Vol.
183,
March
No.
16,
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1992
725-732
CALMODULIN-BINDING PROTEINS OF CALCIUM-INDEPENDENT TYPE IN RAT BRAIN SYNAPTOSOMAL MEMBRANES: THEIR LOCALIZATION AND PROPERTIES Naoki National
1* , Akiko
Natsukari
Institute for Transport,
IDivision
Miwa,
Physiological Myodaiji,
Sciences, Okazaki 444,
of Neuropsychopharmacology, Pennsylvania, Philadelphia,
'Department of Biochemistry, Medicine, 3600 Handa-cho,
Received
and Michiya
January
31,
2
Fujita Division Japan
of Active
The Medical PA 19129
College
Hamamatsu University Hamamatsu 431-31,
School Japan
of of
1992
Some properties of calmodulin(CaM)-binding proteins (CaMBPs) of the Ca2+-independent type were investigated in the synaptosomal membrane (SM) from rat brain using the [1251]CaM gel overlay method. When SM was prepared in the presence of Ca", Ca"-independent CaM binding was decreased, whereas the Cazt-dependent type was not altered. All Cazt-independent-type CaMBPs were membrane-bound and scarcely present in the soluble fractions. When SM was heat-denatured, the 24/22.5-kDa CaMBPs could no longer be detected by [l"]CaM binding and a new component with higher molecular mass (>ZOOkDa) was shown to bind CaM in a Cazt -independent manner. A possible effect of CAMP- and Ca2+/CaM-dependent phosphorylation on CaM binding was also examined.
Calmodulin(CaM) closely Although of the
the
with
intracellular
the
located
will
regulatory
(see
ref.
of CaM has been is
help
the
protein a review).
studied
not well-known.
us understand
localizations
1 for
(2).
Knowing
that where
relationship(s)
of CaM and CaMBPs and their
functions. showed
[ lz51]CaM
synaptosomal
CaMBPs of the
should
CaM,calmodulin; membrane.
gel
membranes
CaPt-dependent
*To whom correspondence Abbreviations: SM,synaptosomal
calcium-binding activities
proteins(CaMBPs)
We previously of EGTA-washed
neuronal
intracellular
physiological
the
a ubiquitous distribution
CaM-binding
CaMBPs are
between
is
associated
type
profiles
overlay
(SM)
from
were
identified
rat
of the brain
Some of
by immunological
be addressed. CaMBP,CaM-binding
(3).
CaMBPs
protein;
Vol.
183, No. 2, 1992 and enzymic comparing found
properties effects
analyses, them with
In this are
BIOCHEMICAL
report, only
in
AND BIOPHYSICAL
and others those
from
we show that the membrane
different of phosphorylation
from
those
were
tentatively
other
identified
by
tissues.
CaMBPs of the fraction
Ca 2t -independent
and have
of the
on their
RESEARCH COMMUNICATIONS
physico-chemical
Ca2'-dependent
CaM-binding
type
type.
properties
Possible were
also
examined.
MATERIALS
AND METHODS
Bovine brain CaM was purchased from Sigma Chemical Co. [ 125 IJCaM (92.5 uCi/ug, bovine brain) was purchased from New England Nuclear. All other chemicals were of reagent-grade. Male Sprague-Dawly rats (125-150 g) were obtained from Shizuoka Laboratory Animal Center (Hamamatsu). The EGTA-washed lysed SM was prepared from rat brain cortex and cerebellum by a modified method of Malnoe et al. (4) as described previously (5). Briefly, the brain was homogenized in medium A (2 mM EGTA,SO ug/ml PMSF, 0.2 ug/ml leupeptin, 0.5 mMDTT.5 mMTris-Xl, pH 7.5) containing 0.32 M sucrose and centrifuged at 1,OOOxg for 20 min. The supernatant was further centrifuged at 10,OOOxg for 15 min and the The pellet was suspended in medium A supernatant (S2) was recovered. containing 0.32M sucrose, diluted with medium B (medium A at pH 8.1) and centrifuged at 27,OOOxg for 30 min. The pellet was resuspended and centrifuged at 27,OOOxg for 30 min. The final pellet was suspended in medium C (60 mM Tris-HCl, pH 7.5, 5.0 mM MgS04, 0.5 mM DTT) (P,), and stored at -80°C. In addition to EGTA-washed SM "untreated" and "Ca"-treated" SMs were prepared according to the same procedures as EGTA-washed SM except that EGTA was excluded from or 100 uM CaC12 was added to the medium, respectively. [1251]CaM gel overlay was performed by a modified procedure of Hertzberg et a1.(6) as described previously (3). The membrane preparations (2-3 mg protein/ml) were mixed with l/3 vol. of the sample buffer consisting of 125 mM Tris-HCl, pH 6.6, 3X(w/v) SDS, lOX(w/v) glycerol and S%(v/v) 2-mercaptoethanol. A sample (15-25 ug protein) of each membrane preparation was immediately subjected to SDS-PAGE(12X gel) in the Laemmli system (7) without heat-denaturation unless otherwise stated. The final gels after pretreatment were incubated in the medium (50 mM Tris-HCl. pH 7.5, 0.2 M NaCl) containing 0.02% NaN3, 10 n&l [1251]CaM (1.8~10~ cpm/ml) and either 100 uM CaC12 or 1.0 rnkfEGTA. After the gels were exhaustively washed, they were stained, destained, dried and then exposed to X-ray films for 24-80 h with an enhancing screen. Phosphorylation was performed according to the conventional procedures. The reaction mixture contained 60 mM Tris-HCl,pH 7.5, 5.0 ug protein of SMs, 1.0 mM mu MgS04, 0.5 mM DTT, 100 uM ATP, 110-125 EGTA and 0.943 mM CaC12 (pCa 6.0) and either 1 uM CaM or 40 uM CAMP in a total volume of 100 ul. The reaction was initiated by adding 30 ul of SM and allowed to proceed for 1 min at 3O'C. The reaction mixture then received 25 ul of the sample buffer, followed by heating 726
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
for 2 min at 100 'C, and a 20-ul aliquot of the mixture was subjected to SDS-PAGE (12X gel), followed by [ 1251]CaM gel overlay as described above. Protein content was determined by the method of Lowry et a1.(8).
RESULTS CaMBPs of cerebral
cortical
classified
by their
those
mass higher
with
with
mass lower The bigger
1351127 1, panel
k,
of proteins
l-4)
1, panel
(S2),
A, lanes
cerebellar
their
with
1).
molecular
variously
relative
enriched based
cerebral
In the
k = 58 k > soluble
was 58 k = 49 k >> 235K = 1351127
A kDa
12345676
235135/127g=
B kDa
Fig.1. [ 1251]CaM and Ca2+-treated
rzel overlay SMs (lanes
for CaMBPs. 2 and 4) and
EGTA-washed corresponding
(lanes 1 and soluble
3)
fractions (lanes 5-8) were obtained from cerebral cortex (lanes 1.2,s and 6) and cerebellum (lanes 3.4.7 and 8) as described in the Method. T:z5same amount of protein (18 ug) was subjected to SDS-PAGE (12%) and I]CaM gel overlay was performed [ CaC12 (A) or 1 mM EGTA (B), followed a representative of six experiments
in
the presence by autoradiography.
with
three
of
different
in
5-8).
abundance
> 235 kDa for 3 and 4).
masses
(lanes
1 and 2) and 1351127
SMs (lanes
abundance
and those
fractions
their
was 49 k > 58 k > 135/127k
SMs (Fig.
two groups: (Fig.
They were
and soluble
of Ca2+,
tentatively
components")
components")
58 k, and 49 kDa. presence
235 k = 49 kDa for fractions
mass on SDS-PAGE into
consisted
A, lanes
SMs were
49 kDa ("bigger
34 kDa ("smaller
in the
on CaM binding cortical
than
components
of 235 k,
When assayed
molecular
than
SMs (Fig.
and cerebellar
either The
100 )H data is
preparations.
kDa
Vol.
183,
No.
2, 1992
for
the
cerebral
= 49 kDa for or Cazt
cortex
the
in the
dependent "untreated not k, while the
22.5
24-kDa
Soluble
were
assayed
lanes
l-8).
devoid There
panel
A),
the
enhanced with
presence
binding
the
Ca2+-
final
components
to that
k
EGTA
of an
of EGTA-washed
SM
A, lanes
in
bigger
Heating definite component
and 22.5-kDa heating
Ca2+ -independent Fig.
was prepared
cortex
and
(Fig.
[ 1251]CaM
1, panels
binding
and CaPt-treated 1, panel
of the
preparations A, four
31.5
A and
by the
k-kDa
pairs
of
and 24-kDa
the Ca 2t-treated
cerebellar
CaM binding
SM (Fig.
of Ca 2t was characteristic
1,
of the
B).
from
both
no 24-kDa
the cerebral component
in the
a 22.5-kDa
presence
component
when
assayed
in
cortex
and
was detected
in
of Ca 2t (Fig.
1,
and ones with the
was
presence
the
lower of EGTA
1 and 2). on the
of the
components
(lanes
(filled
arrows). activity
electrophoresis
overlay
pattern
CaM binding
2, panels 24-kDa
staining not
was detected
B).
728
(Fig.
of the
(cerebellum;
(data
235 kDa component
had a
(Fig.
3-8;
panel
no longer could shown). at the 2, panel
2, panels
A
235-kDa
A and B, lanes
1 and 2) could Protein
aggregation
binding 1, panel
before
gel
and 3-8). (Fig.
those
to the
samples
[125 I]CaM l-2
was due to protein
corresponding
cerebral
in
in
assayed
detected
lanes
whereas
cerebellar
same membrane
components
absence
Although
was increased
arrows),
1 and
In fact, it was markedly 2t of EGTA. No Ca -independent CaM binding
of the membrane effect
of 34 k,
kDa
1. lanes
in EGTA-washed
of Ca *+(Fig.
the
masses
The 31.5
SM (Fig.
the
the
smaller
1, panel
1 and 2),
and B; compare
B).
to the Ca 2t-dependent
SM when
mass were B, lanes
both
components
B).
by molecular
4).
decreased
presence
(panel
when
CaM bindings
(Fig.
cortical
molecular
from
In contrast
the
cerebellum
compare
in
1, panel
EGTA-washed
markedly
by the
cerebral
this
on the
cortical
(lane
of the
the
components
(Fig.
was no difference
the
were
3 and 4).
Ca '+
between
in
(lane
after
influence
characterized
lower
absent
(S2)
However,
components
(panel
similar
was predominant
of added
5-8).
when
panel
of either
The corresponding
were
component
components
found
COMMUNICATIONS
Presence
components
in cerebral
fractions
cerebellum B. lanes
smaller
bigger A).
or
3) but was almost presence
bigger
kDa
was predominant
SM (lane in
had little
a CaM binding
components
24 k,
component Z),
RESEARCH
shown).
The smaller 31.5
of the
SM" showed
7 and 8).
medium
1, panel
BIOPHYSICAL
5 and 6) and 58 k >> 235 k = 135/127 (lanes
preparation (Fig.
AND
(lanes
cerebellum
CaM binding
preparations (data
BIOCHEMICAL
open
B, lane
2)
be observed not
determine
Unexpectedly.
a
position B, lanes
if
3-8;
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A 12345676
kDa
31.524-
4
6 12345678
kDa
gel overlay for heat-denatured,& phosphorylated Fig.2. [125 I]CaM CaMBPs EGTA-washed SMs were prepared from --and then heat-denatured cerebral cortex (lanes l=and 7) or cerebellum (lanes 2,4,6, and 8) as described in the Method. Membrane proteins were subjected to SDSPAGE either after (lanes 3-8) or without (lanes 1 and 2) heatgel overlay was performed in the presence of denaturation. [ 1251]CaM either 100 p CaC12 (A) or 1 mM EGTA (B), followed by autoradiography. When indicated, proteins were phosphorylated in a CAMP- (lanes 5 and 6) and a Ca2+/CaM(lanes 7 and 8) dependent manners. To terminate the phosphorylation reaction, SMs were heat-denatured. The conditions of endogenous phosphorylation were described in the Method. On heating CaM binding of 235 kDa (open arrows) increased but that of 24/22.5 kDa bands (filled arrows) was lost. The data was a representative of two independent experiments
Membrane
proteins
dependent then
manner
tested
However, except
for
A,
lane
in
component
7).
were
their
CaM-binding
blur
between
58
also
slightly
noted
increased
that on
(Fig.
CaM
CaM
2,
binding
k-Da
in
or
a Ca 2+ICaM-
electrophoresed
and
49-kDa
this
and
lanes
profile
manner,
heterogeneity
was
a CAMP-
capacities in
a Ca2+/CaM-dependent
molecular It
in
heat-denatured,
alteration
a slight
in
They
remarkable
phosphorylated increase
phosphorylated
(3).
for no
were
5-8). was
which
bands
indicating mass
binding
range by
phosphorylation
the (lanes
detected
were
some (Fig. 235
2.
panel
kDa 5-8).
DISCUSSION Some
Ca2+ ions from
the
speculations
can
demonstrated membrane
in to
the
be made the soluble
about
present
the work.
compartment
729
of CaMJ3Ps to
susceptibility 1.
CaMBPs were
mobilized
as
reported
Saitoh
by
and
Vol.
183, No. 2, 1992
Schwartz
(9),
modified
the
induced
BIOCHEMICAL
2. EGTA stabilized activities
The abolition
the
possibility study.
could It
their
could
not also
CaM-binding
denaturation
smaller
corresponded,
70-,
235-kDa
calspectin 24/22.5-kDa
Synapsin
a Ca2+-dependent
which
tentatively There
three
Neuromodulin
(GAP43,
inner
surface
was found
that
CaM in the seemed a higher (15).
the
support affinity
It
B50, 24-kDa
previous
for
CaM in
was assumed
neuronal
cell
membrane
response
to an increase
phosphorylation rich
C kinase
independent
that in
by protein substrates type,
were
protein
4.1
The 58-
and 49-kDa
heterogeneity
subunits.
Mr=24,000) (11).
kinase
presence
C
sequester
more
This
in
with
work
SM bound
neuromodulin
of Ca2+ than could
associated present
of Ca2+.
that
absence
is
In the
cerebellar
observation
finding exhibited
its
presence
CaM at the
Ca 2t conditions and release 2t intracellular Ca and/or to
it
kinase
alanine-
low
(MARCKS)", found
of erythroid
on phosphorylation protein
of the
in the
were
and neurogranin.
Fl;
neuromodulin
under
84/82-kDa
from
membrane
the
protein,
distinct
pp46,
of Ca2+ than
aggregated
which
component
the
the
(14)
brain-specific
~57,
or not
such as
membrane
II
I,
a slightly
including
I and a related
plasma
synapsin
whether
components
These
The high
component
MARCKS protein,
of the
absence
to
was uncertain
for
which
to calspectin,
as CaM kinase
neuromodulin,
components
CaM binding.
well-known
(3).
on phosphorylation,
an increased
identified
are
substrates: the
showed
by heat-
substrates
respectively.
it
synaptosomal
membrane components
present
obtained
kinases
and 49-kDa
of multiple
and enzymically
showed
caused
contrary,
protein
showed,
However,
components. of bovine
this
in the
endogenous
mass basis,
immunologically
were
58-,
band
or a mixture
protein,
identified
was made up of a single
component
still
CaMBPs lost
change
on the
and beta-subunits,
CaM binding.
235-kDa
CaMBPs,
by
activity.
235-,
alpha-
weight
activated
astructural
larger
on the molecular II
molecluar
through
the
while
However,
staining
Ca*+ /CaM-dependent
included
CaM kinase
activity.
that
the
and
mass region
by protein
CaMBPs of SMs were
CAMP-dependentsubstrates
molecular
components and their
be supported
CaM-binding
Among the
24/22.5-kDa
be hypothesized
and that
and 3. Ca2+
such as Ca 2+-
an aggregation
CaM-binding
activity
Ca*+ -independent
of the
higher
Cazt -independent
of CaMBPs,
degradation.
indicate
to the
RESEARCH COMMUNICATIONS
a mechanism(s)
CaM binding
seemed to migration
retaining
structure
or proteolytic
of the
heat-denaturation
the
of CaMBPs by
phosphorylation
subsequent
AND BIOPHYSICAL
C (16).
The
a class
to release 730
"myristoylated
of CaMBPs of Ca'+-
CaM when
they
were
in
it
Vol.
183,
No.
2, 1992
BIOCHEMICAL
phosphorylated
by protein
AND
kinase
C (lO).It
(~17;
Mr=15,000-19,000)
also
(17).
These
neuromodulin
classified based
two CaMBPs, into
the
on both
binds
"smaller
BIOPHYSICAL
is
known
to CaM in the
that
defined
mass and the
COMMUNICATIONS
neurogranin
absence
and neurogranin,
components"
the molecular
RESEARCH
of Ca2+
can be in the
present
Ca2+-independent
work
manner
of
CaM binding. Another
CaMBP of synaptosomal
was reported (18).
to bind
However,
support
the
to calspectin
in the
present
assumption
corresponded
the
to cytosynalin.
More
was phosphorylated
CaM kinase
II.
Very
candidates
for
smaller
Identification
there
34-kDa
was found
cytosynalin
and interact
work
that
ACAMP-81(Mr=81,000), protein
membrane,
in the
by both
with
evidence in the
a new,
bovine
brain
histone type
is
in
(19).
protein
itself
is
work
CaMBP,
membrane
CaMBPs of Ca 2t-independent
of CaMBPs of this
to
present
acidic
CAMP-dependent
interestingly,
cytoskeleton
was little component
recently,
(Mr=35,000),
This
kinase
one of
the
(12,
13).
type
and
progress.
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COMMUNICATIONS
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