molecular
size
markers
were
those
described
in
the
legend
to
Fig.
5.
Finally,
we
examined
the
effect
of
the
activating
Cys-
345->Ser-345
mutation
on
the
tyrosine
protein
kinase
activ-
ity
of
the
trk
proto-oncogene
product.
Cell
lysates
from
normal
NIH
3T3
cells
expressing
the
trk
proto-oncogene
(E25-427
cells),
NIH
3T3
cells
transformed
by
the
recon-
structed
trk5
oncogene
(B38-91
cells),
and
NIH
3T3
cells
transformed
by
pRK26
DNA
(B38-42
cells)
were
immuno-
precipitated
with
a
rabbit
antiserum
raised
against
a
peptide
corresponding
to
the
carboxy-terminal
domain
of
the
trk
proto-oncogene,
in
either
the
presence
or
absence
of
com-
peting
peptide.
The
resulting
immunoprecipitates
were
then
incubated
with
[-y-32P]ATP
in
the
presence
of
divalent
cat-
ions.
As
shown
in
Fig.
7,
the
product
of
the
trk
oncogene
activated
by
the
Cys-345-*Ser-345
miscoding
mutation
ex-
hibits
a
kinase
activity
comparable
to
that
of
the
product
of
the
trkS
oncogene
gpl2O/gp95rks
and
the
trk
proto-oncogene
gpl40/gpllork.
Similar
findings
were
obtained
with
a
poly-
clonal
antibody
raised
against
the
bacterially
expressed
trk
oncogene
product
p7Otrk
(12)
(data
not
shown).
These
results
indicate
that
the
product
of
the
in
vitro-engineered
trkS345
oncogene
retains
kinase
activity.
DISCUSSION
The
trk
locus
codes
for
a
receptorlike
molecule
whose
transcripts
appear
to
be
exclusively
localized
in
certain
ganglia
of
the
peripheral
nervous
system
(9a).
These
obser-
vations
suggest
that
this
proto-oncogene
may
have
a
highly
specialized
role
in
the
nervous
system.
Yet,
the
trk
proto-
oncogene
can
undergo
genetic
rearrangements
that
activate
it
as
a
transforming
oncogene
in
at
least
two
types
of
human
MOL.
CELL.
BIOL.
ACTIVATION
OF
HUMAN
trk
ONCOGENES
4209
malignancies,
colon
carcinoma
and
papillary
thyroid
cancer
(4,
10).
All
human
trk
oncogenes
characterized
so
far
result
from
the
fusion
of
the
kinase
domain
to
sequences
derived
from
unrelated
loci.
Two
of
the
genes
known
to
participate
in
the
generation
of
human
trk
oncogenes
have
been
identified
as
those
coding
for
a
nonmuscle
tropomyosin
and
the
ribosomal
protein
L7a
(10,
22).
The
latter
corresponds
to
a
trk
oncogene
activated
during
transfection
of
human
breast
carcinoma
DNA
(8).
The
contribution
of
these
unrelated
genes
to
the
malignant
activation
of
the
trk
kinase
is
likely
to
involve
not
only
those
coding
sequences
present
in
the
chimeric
oncogene
product
but
also
their
regulatory
ele-
ments.
This
property
will
allow
the
ectopic
expression
of
the
trk
tyrosine
kinase
outside
its
restricted
physiological
envi-
ronment,
a
property
required
for
the
involvement
of
trk
oncogenes
in
tumors
of
epithelial
origin
(4,
10).
The
mechanism
by
which
foreign
coding
sequences
con-
tribute
to
the
activation
of
the
trk
kinase
is
not
well
under-
stood
(5).
Preliminary
analysis
of
the
tropomyosin
and
L7a
ribosomal
protein-derived
domains
present
in
two
human
trk
oncogenes
did
not
reveal
any
obvious
common
structural
features.
In
both
cases
however,
the
tropomyosin
and
L7a
sequences
replaced
the
extracellular
domain
of
the
trk
receptor
(10,
22).
This
results
in
chimeric
molecules
that
cannot
interact
with
cell
membranes.
However,
cytoplasmic
localization
is
not
sufficient
to
confer
neoplastic
properties
to
trk
oncogenes,
since
cytoplasmic
trk
chimeras
in
which
tropomyosin
sequences
were
replaced
by
those
of
actin
or
globin
did
not
exhibit
transforming
activity
(5).
The
human
trk
proto-oncogene
frequently
becomes
acti-
vated
as
a
transforming
gene.
Transfection
of
NIH
3T3
cells
with
either
the
trk
catalytic
domain
(with
or
without
pro-
moter
sequences)
or
the
entire
trk
proto-oncogene
results
in
the
generation
of
trk
oncogenes
in
1
out
of
3,000
to
1
out
of
10,000
transfected
cells.
Molecular
characterization
of
some
of
these
in
vitro-generated
transforming
genes
has
revealed
additional
mechanisms
by
which
the
trk
proto-oncogene
can
acquire
transforming
properties.
Partial
nucleotide
sequence
analysis
of
cDNA
clones
derived
from
two
of
these
onco-
genes,
trk2
and
trk4,
revealed
that
association
of
the
trk
kinase
with
heterologous
sequences
is
not
essential
to
be-
come
oncogenic.
Both
trk2
and
trk4
code
for
proteins
that
consist
of
a
head-to-tail
tandem
arrangement
of
two
tyrosine
kinase
catalytic
domains.
We
have
previously
shown
that
expression
of
a
single
trk
catalytic
domain
possessing
a
structure
identical
to
the
carboxy-terminal
half
of
the
prod-
ucts
of
the
trk2
and
trk4
oncogenes
results
in
an
enzymati-
cally
active
but
nontransforming
protein
(13).
Therefore,
it
is
likely
that
the
amino-terminal
half
of
the
p67trk2
and
p69trk4
oncoproteins
can
activate
the
carboxy-terminal
kinase
by
the
same
mechanism
with
which
tropomyosin
and
L7a
sequences
activate
their
respective
trk
oncogenes.
One
such
mechanism
may
involve
trans-phosphorylation
of
their
kinase
domains.
In
the
case
of
the
colon
carcinoma
trk
oncogene,
the
tropomyosin
sequences
may
induce
dimer-
ization
of
its
gene
product
(1),
leading
to
the
formation
of
homodimers
in
a
fashion
reminiscent
of
the
oligomerization
of
growth
factor
receptors
induced
by
their
cognate
ligands
(17).
In
the
case
of
the
trk2
and
trk4
oncogene
products,
the
proposed
trans-phosphorylation
may
simply
occur
intramo-
lecularly.
In the
case
of
the
trk
oncogene
activated
by
the
L7a
ribosomal
protein,
the
amino
acid
sequence
is
not
sufficiently
informative
to
predict
the
formation
of
ho-
modimers.
The
validity
of
such
a
model
must
await
direct
experimental
analysis.
Molecular
characterization
of
oncogenes
generated
by
transfection
of
the
entire
trk
cDNA
clone
indicated
that
the
trk
proto-oncogene
can
also
become
activated
without
losing
its
basic
receptor
structure
(11).
The
nucleotide
sequence
of
a
partial
cDNA
clone
of
the
trk5
oncogene
revealed
a
153-bp
deletion
in
the
region
coding
for
the
extracellular
domain.
The
missing
sequences
are
likely
to
have been
looped
out
during
transfection,
a
mutation
that
may
have been
facili-
tated
by
the
presence
of
an
8-nucleotide-long
direct
repeat
flanking
the
deleted
sequences.
The
51
amino
acid
residues
coded
for
by
these
deleted
sequences
encompass
a
stretch
of
26
amino
acids,
of
which
20
are
also
present
in
the
corre-
sponding
region
of
trkB,
a
highly
related
neurogenic
receptor
(7).
This
51-amino-acid-long
domain
also
contains
3
of
the
13
putative
N-glycosylation
sites
(two
are
conserved
in
the
trkB
product)
and
1
of
the
9
cysteines
shared
between
the
trk
and
trkB
receptors
(7,
11).
Thus,
this
deleted
region
is
likely
to
play
an
important
role
in
regulating
the
trk
receptor.
Whether
deletion
of
other
sequences
within
the
extracellular
domain
of
the
trk
proto-oncogene
product
also
results
in
its
malignant
activation
awaits
the
molecular
characterization
of
other
pDM38-derived
trk
oncogenes.
The
conserved
nature
of
cysteine
residues
present
in
the
ligand-binding
domain
of
tyrosine
protein
kinase
receptors
has
underscored
their
important
role
in
maintaining
the
overall
structure
of
these
receptors
(17,
21).
Therefore,
it
was
not a
complete
surprise
that
substitution
of
serine
for
Cys-345,
the
conserved
cysteine
residue
deleted
in
the
trk5
oncogene,
resulted
in
its
malignant
activation.
Activation
of
tyrosine
protein
kinase
receptors
by
miscoding
mutations
has
been
previously
documented
for
the
neu
(2,3)
and
CSF-1
receptors
(15).
In
the
case
of
the
neu
gene
product,
only
certain
substitutions
within
its
transmembrane
domain
ap-
pear
to
have
neoplastic
consequences
(3).
In
the
case
of
the
CSF-1
receptor,
the
activating
mutation
present
in
its
trans-
forming
allele
v-fms
has
been
mapped
in
the
extracellular
domain
but
involved
a
leucine
residue
(15).
Whereas
the
transmembrane
mutation
renders
the
neu
gene
a
fully
trans-
forming
oncogene,
the
extracellular
mutations
present
in
v-fms
and
engineered
in
the
trk
cDNA
clone
confer
only
moderate
transforming
activities.
Nevertheless,
these
re-
sults
demonstrate
that
subtle
changes
in
the
extracellular
domain
of
the
trk
receptor
can
have
profound
consequences
in
its
ability
to
induce
malignant
transformation.
In
summary,
our
studies
indicate
that
the
human
trk
locus
can
become
an
oncogene
by
a
variety
of
mutations.
Forma-
tion
of
chimeric
molecules
expressed
under
the
control
of
heterologous
promoters
appears
to
be
the
most
favored
mechanism
encountered
in
human
tumors
(4,
10).
These
observations
may
reflect
the
selective
advantage
of
such
rearrangements,
considering
the
otherwise
limited
range
of
expression
of
the
endogenous
trk
promoter
(9a).
However,
the
high
transforming
activity
of
the
trk5
oncogene
illustrates
that
small
mutations
within
this
locus
can
also
lead
to
its
malignant
activation.
Whether
such
putative
mutations
are
involved
in
the
development
of
neural
tumors
or
in
some
other
type
of
neurological
abnormalities
remains
to
be
de-
termined.
Finally,
the
recent
results
of
Bongarzone
et
al.
describing
the
presence
of
trk
oncogenes
in
a
significant
fraction
of
papillary
thyroid
carcinomas
(4)
demonstrate
that
malignant
activation
of
trk
sequences
in
human
cancer
is
not
limited
to
sporadic
cases
(10).
Considering
the
multiple
mechanisms
by
which
this
tyrosine
protein
kinase
proto-
oncogene
can
acquire
neoplastic
properties,
its
contribution
to
human
neoplasia
may
be
wider
than
previously
suspected.
VOL.
10,
1990
4210
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