apparent presence of ser133-phosphorylated cyclic amp res · apparent presence of...
TRANSCRIPT
Apparen
tp
resence
of
Ser1
33
-ph
osp
ho
rylated
cy
clicA
MP
respon
seelem
ent
bin
din
gp
rotein
(pC
RE
B)
inb
rainm
itoch
ond
riais
due
tocro
ss-reactivity
of
pC
RE
Ban
tibo
dies
with
py
ruvate
deh
yd
rogen
ase
Jan
Pla
ten
ik,V
lad
imir
J.
Ba
lcar
,Y
ukio
Yo
ne
da
,B
arb
ara
Mio
dusze
wska
,R
ich
ard
Buch
al,
Ra
do
van
Hyne
k,
Lu
kasz
Kilia
ne
k,N
obu
yukiK
ura
moto
,G
rze
go
rzW
ilczynski,
Kiy
oka
zu
Ogita
,Y
oic
hiN
akam
ura
,a
nd
Le
szek
Kaczm
are
k
1,2
,32
23
14
52
36
23
99
.12
12
3
45
6
Inst.M
ed.B
ioch
em
.,1
st
Fac.
Me
d.,
Cha
rles
Un
iv.,P
rah
a2,
CZ
EC
HR
EP
UB
LIC
.La
b.
Mol.
Pha
rma
co
l.,D
iv.P
ha
rm.
Sci.,
Ka
nazaw
aU
niv.,
Kan
azaw
a,JA
PA
N.
De
pt.
Mo
l.C
ell.
Neu
robio
l.,N
en
ckiIn
st.,
Wa
rsaw
,P
OL
AN
D.
Dep
t.B
ioche
m.&
Mic
rob
iol.,
Inst.
Ch
em
.Te
ch
no
l.,P
raha
,C
ZE
CH
RE
PU
BL
IC.
La
b.C
on
foca
lM
icro
scop
y,N
enckiIn
st.,
Wars
aw
,P
OL
AN
D.
De
pt.
Ph
arm
aco
l.,S
ets
una
nU
niv.,
Osa
ka
,JA
PA
N.
Th
ecy
clic
AM
Pre
spon
se
ele
me
nt
bin
din
gp
rote
in(C
RE
B)
isan
ubiq
uito
usly
and
cons
titutiv
ely
expre
ssed
transcrip
tion
facto
rof
the
ba
sic
/leucin
ezip
pe
rfa
mily
tha
tre
cog
niz
es
aD
NA
consen
sus
sequ
ence
calle
dC
RE
(cyclic
AM
P/C
a2
+re
sponse
ele
men
t).Its
ability
toactiv
ate
tran
scrip
tion
of
the
regu
late
dg
en
ecritic
ally
dep
end
so
nph
ospho
rylatio
no
fa
serin
ere
sid
ue
num
ber
13
3in
itstra
nsa
ctivatio
ndo
main
,w
hich
isd
on
eby
varie
tyo
fp
rote
inkin
ases
inre
spon
seto
ele
vatio
nofin
trace
llula
rca
lcium,
cAM
Por
neu
rotro
ph
insig
nalin
g.
With
over
500
0re
ports
rela
ted
toC
RE
Bin
Me
dlin
eto
date
this
pro
tein
ha
sa
lso
be
com
eo
ne
of
the
mo
ststu
die
dtra
nsc
riptio
nfa
ctors
.In
neuro
scie
nce
the
CR
EB
has
bee
nw
ide
lyim
plica
ted
inthe
synap
ticpla
stic
ityund
erlyin
glo
ng
-term
me
mory
con
solid
ation
,an
dals
oin
the
neu
rona
lpro
-surv
iva
lsig
nalin
gpa
thw
ay
s.
Th
epre
vailin
gvie
wis
thatC
RE
Bis
perm
an
ently
and
exclu
sive
lyp
resent
inth
ecell
nucle
us.
Ho
wev
er,
there
have
be
ensevera
lmutu
ally
contro
versia
lre
po
rtsd
escrib
ing
an
extra
nu
clear
loca
liza
tion
of
CR
EB
.S
pecific
ally,
syna
ptic
,cy
toso
lic,
and
inp
artic
ula
rm
itoch
on
dria
llo
ca
liza
tion
wa
sre
po
rted.
He
rew
epre
sentre
sults
ofo
ur
ow
nstu
dy
on
sub
cellu
lar
loca
liza
tion
ofC
RE
B.
Intro
du
ctio
n
Me
tho
ds
Th
esu
bce
llula
rlo
ca
lizatio
nof
CR
EB
wa
sexam
ine
dby
mea
ns
of:
-S
ubc
ellu
lar
fractio
nation
ofa
du
ltra
tb
rain
corte
xfo
llowe
dby
We
ste
rnim
uno
blo
tting
with
CR
EB
/pC
RE
Ba
ntib
odie
sand
ge
lshift
with
CR
Ep
rob
e.S
uita
ble
nucle
ar,
mito
chon
dria
lan
dsyn
aptic
ma
rkers
als
oe
mplo
yed
.
-Im
mu
no
fluo
rescen
cesta
inin
gand
con
foca
lmicro
scop
yof
cultu
red
ratb
rain
ne
uro
nalan
dastro
glia
lce
lls
-Im
mu
no
pre
cip
itatio
nofC
RE
Bor
pC
RE
BIR
from
nucle
ar
or
mito
ch
on
drial
frac
tions,
follo
wed
by
We
stern
blottin
gan
alysis
and
identific
ation
ofp
recip
itate
dprote
ins
by
MA
LD
I-TO
Fm
as
sspectro
me
try
The
follow
ing
comm
ercialC
RE
B/pC
RE
Ba
ntibo
die
s(A
b's)
we
reu
sed
:
Pho
sph
o-C
RE
B(S
er13
3)A
b,
Ce
llSig
nallin
gT
ech
no
log
y(C
ST,
#9
191
)P
ho
sph
o-C
RE
BA
bfro
mU
psta
teB
iotech
no
log
y(#
06-5
19)
CR
EB
an
tibod
y(a
gain
sta
nep
itope
at
the
CR
EB
C-term
inus
),CS
T(#
919
2)
C-2
1C
RE
BA
b(a
gain
stC
RE
BD
NA
-bin
din
gd
om
ain
),S
anta
Cru
zB
iotech
nolo
gy
(sc-18
6).
Hm
CR
EB
antib
od
y(fro
m"h
om
e-m
ad
e")
was
raise
din
Y.
Yon
eda
'sla
bo
rato
rya
gain
stp
ep
tide
KR
RE
ILS
RR
PS
YR
KC
(resid
ues
123
-136
of
mou
se
or
rat
CR
EB
,en
comp
assing
unp
ho
sph
oryla
tedS
er13
3),a
nd
affin
ity-p
urifie
dus
ing
the
sam
ep
eptid
e.
(pCR
EB
...C
RE
Bp
hosp
hory
lated
at
Ser1
33,
IR...
imm
un
ore
activity,
MA
LD
I-TOF...
matrix-as
siste
dlase
rde
sorp
tion/io
nisa
tiontim
e-o
f-flight)
1)
Su
bce
llula
rfra
ctio
natio
np
roc
ed
ure
Fra
ctio
ns
ob
tain
ed:
HP1
P2
SP3
ABCDE
(orig
inalh
om
og
ena
te)
(nucle
ar
fractio
n,
but
als
oco
nta
inin
ga
lot
of
mito
cho
nd
riafro
mce
llde
bris)
(cru
de
mito
ch
ond
ria/sy
na
pto
so
me
s)
(cyto
sol)
(mic
roso
mal)
(pro
ba
bly
cell
me
mbra
nes,
Go
lgi)
(syn
apto
so
mes)
(no
n-sy
na
ptic
mito
ch
ond
ria)
(syn
aptic
mem
bra
ne
s,b
ut
als
ocon
tainin
ga
lot
of
syna
ptic
mito
cho
nd
ria)
(syn
aptic
mito
ch
ond
ria)
Pro
tein
con
ce
ntra
tion
and
yie
ldW
este
rnb
lotting
for
CR
EB
,p
CR
EB
,N
MD
Are
cep
tor
2B
sub
un
itC
yto
ch
rom
eo
xida
se
activity
Ge
lsh
iftfo
rC
RE
DN
Ab
ind
ing
activ
ity
Ineac
hfra
ctio
nm
ea
su
red
:
2)
pC
RE
BIR
isp
rom
ine
nt
inm
itoch
on
dria
lfra
ctio
ns
,b
ut
CR
EB
IRis
no
t
A),
C),
D):
repre
senta
tiveW
este
rnblo
tsofth
esubcellula
rfra
ctions
pro
bed
with
vario
us
pC
RE
Band
CR
EB
antibo
die
sas
indic
ate
d.B
):D
ensito
me
trice
valu
atio
no
fth
epC
RE
BAb
CS
TIR
(inse
t)in
com
pariso
nto
dis
tributio
no
fN
MD
Are
cep
tor
2B
subunit
IR(s
ynaptic
marke
r)an
da
zide
-inhib
itable
cytoch
rom
eoxid
ase
en
zym
ea
ctivity(m
itoch
ondria
lm
ark
er).
Me
an
±S
D,
N=
4in
dep
ende
ntexperim
en
ts,only
for
cyto
chro
me
oxid
ase
N=2
ind
epen
dente
xperim
ents.
*…p<0
.05
,**…
p<0.0
1vs.
orig
inalhom
ogen
ate
),o
ne-w
ay
AN
OVA
with
Dunn
ett's
pos
tte
st.
Th
es
ign
al
with
two
diffe
ren
tp
CR
EB
Ab
'sw
as
stro
ng
inm
itoc
ho
nd
ria-en
rich
ed
frac
tion
s(C
,D
,E
inp
artic
ula
r),a
nd
qu
an
titativ
ed
istrib
utio
no
fp
CR
EB
IRc
orre
late
dw
ithth
em
itoc
ho
nd
rial
ma
rker
ra
the
r
tha
ns
yn
ap
tico
ne
.In
co
ntra
st,
the
ba
nd
dete
cte
dw
ithtw
oC
RE
Ba
ntib
od
ies
ag
ain
st
diffe
ren
te
pito
pe
sw
as
se
en
mo
stly
inn
uc
lea
rfra
ctio
n,
so
me
inS
an
dP
3,
bu
ta
lmo
st
ab
se
nt
from
mito
ch
on
dria
lfra
ctio
ns
.
3)
Ge
lsh
ift:m
itoc
ho
nd
rial/s
yn
ap
ticC
RE
DN
Ab
ind
ing
activ
ityd
iffere
nt
from
nu
cle
ar
CR
EB
Gelsh
iftw
ith32P
-labelle
dC
RE
pro
be
perfo
rmed
with
allth
esu
bce
llula
rfra
ctio
ns,
for
nu
clear
and
mito
chond
rial
fractio
ns
also
nclu
din
gsu
persh
iftw
ithva
rious
CR
EB
/pC
RE
BA
b's
as
indic
ate
d).
The
dom
ina
ntlo
wer
band
appearin
gin
the
nucle
ar
fractio
nw
as
su
pers
hifte
db
ytw
oC
RE
BA
b's
which
qua
lifies
this
band
as
auth
entic
CR
EB
.In
con
trast,
the
CR
E-
bin
din
gactiv
ityin
mito
chon
dria
l/sy
nap
ticfra
ctio
ns
diffe
redin
ele
ctro
pho
retic
mobility
an
dw
as
una
ffecte
dby
CR
EB
Ab's.
4)
Nu
cle
ar
co
nta
min
atio
nof
P2
fractio
nw
as
low
bu
td
ete
cta
ble
DA
PI
sta
ino
ffre
sh
lyp
rep
are
dP
1an
dP
2fra
ctio
nre
veale
da
ple
nty
of
nu
cle
ia
nd
blo
od
ves
selfra
gm
en
tsin
the
P1,b
ut
only
very
few
nu
cle
iin
the
P2.
Lik
ew
ise,p
rese
nce
of
his
ton
eH
3d
ete
cte
dw
ithW
este
rnim
mu
no
blo
tting
was
res
tricte
dto
the
P1
fractio
n.S
eve
ral
dilu
tion
so
fP
1fra
ctio
nw
ere
inclu
ded
tosh
ow
that
the
fain
th
isto
ne
ban
din
P2
actu
ally
did
no
tre
pre
se
nt
mo
reth
an
ab
ou
t1%
of
his
ton
eco
nce
ntra
tion
inP
1.
5)
Ele
ctro
ph
ore
ticm
ob
ilityo
fm
itoc
ho
nd
rial
pC
RE
BIR
ma
rked
lyd
iffere
dfro
mn
uc
lea
rp
CR
EB
/CR
EB
IR
A):
The
CR
EB
IRo
nW
este
rnblo
tsde
tecte
dw
ithth
eC
RE
BA
bC
ST
inth
enuc
lear
fractio
nconsiste
ntly
app
eare
da
sa
double
ban
d,
likely
rep
resentin
gtw
oC
RE
Bsp
licin
gvaria
nts,
alp
ha
(the
upp
er
ban
d)
and
delta
(the
low
er
band
).Allo
the
rfra
ctions
app
are
ntly
con
tain
ed
only
delta
CR
EB
.T
heblo
tsdete
cte
dw
ithpC
RE
BA
bU
psta
teals
odis
pla
yed
ano
n-consta
ntdou
ble
band
inth
en
ucle
ar
P1
fractio
n,w
hile
inth
em
itochond
rialP
2fra
ctio
nth
ere
wa
salw
ays
asin
gle
band
with
positio
ncorre
spondin
gto
the
P1
low
erban
d.
B)
2-D
ele
ctroph
ore
sis
ofP
1(u
pper
pa
nel)
and
P2
(mid
dle
pan
el)
fractio
ns
follo
wed
by
We
stern
imm
uno
blo
tting
with
pC
RE
BA
bU
psta
te.
The
P1
blo
tth
en
subje
cted
tostrip
pin
gpro
cedure
,develo
pe
dw
ithsecondary
antib
od
yto
verify
com
ple
tere
mova
lof
the
pC
RE
BA
b,an
dre
pro
bed
with
the
CR
EB
Ab
CS
T(b
otto
mpan
el).
Mo
st
imp
orta
ntly,
the
po
sitio
ns
of
nu
cle
ar
do
ub
leb
an
ds
dete
cte
dw
ithC
RE
BA
bC
ST
an
dp
CR
EB
Ab
Up
sta
ted
idn
ot
co
rresp
on
dto
eac
ho
the
r.R
ath
er,
the
ban
dd
ete
cte
dw
ithC
RE
BA
bC
ST
co
rresp
on
ded
toth
eb
an
dd
ete
cte
dw
ithp
CR
EB
Ab
Up
sta
te.
2-D
ele
ctro
ph
ore
sis
reso
lve
dth
en
uclea
ran
dm
itoch
on
dria
lp
CR
EB
IR's
into
two
sp
ots
with
su
ch
ab
igd
iffere
nce
in
ap
pare
nt
isoe
lec
tricp
oin
ts,
that
the
ym
ust
be
co
mp
lete
lyd
iffere
nt
pro
tein
s.T
he
mito
cho
nd
rial
pC
RE
BIR
infa
ct
rep
resen
tsan
oth
er
mito
ch
ond
rial
pro
tein
,w
hic
hc
ross-
rea
cts
with
the
pC
RE
BA
b's
.
low
er
up
per
6)
pC
RE
BA
b’s
sta
ine
dm
itoch
on
dria
,in
ad
ditio
nto
nu
cle
i,o
fg
lialc
ells
inp
rima
ryc
ortic
alc
ultu
res
The
prim
ary
neu
rona
lcultu
res
were
made
ofnew
-born
ratcortice
s,cyto
sine
ara
bin
osid
ea
dded
atd
ay
2;a
nd
cultu
res
used
for
imuno
fluores
cence
stain
ing
at
day
3-6
.M
ostof
the
cells
were
neu
rone
s,w
hile
glia
le
lem
ents
acc
oun
ted
forle
ss
than
5%
.A
ntib
odie
sag
ain
stpC
RE
B/C
RE
B,
cyto
chro
me
oxid
ase
(CO
X)
or
astro
cytic
marke
rG
FA
Pw
ere
used
for
stainin
g,a
nd
the
pre
para
tions
were
exa
min
ed
by
confo
calm
icrosc
opy.
pC
RE
Bantib
od
yU
psta
testa
ined
nu
clei
ofa
llcells,
buta
lso
mito
chondria
ofglia
lelem
ents,
where
itco
-loca
lized
with
CO
X.T
he
mito
cho
ndria
lsig
nalw
as
evid
en
teve
nin
unstim
ula
ted
cultu
res
(A),
bu
tw
as
stron
ger
wh
en
the
cultu
res
were
challe
nge
dw
ithfo
rsko
lin(a
ctivato
ro
fpro
tein
kin
ase
A)
prior
tofix
atio
n(B
,C).
IfC
RE
BA
bw
as
used
for
stain
ing
,only
fain
tnu
clear
sign
alc
ould
be
ob
taine
din
this
experim
enta
lsy
stem
.
7)
Th
ree
pro
tein
sp
rec
ipita
ted
by
pC
RE
BA
bfro
mP
2fra
ctio
nw
ere
iden
tified
as
PD
H
Su
cc
es
sfu
lim
mu
no
pre
cip
itatio
no
fth
e'fa
lse'p
CR
EB
IRfr
om
P2
was
co
nsis
ten
tlya
sso
cia
ted
with
3C
oo
ma
ssie
-de
tec
tab
lep
rote
inb
an
ds
of
ap
pare
nt
MW
65
.5,
42
an
d3
1.5
kD
a.
Th
ese
3b
an
ds
we
rec
on
clu
sive
lyid
en
tified
by
MA
LD
I-TO
FM
Sas
the
E2
,E
1a
lpha
su
bu
nit,
an
dE
1b
eta
su
bu
nit
co
mp
on
en
ts,
res
pe
ctiv
ely
,o
fth
ew
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no
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mito
ch
on
dria
le
nzy
me
co
mp
lex
py
ruv
ate
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ydro
ge
na
se
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Ine
xperim
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A)
we
achie
ved
identific
atio
nofth
e65.5
kDa
band
(MA
LD
I-TO
FM
Ssp
ectru
msh
ow
n).
B)
isan
oth
er
exp
erim
entw
here
imm
unop
recip
itatio
npro
cedure
was
impro
ved
an
dth
eth
ree
bands
were
sentto
Alp
haly
seA
/S,(O
dense,D
enm
ark,
http
://ww
w.p
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),w
hich
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tein
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ntifica
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mercia
lservice
;on
the
right
issu
mm
ary
ofre
sults
pro
vide
db
yth
eA
lph
alys
e.
8)
pC
RE
BA
bre
ac
tsw
ithp
urifie
dp
yru
vate
deh
yd
rog
en
as
e
Pyru
va
tede
hyd
rogen
ase
(PD
H)
pu
rified
from
po
rcin
eh
eart
(Sig
ma
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2),
su
bje
cte
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rnim
mu
no
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ps
tate
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ou
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o-S
er300
issim
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Bsequ
ence
aro
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33
.
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lshow
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igm
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ction
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ath
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or
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icate
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ple
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ithpurifie
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1fra
ctio
n,
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sam
enon
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sph
ory
late
dpe
ptid
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ss
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pe
isp
ho
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ho
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on
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nc
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eha
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un
da
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ver,
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ytu
rned
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vate
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hydro
gen
ase,
rath
er
tha
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entic
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EB
.
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isfin
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gca
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lain
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exp
erim
en
tale
vide
nce
for
mito
chon
dria
lloca
lizatio
no
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B,pu
blish
ed
pre
viously
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ke
nth
isin
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nt
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no
tse
em
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een
ough
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efo
rm
itoch
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ec
om
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rcia
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odie
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cte
dagain
st
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pho
sp
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er1
33
cross-re
act
with
phospho
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00
on
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un
ito
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vate
de
hydro
gen
ase.
The
antib
odie
ssho
uld
be
use
dw
ithcau
tion
an
ds
om
eo
fth
epre
vio
us
data
bas
ed
on
the
irusag
em
ay
ne
ed
tob
ere
vis
ited.
Th
isw
ork
wa
sp
ub
lish
ed
inJo
urn
alo
fN
eu
roch
em
istry,
Vo
l.95
,200
5,
pp
.1
446
-1460
J.P.
was
Fello
wof
theJa
pan
So
ciety
forth
eP
rom
otion
ofS
cience
wh
iledo
ing
this
work
inJa
pan
,a
ndF
ellow
of
theIn
tern
ation
alBrain
Res
ea
rch
Fou
nda
tion
(IBR
O)
wh
ileco
ntinu
ingth
iss
tud
yin
Warsa
w.
InC
zech
Rep
ub
lics
upp
orte
dby
agra
ntfro
mth
eG
rantA
gen
cy
of
the
Cze
ch
Re
pub
licN
o.
303
/02/1
282
,an
dgra
nts
from
the
Min
istryof
Edu
ca
tion
,Yo
uth
an
dS
ports
ofth
e
Cze
ch
Re
pub
licN
o.M
SM
1111
000
01
,an
dN
o.M
SM
00
216
208
06.
The
(PD
H)
co
nsis
tso
fth
ree
ma
jorco
mp
one
nts:
pyru
vate
dehy
drog
enas
e/de
carb
oxyla
se
(E1)
(EC
1.2
.4.1
.),d
ihydro
lipo
am
ide
ac
etyltra
nsfe
ras
e(E
2)
(EC
2.3
.1.1
2.)
lipoa
mide
deh
ydro
gena
se
(E3
)(E
C1
.6.4
.3.),
with
stoich
iom
etry
20-3
0alp
ha
2-be
ta2
tetra
mers
+6
hom
odim
ers
of
E3
+60
cop
ies
ofE
2(S
wis
spro
ten
tryP
084
61
).
pyruv
ate
de
hyd
roge
nas
e
(Fig
ure
from
Lod
ish
et
al.
Mo
lecu
lar
Cell
Biolo
gy,
5th
ed
.,W
.H.F
ree
man
Co.,N
.Y.
20
04
)