Calmodulin dependent multifunctional protein kinase in Aspergillus nidulans

Abstract

A Ca2+/calmodulin (CaM)-dependent multifunctional protein kinase has been isolated from Aspergillus nidulans and purified to homogeneity. Unlike any CaM-dependent multifunctional protein kinase described previously, the native enzyme from Aspergillus behaves as a monomer. The calculated molecular weight is 41,200. NaDodSO4/PAGE reveals a single protein band with an apparent Mr of 51,000. Two-dimensional isoelectric focusing/NaDodSO4/PAGE of the purified enzyme showed one major and one minor more acidic Coomassie blue-stained spot, both of which bind 125I-labeled calmodulin in a calcium-dependent manner. The kinase is autophosphorylated in a calcium- and CaM-dependent manner, yielding an increase in the amount and number of more acidic forms of the enzyme. The Aspergillus kinase catalyzes the Ca2+/CaM-dependent phosphorylation of known substrates of type II Ca2+/CaM-dependent protein kinases, including glycogen synthase, microtubule-associated protein 2, synapsin, tubulin, gizzard myosin light chain, and casein. Cross-reactivity between antiserum raised against native rat brain protein kinase II and 125I-labeled Aspergillus kinase has been detected. Two forms of CaM have been isolated from Aspergillus nidulans, both of which activate the Aspergillus kinase at lower concentrations than that required for activation by bovine brain CaM.

Full text

Proc.
Natl.
Acad.
Sci.
USA
Vol.
85,
pp.
3279-3283,
May
1988
Biochemistry
Calmodulin-dependent
multifunctional
protein
kinase
in
Aspergillus
nidulans
(protein
kinase
iI/fungal/calmodulins)
DIANA
C.
BARTELT*,
SETH
FIDELt,
LEN
H.
FARBERt,
DONALD
J.
WOLFF,
AND
ROBIN
L.
HAMMELL
Department
of
Pharmacology,
University
of
Medicine
and
Dentistry
of
New
Jersey,
Robert
Wood
Johnson
Medical
School,
675
Hoes
Lane,
Piscataway,
NJ
08854
Communicated
by
Aaron
J.
Shatkin,
January
4,
1988
ABSTRACT
A
Ca2e/calmodulin
(CaM)-dependent
mul-
tifunctional
protein
kinase
has
been
isolated
from
Aspergillus
nidulans
and
purified
to
homogeneity.
Unlike
any
CaM-
dependent
multifunctional
protein
kinase
described
previ-
ously,
the
native
enzyme
from
Aspergillus
behaves
as
a
mono-
mer.
The
calculated
molecular
weight
is
41,200.
NaDodSO4/
PAGE
reveals
a
single
protein
band
with
an
apparent
Mr
of
51,000.
Two-dimensional
isoelectric
focusing/NaDodSO4/
PAGE
of
the
purified
enzyme
showed
one
major
and
one
minor
more
acidic
Coomassie
blue-stained
spot,
both
of
which
bind
1251-labeled
calmodulin
in
a
calcium-dependent
manner.
The
kinase
is
autophosphorylated
in
a
calcium-
and
CaM-depen-
dent
manner,
yielding
an
increase
in
the
amount
and
number
of
more
acidic
forms
of
the
enzyme.
The
Aspergillus
kinase
catalyzes
the
Ca2
4/CaM-dependent
phosphorylation
of
known
substrates
of
type
II
Ca2+/CaM-dependent
protein
kinases,
including
glycogen
synthase,
microtubule-associated
protein
2,
synapsin,
tubulin,
gizzard
myosin
light
chain,
and
casein.
Cross-reactivity
between
antiserum
raised
against
native
rat
brain
protein
kinase
II
and
'251-labeled
Aspergillus
kinase
has
been
detected.
Two
forms
of
CaM
have
been
isolated
from
Aspergillus
nidulans,
both
of
which
activate
the
Aspergilius
kinase
at
lower
concentrations
than
that
required
for
activation
by
bovine
brain
CaM.
There
is
increasing
evidence
that
Ca2
+
mediates
cell
function
through
Ca2
+-dependent
phosphorylation
of
regulatory
en-
zymes
and
proteins
(1,
2).
A
Ca2
+/calmodulin
(CaM)-regu-
lated
protein
kinase
capable
of
phosphorylating
and
coordi-
nately
modifying
the
activities
of
several
regulatory
enzymes
and
structural
proteins
could
play
a
pivotal
role
in
cellular
responses
to
stimuli
that
are
mediated
by
Ca2
.
A
class
of
Ca2
+/CaM-independent
multifunctional
kinases
(kinase
II)
was
detected
first
in
rat
brain
(3,
4)
and
later
in
several
tissues
of
both
mammalian
and
nonmammalian
origin
(5-7).
This
class
of
Ca2+
/CaM-regulated
protein
kinases
is
unique
among
Ca2
+
/CaM-dependent
protein
kinases
in
that
it
is
the
only
one
capable
of
in
vitro
phosphorylation
of
a
broad
range
of
protein
substrates
(8).
The
most
well-characterized
Ca2
+/CaM-dependent
kinase
II
(CaM-PK
II),
the
rat
brain
enzyme,
is
a
heteropolymer
with
a
native
molecular
weight
of
-600,000
and
is
present
in
forebrain
neurons
in
high
con-
centrations
(3,
4).
Evidence
has
been
presented
to
implicate
CaM-PK
II
in
Ca2+-dependent
neurotransmitter
release
(3,
4, 9,
10).
Recently,
the
genes
encoding
both
the
a
and
P
subunits
of
rat
brain
CaM-PK
II
have
been
cloned
and
sequenced
(11,
12).
The
study
of
the
in
vivo
function(s)
of
multifunctional
CaM-dependent
protein
kinases
could
be
greatly
facilitated
by
the
capacity
to
manipulate
the
gene(s)
encoding
the
enzyme.
Gene
disruption
by
site-specific
integrative
trans-
formation
has
been
accomplished
in
several
organisms
(13,
14),
including
Aspergillus
nidulans
(15).
We
examined
A.
nidulans
for
the
presence
of
a
multifunctional
CaM-depen-
dent
protein
kinase
with
a
view
toward
studying
its
function
in
vivo
in
A.
nidulans
by
gene
manipulation.
We
report
here
the
characterization
of
a
multifunctional
CaM-dependent
protein
kinase
from
A.
nidulans
(ACMPK).
EXPERIMENTAL
PROCEDURES
Materials.
Centricon
and
Centriprep
microconcentrators
were
purchased
from
Amicon.
Triton
X-100
and
insoluble
protein
A
were
obtained
from
Sigma.
Glycogen
synthase,
Syntide
2
(16),
rat
brain
Ca2
+
/CaM-PK
II
and
goat
preimmune
and
antiserum
raised
against
native
rat
brain
CaM-PK
II
were
the
generous
gifts
of
Thomas
Soderling
and
Roger
Colbran
(Vanderbilt
University
and
the
Howard
Hughes
Institute,
Nashville,
TN).
Bovine
brain
synapsin
I
was
the
generous
gift
of
Mary
Kennedy
(California
Institute
of
Technology,
Pasa-
dena,
CA).
All
other
materials
were
obtained
from
sources
or
prepared
by
methods
described
in
ref.
17.
Methods.
Proteins
tested
as
substrates
for
ACMPK
were
prepared
by
procedures
described
or
cited
in
ref.
17.
Bovine
brain
CaM
and
ACMPK
were
labeled
with
1251I
as
described
(17).
Two
forms
of
CaM
were
prepared
from
A.
nidulans
by
a
modification
of
the
method
of
Gopalakrishna
and
Anderson
(18)
and
resolved
by
preparative
PAGE
under
nondenaturing
conditions
(19).
One-dimensional
NaDodSO4/PAGE
was
performed
on
7.5-15%
acrylamide
gradient
gels
by
the
procedure
of
Laemmli
(20).
Two-dimensional
separation
of
proteins
was
achieved
by
the
method
of
O'Farrell
(21).
CaM-binding
proteins
were
detected
after
two-dimensional
separation
and
electrophoretic
transfer
of
proteins
to
nitro-
cellulose
(22)
by
binding
of
1251I-labeled
CaM
(1251I-CaM)
as
described
(23).
Samples
containing
purified
ACMPK
were
subjected
to
sucrose
density
gradient
ultracentrifugation
as
described
(17, 24).
The
molecular
weight
of
the
kinase
was
calculated
as
described
(25).
Isoelectric
focusing
in
poly-
acrylamide
tube
gels
under
nondenaturing
conditions
was
performed
as
described
by
O'Farrell
(21)
with
urea
omitted
from
the
gel
and
sample.
Triplicate
gels
were
focused
and
then
sliced.
Protein
was
eluted
from
one
gel
by
diffusion
into
25
mM
Tris
HCl,
pH
7.5/100
mM
NaCl/1
mM
MgCl2/1
mM
EGTA/0.2
mM
diisopropyl
fluorophosphate/leupeptin
(3
,g/ml)/5%
(vol/vol)
glycerol/0.05%
Tween
20
(buffer
A),
and
the
eluates
were
assayed
for
protein
kinase
activity.
The
Abbreviations:
CaM,
calmodulin;
CaM-PK
II,
Ca2
/CaM-depen-
dent
protein
kinase
II;
ACMPK,
Aspergillus
Ca2"/CaM-dependent
multifunctional
protein
kinase;
MAP,
microtubule-associated
pro-
tein;
MLC,
myosin
light
chain;
1251I-CaM,
125I-labeled
CaM;
125i-
ACMPK,
125I-labeled
ACMPK.
*To
whom
reprint
requests
should
be
addressed.
tPresent
address:
Department
of
Surgery,
Children's
Hospital,
Boston,
MA
02115.
tPresent
address:
Scripps
Clinic
and
Research
Foundation,
Division
of
Preclinical
Neuroscience
and
Endocrinology,
La
Jolla,
CA
92037.
3279
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
slices
of
the
second
gel
were
subjected
to
NaDodSO4/PAGE
to
locate
the
position
of
protein
bands.
The
pH
gradient
of
the
isoelectric
focusing
gel
was
determined
after
incubating
slices
of
the
gel
in
H20.
Assay
of
CaM-Dependent
Protein
Phosphorylation.
Phos-
phate
(32p)
incorporation
into
25
,g
of
gizzard
myosin
light
chain
(MLC)
catalyzed
by
ACMPK
was
measured
at
30TC
in
a
final
vol
of
50
1ul
of
25
mM
Tris-HCl,
pH
7.5/10
mM
MgCl2/100
uM
(y-32PJATP
(500
cpm/pmol),
and
either
0.5
mM
EGTA
or
1
mM
CaCI2,
in
the
absence
or
presence
of
CaM
(10
,ug/ml).
The
reactions
were
terminated
and
phos-
phorylation
was
quantified
as
described
(17).
Proteins
were
tested
as
substrates
for
ACMPK
at
concentrations
of
5-25
,ug
per
25-,ul
assay
mixture.
Protein
concentrations
were
mea-
sured
as
described
(26,
27).
When
comparing
the
substrate
specificities
of
the
ACMPK
to
that
of
rat
brain
CaM-PK
II,
assays
were
conducted
as
described
by
Schworer
et
al.
(16).
Phosphorylation
of
peptide
substrates
was
quantitated
as
described
(17).
In
the
autophosphorylation
experiment
shown
in
Fig.
2,
4
,ug
of
ACMPK
was
incubated
for
15
min
at
30°C
under
the
conditions
described
above.
Reactions
were
terminated
by
the
addition
of
200
1,u
of
lysis
buffer
(21).
Buffer
and
free
[-32P]ATP
were
removed
and
samples
were
concentrated
by
ultrafiltration
prior
to
application
to
isoelec-
tric
focusing
gels.
Experiments
testing
the
effect
of
autophos-
phorylation
on
CaM
dependence
of
both
rat
brain
protein
kinase
II
and
ACMPK
were
performed
as
described
(16).
Immunoprec~ipitation
of
Aspergillus
CaM.
Dilutions
of
goat
preimmune
and
antiserum
raised
against
native
rat
brain
CaM-PK
II
were
incubated
with
0.2-1.4
Ig
of
1251-labeled
ACMPK
(125I-ACMPK)
(1.5
x
i05
cpm/,ug)
for
30
min
at
30°C.
Immunoprecipitates
were
collected
by
using
insoluble
protein
A
in
a
modification
of
the
method
described
in
ref.
28.
Precipitated
antigen
was
eluted
from
protein
A
and,
after
centrifugation,
the
samples
were
concentrated
and
aliquots
were
subjected
to
NaDodSO4/PAGE.
Protein
bands
of
M,
51,000
were
cut
from
the
gels
and
counted.
Immunoblot
analyses
were
performed
as
described
by
Farber
et
al.
(29).
Purification
of
CaM-Dependent
Multifunctional
Protein
Ki-
nase
from
A.
nidulans.
Eight
1-liter
cultures
of
A.
nidulans
(R-153)
were
prepared
by
inoculation
of
YG
medium
(15)
with
8'
x
108
spores
per
liter
and
incubating
for
16
hr
at
37°C
with
shaking.
Aspergillus
mycelia
were
harvested
by
filtration
through
Miracloth,
and
pressed
dry,
yielding
115
g
(wet
weight)
of
cells.
The
mycelia
were
rehydrated
in
3
vol
of
25
mM
Tris-HCI,
pH
7.5/50-
mM
NaCl/1
mM
MgC12/1
mM
EGTA/0.2
mM
diisopropylfluorophosphate/0.1
,uM
pepstat-
in
A/leupeptin,
(3
,ug/ml),
and
disrupted
in
a
French
pressure
cell
at
14,000
psi
(1
psi
=
6.89
kDa).
The
lysate
was
centrifuged
at
80,000
x
g
for
30
min
and
the
pH
of
the
supernatant
liquid
was
adjusted
to
6.8.
The
extract
was
chromatographed
on
a
column
of
phosphocellulose
(2.5
x
25
cm)
equilibrated
with
25
mM
Pipes-KOH,
pH
6.8/50
mM
NaCl/1
mM
MgCl2/1
mM
EGTA/0.2
mM
diisopropylfluo-
rophosph-ate/0.1
tLM
pepstatin
A/leupeptin
(3
,ug/ml).
The
fraction
containing
CaM-dependent
protein
kinase
activity
was
eluted
in
the
buffer
described
above
made
250
mM
in
NaCl.
The
phosphocellulose
eluate
was
adjusted
to
pH
7.5
and
made
1.5
mM
in
CaC12.
The
sample
was
applied
to
a
column
of
CaM
Affi-Gel
(1.5
x
10
cm)
equilibrated
with
25
mM
Tris-HCl,
pH
7.5/100
mM
NaCI/1
mM
MgCI2/0.5
mM
CaCI2/0.2
mM
diisopropyl
fluorophosphate/leupeptin
(1
,ug/ml)/5%
(vol/vol)
glycerol/0.05%
Tween
20.
The
fraction
containing
CaM-dependent
protein
kinase
activity
was
eluted
with'buffer
A.
Proteins
eluted
from
CaM
Affi-Gel
in
buffer
A
were
concentrated
by
ultrafiltration
and
applied
to
a
column
of
Ultrogel
AcA
34
(1
x
55
cm)
equilibrated
in
buffer
A.
The
column
had
been
calibrated
as
described
(17).
Fractions
containing
CaM-dependent
protein
kinase
activity
were
com-
bined
and,
after
concentration,
were
stored
at
-
70°C.
RESULTS
Purification
of
CaM-Dependent
Multifunctional
Protein
Ki-
nase
from
A.
nidulans.
A
representative
scheme
for
the
purification
of
ACMPK
from
A.
nidulans
has
been
described
and
is
summarized
in
Table
1.
ACMPK,
like
rat
brain
CaM-PK
II,
binds
to
phosphocellulose
at
pH
6.8
(30).
Most
of
the
Ca2
-independent
protein
kinase
activity
as
well
as
the
two
endogenous
forms
of
CaM
are
not
bound
by
phospho-
cellulose
and
are
thus
separated
from
the
enzyme,
permitting
chromatography
of
the
ACMPK
on
CaM
Affi-Gel.
Elution
of
the
CaM
Affi-Gel
column
with
buffer
containing
EGTA
resulted
in
the
release
of
a
peak
containing
a
complex
mixture
of
proteins,
followed
by
effluent
containing
predominantly
a
Mr
51,000
protein
and
CaM-dependent
protein
kinase
activity
(data
not
shown).
Gel
filtration
of
the
CaM-dependent
kinase
activity
after
concentration
yielded
a
single
symmetrical
peak
of
CaM-dependent
kinase
activity
containing
>77%
of
the
activity
applied
was
eluted
(Fig.
LA).
Physical
Properties
of
ACMPK.
Upon
gel
filtration,
ACMPK
is
eluted
in
a
position
corresponding
to
a
protein
with
a
Stokes
radius
of
25.5
A
(Fig.
LA).
During
sucrose
density-gradient
ultracentrifugation
of
the
purified
enzyme,
>70o
of
the
CaM-dependent
kinase
activity
applied
sedi-
ments
between
CaM
and
hemoglobin
with
an
S
value
-of
3.9
(Fig.
1B).
When
aliquots
of
these
fractions
are
subjected
to
NaDodSO4/PAGE,
the
intensity
of
a
Coomassie
blue-stained
protein
band
of
Mr
51,000
correlates
with
kinase
activity
(data
not
shown).
Using
the
Stokes
radius
and
S
value
in
the
equations
given
in
ref.
25,
an
Mr
of
41,200
and
a
frictional
coefficient
of
1.12
were
calculated
for
ACMPK.
After
non-
denaturing
isoelectric
focusing
of
ACMPK,
a
peak
of
CaM-
dependent
kinase
activity
was
eluted
from
gel
slices
corre-
sponding
to
pH
6.0-6.4,
with
the
peak
of
activity
at
pH
6.2.
A
comparison
of
the
physical
properties
of
ACMPK
with
those
of
the
rat
brain
CaM-PK
II
is
given
in
Table
2.
Two-dimensional
isoelectric
focusing/NaDodSO4/PAGE
of
purified
ACMPK
reveals
one
major
and
one
minor
more
acidic
protein
spot
with
apparent
Mrs
of
51,000
when
the
gel
is
stained
with
Coomassie
blue
(Fig.
2A).
Upon
silver
staining
of
the
same
gel,
three
additional
more
acidic
Mr
51,000
protein
spots
are
visible
(data
not
shown).
The
number
and
intensity
of
these
minor
spots
yaries
with
the
preparation
of
ACMPK.
Upon
incubation
of
a
nitrocellulose
transfer
of
a
Table
1.
Purification
of
Aspergillus
CaM-dependent
multifunctional
protein
kinase
CaM-dependent
Purifi-
Protein,
MLC
kinase,
Specific
activity,
Yield,
cation,
mg*
nmol/min
nmol-min-
'-mg1
%
-fold
80,000
x
g
supernate
3100
ND
Phosphocellulose
eluate
182
5.184
0.028
(100)
(1.00)
CaM-Affi-Gel
EGTA
eluate
0.194
2.791
14.39
53.9
514
Ultrogel
AcA-34
0.041
2.148
52.28
41.5
1867
Starting
material
was
115
g
(wet
weight)
of
A.
nidulans.
ND,
not
detectable.
Numbers
in
parentheses
are
arbitrarily
set
values
for
starting
material.
*Determined
by
the
method
of
Schaffner
and
Weissmann
(27).
3280
Biochemistry:
Bartelt
et
al.

Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
3281
U-)
t
I
0
*
uL
-
>
;T
rOC'J
200
-
\
°
50I\
E
I00
A
1f
0-
E
0
50
E
C
10
20
30
40
°
Elution
Volume,
ml
u
II(
B
9
13
17
21
Fraction
FIG.
1.
Estimation
of
the
M,
of
ACMPK.
(A)
Purified
ACMPK
was
subjected
to
gel
filtration
on
Ultrogel
AcA
34
as
described.
Arrows
mark
the
void
volume
of
the
column
(VO)
and
the
elution
positions
of
protein
standards
with
Stokes
radii,
which
are
given
above
the
arrow.
(B)
Sucrose
density-gradient
ultracentrifugation
of
purified
ACMPK
was
performed
as
described.
Arrows
mark
the
sedimentation
positions
of
proteins
with
known
sedimentation
co-
efficients,
which
are
given
above
the
arrows.
duplicate
two-dimensional
gel
with
`25I-CaM,
Ca2+-depen-
dent
CaM-binding
is
detected
only
in
a
position
correspond-
ing
to
the
Coomassie
blue-stained
material
(Fig.
2B).
Autophosphorylation
of
ACMPK.
Incubation
of
ACMPK
with
[y-32P]ATP
followed
by
two-dimensional
separation
by
isoelectric
focusing/NaDodSO4/PAGE
reveals
incorpora-
tion
of
32P
into
the
M,
51,000
protein.
Fig.
2C
shows
a
Coomassie
blue-stained
gel
and
Fig.
2D
is
the
corresponding
autoradiogram
of
ACMPK
after
autophosphorylation
for
15
min
in
the
presence
of
Ca2"
and
CaM.
In
the
autophosphory-
lated
sample,
there
is
an
increase
in
the
amount
of
the
more
acidic
spot
on
the
left
(Fig.
2C)
as
compared
to
the
unphos-
phorylated
sample
(Fig.
2A).
Inclusion
of
Ca2"
and
CaM
in
the
incubation
increased
the
amount
of
32P
incorporated
into
all
forms
of
ACMPK
5.7-fold
with
the
majority
of
the
32P
incorporated
into
the
more
acidic
of
the
forms
of
ACMPK
(Fig.
2D).
When
autophosphorylation
was
allowed
to
pro-
ceed
for
150
min,
three
additional
more
acidic
labeled
proteins
were
detected
(Fig.
2E)
with
a
3-fold
increase
in
total
Table
2.
Comparison
of
physical
properties
of
multifunctional
kinases
-31
>
E2
.
IV
T
.
V
-14
+
-
-
FIG.
2.
125I-CaM
binding
to
and
autophosphorylation
of
ACMPK.
Two-dimensional
denaturing
isoelectric
focusing/NaDod-
S04/PAGE
analysis
of
ACMPK
purified
through
gel
filtration
before
(A
and
B)
and
after
(C-E)
autophosphorylation.
(A
and
C)
Coomassie
blue-stained
gels.
(B)
Autoradiogram
of
nitrocellulose
transfer
of
a
duplicate
of
the
gel
shown
in
A
after
incubation
with
'251-CaM
in
the
presence
of
calcium.
Binding
of
125I-CaM
was
calcium
dependent.
(D)
Autoradiogram
of
gel
in
C,
showing
32p
incorporation
into
ACMPK
kinase
after
incubation
of
the
enzyme
with
[Y-32P]ATP
in
the
presence
of
calcium
and
CaM
for
15
min.
Arrowheads
in
C
and
D
mark
the
position
of
the
two
Coomassie
blue-stained
spots.
(E)
Portion
of
an
autoradiogram
of
ACMPK
after
autophosphorylation
for
150
min.
Arrowheads
in
E
point
to
positions
of
phosphorylated
forms
of
ACMPK.
Arrowheads
between
panels
indicate
the
migra-
tion
of
protein
standards
of
Mr
values
x
10
-I
given
at
the
positions
of
the
arrows.
32P
incorporation
over
the
15-min
incubation.
The
effect
of
autophosphorylation
on
the
CaM
dependence
of
kinase
activity
of
both
rat
brain
CaM-PK
II
and
ACMPK
was
studied.
Autophosphorylation
of
rat
brain
CaM-PK
II
led
to
an
increase
in
CaM-independent
activity
from
11%
for
the
native
enzyme
to
88%
for
the
phosphorylated
form.
Auto-
phosphorylation
of
ACMPK
under
identical
conditions
yielded
no
increase
in
CaM-independent
activity;
however,
phosphate
incorporation
was
limited
to
0.003
mol
per
mol
of
ACMPK.
Aspergillus
and
rat
brain
CaM-dependent
Rat
Property
Method
Aspergillus
brain*
Stokes
radius,
A
Gel
filtration
25.5
94.7
Sedimentation
Density-gradient
3.9
16.4
coefficient,
S
ultracentrifugation
Frictional
coefficient,
f/fo
Calculationt
1.12
1.67
Subunit
molecular
weight
NaDodSO4/PAGE
a
51,000
51,000
B'
58,000
B
-
60,000
Native
molecular
weight
Gel
filtration
44,500
Calculationt
41,200
650,000
Isoelectric
point
Nondenaturing
6.2
isoelectric
focusing
*Data
are
from
Table
II
in
ref.
3.
tCalculated
according
to
the
equation
given
in
ref.
25.
A
C
B
-
200-
-
93
>
-
66
-45
-
-
31
-
-
21
.
-
-
14
-+
D
-200
-93
-66-
-45
-
Biochemistry:
Bartelt
et
al.
0-

Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
Substrate
Specificity
of
ACMPK.
A
comparison
of
the
substrate
specificities
of
ACMPK
and
rat
brain
CaM-PK
II
for
nine
substrates,
assayed
as
described
(16),
is
presented
in
Table
3.
Data
for
the
phosphorylation
of
four
additional
substrates
by
ACMPK
assayed
as
described
in
Experimental
Procedures
are
also
presented.
All
nine
substrates
tested
in
parallel
were
phosphorylated
in
a
Ca2
/CaM-dependent
manner
by
both
the
rat
brain
and
Aspergillus
enzymes.
Two
of
the
four
additional substrates,
phosphorylase
b
and
fodrin,
which
have been
reported
not
to
be
phosphorylated
by
CaM-PK
II
(4,
31),
are
not
phosphorylated
by
ACMPK.
Skeletal
muscle
MLC
and
histone
2b,
which
have
been
reported
to
be
phosphorylated
slowly
by
CaM-PK
II
(31,
32),
are
also
phosphorylated
by
ACMPK.
The
synthetic
peptides
containing
phosphorylation
site
2
of
glycogen
synthase'(Syn-
tide
2)
and
gizzard
MLC
(Kemptamide)
are
the
preferred
substrates
for
both
the
rat
brain
and
Aspergillus
enzymes,
and
microtubule-associated
protein
(MAP)
2,
glycogen
syn-
thase,
and
gizzard
MLC
are
the
three
protein
substrates
phosphorylated
at
the
highest
rates
by
both
of
the
enzymes.
CaM
Dependence
and
Kinetic
Properties
of
ACMPK.
Two
forms
of
CaM
having
apparent
Mrs
of
23,000
(form
1)
and
20,500
(form
2)
were
purified
from
A.
nidulans
and
separated
by
nondenaturing
PAGE
(D.J.W.
et
al.,
unpublished
obser-
vation).
Aspergillus
CaMs
1
and
2
were
compared
with
each
other
and
with
bovine
brain
CaM
for
their
ability
to
activate
ACMPK.
As
shown
in
Fig.
3,
all
three
CaMs
stimulate the
activity
of
ACMPK
at
least
25-fold.
Higher
concentrations
of
all
three
CaMs
inhibit
the
enzyme.
Both
forms
of
Aspergillus
CaM
appear
to
have
a
higher
affinity
than
bovine
brain
CaM
for
ACMPK,
with
Aspergillus
CaM
1
having
a
higher
affinity
but
lower
efficacy
than
CaM
2.
The
Ko.5
values
calculated
from
these
data
are
reported
'in
Table
4.
The
kinase
activity
of
ACMPK
toward
MLC
is
ATP-,
Mg2
+,
and
MLC-concentration
dependent.
A
comparison
of
the
kinetic
properties
of
ACMPK
with
those
reported
for
rat
brain
CaM-PK
II
(4)
is
presented
in
Table
4.
Due
to
the
lower
affinity
of
ACMPK
for
MLC,
it
was
not
possible
to
determine
the
Vman
of
the
enzyme
for
this
substrate.
Immunoreactivity
of
Antiserum
to
Rat
Brain
CaM-PK
II
with
ACMPK.
Cross-reactivity
between
antibody
to
native
rat
brain
CaM-PK
II
and
ACMPK
was
detected
by
incubation
of
125I-ACMPK
with
goat
preimmune
serum
and
antiserum
raised
against
native,
rat
brain
CaM-PK
II
followed
by
precip-
itation
with
insoluble
protein
A.
Between
1%
and
2%
of
the
total
1"I-ACMPK
was
precipitated
by
protein
A
derived
from
incubations
with
antiserum,
while
protein
A
precipitates
from
incubations
with
preimmune
serum
contained
no
net
1251_
Table
3.
Comparison
of
substrate
specificity
of
Aspergillus
and
rat
brain
CaM-dependent
multifunctional
kinases
Aspergillus,
Rat
brain,
Substrate
,uM
nmolfmg-
'min-1
nmol-mg-
l
min-
Syntide
2
150
41.3
2482
Kemptamide
150
28.8
455
MAP-2
3.6
25.8
83.3
Glycogen
synthase
5.3
24.6
121
Gizzard
MLC
40.0
8.7
83.5
Myelin
basic
protein
50.0
5.8
20.4
Tubulin
(a
and
,3)
8.0
2.5
6.7
Synapsin
2.3
1.7
43.7
Casein
7.1
1.6
5.5
Skeletal
muscle
MLC
25.0
1.3*
ND
Histone
2b
27.0
0.2*
ND
Phosphorylase
b
5.4
0.0*
ND
Fodrin
1.3
0.0*
ND
E
la
3.0-
E
-5
E
C-
2.0
0
0.
0
c-
a0
0-0
=
Aspergillus
A-A=Aspergillus
2
*-@=Bovine
Broin
100
300
500
Calmodulin
,
nM
FIG.
3.
Activation
of
ACMPK
by
CaMs
from
Aspergillus
and
bovine
brain.
ACMPK
(575
ng)
was
assayed
for
its
ability
to
phosphorylate
MLC
in
the
presence
of
1
mM
calcium
and
various
concentrations
of
the
two
Aspergillus
CaMs
and
bovine
brain
CaM
as
described.
labeled
material
over
precipitates
incubated
with
buffer.
To
verify
that
the
radioactivity
precipitated
was
125I-ACMPK,
the
protein
A
precipitates
were
treated
as
described
in
Experi-
mental
Procedures.
Precipitation
of
125I-ACMPK
was
quan-
titated
by
counting
labeled
Mr
51,000
protein
bands.
Antise-
rum
precipitated
4%
and
preimmune
serum
precipitated
1.7%
of
the
Mr
51,000
125I-ACMPK.
A
1:50
dilution
of
this
antiserum
will
precipitate
50%o
of
0.6
tug
of
rat
brain
CaM-PK
II
(R.
J.
Colbran,
personal
communication).
A
1:25
dilution
of
this
antiserum,
which
detected
both
the
a
and
13
subunits
of
5
pkg
of
rat
brain
CaM-PK
II
on
an
immunoblot,
failed
to
detect
5
Ag
of
ACMPK
(data
not
shown).
DISCUSSION
CaM-dependent
protein
kinases
are
characterized
not
only
by
their
almost
total
dependence
on
Ca2+
and
CaM
for
activity
but
also
their
high
degree
of
substrate
specificity
(8,
32).
Phosphorylase
kinases
phosphorylate
phosphorylase
b
and,
to
some
extent,
glycogen
synthase
(33).
MLC
kinases
phosphorylate
only
MLCs
(8,
32).
CaM-dependent
protein
kinase
I
phosphorylates
only
synapsin
I
and
protein
III
and
smooth
muscle
MLC
(34).
CaM-dependent
protein
kinase
III
phosphorylates
only
a
Mr
100,000
protein
(35),
which
has
been
identified
as
eukaryotic
elongation
factor
2
(36).
The
exception
to
this
high
degree
of
substrate
specificity
is
a
family
of
ubiquitous
multifunctional
Ca2
+/CaM-dependent
protein
kinases,
now
commonly
denoted
CaM-PK
II
(8,
32).
Table
4.
Comparison
of
kinetic
properties
of
Aspergillus
and
rat
brain
CaM-dependent
multifunctional
kinases
Rat
Parameter
Aspergillus*
brain
Ko.
ATP,
/LM
17
22
KO.5
Mg2+,
mM
4.0
5.0
Gizzard
MLC
KO.5,
AM
246
22
Vmax,
nmol
per
min
per
mg
of
protein
-52.3
185
Ko.
CaM,
nM
Bovine
brain
320
29-620t
Aspergillus
1
55
Aspergillus
2
105
*All
measurements
were
made
using
chicken
gizzard
MLC
as
substrate.
The
preparation
contained
>90%o
phosphorylatable
light
chain.
tData
are
from
Kuret
and
Schulman
(4).
The
values
for
K0.
for
CaM
of
29
and
620
nM
were
obtained
by
using
phosphovitin
and
casein,
respectively,
as
substrates.
Except
where
indicated,
assays
were
performed
as
described
in
ref.
16.
ND,
not
determined.
*Assays
were
'performed
as
described
in
Experimental
Procedures.
3282
Biochemistry:
Bartelt
et
al.

Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
3283
These
enzymes
phosphorylate
a
broad
range
of
the
same
protein
substrates
(32)
and
are
characteristically
composed
of
Mr
50,000-60,000
subunits,
all
of
which
are
catalytic,
are
autophosphorylated
in
a
Ca2
+
/CaM-dependent
manner,
and
bind
CaM
in
a
Ca2'-dependent
manner
(8,
32).
Multifunc-
tional
CaM-PK
Ils
have
been
isolated
from
mammalian
brain
(3,
4),
liver
(5),
pancreas
(37),
and
skeletal
muscle
(38)
as
well
as
the
nonmammalian
tissues
Torpedo
electric
organ
(6),
Aplysia
neurons
(7),
and
sea
urchin
egg
(39).
Cross-reactivity
between
antibodies
to
rat
brain
CaM-PK
II
and
CaM-PK
Ils
from
rabbit
skeletal
muscle
(40)
and
Aplysia
californica
(41)
have
been
reported.
ACMPK
phosphorylates
a
broad
range
of
the
same
protein
substrates
as
CaM-PK
Ils
from
higher
eukaryotes.
In
like
manner
to
CaM-PK
II,
ACMPK
phosphorylates
synthetic
peptides
containing
the
phosphorylation
sites
of
glycogen
synthase
and
MLC
at
rates
significantly
higher
than
the
native
protein
substrates.
It
is
composed
of
a
Mr
51,000
protein,
which
is
autophosphorylated
in
a
Ca2
+
/CaM-
dependent
manner
and
binds
CaM
in
a
Ca2+
-dependent
manner.
It
shows
low
levels
of
cross-reactivity
with
antise-
rum
raised
against
rat
brain
CaM-PK
II.
These
characteristics
preclude
the
classification
of
ACMPK
as
any
type
of
known
Ca2
+
/CaM-dependent
protein
kinase
other
than
CaM-PK
II.
A
feature
that
distinguishes
ACMPK
from
CaM-PK
Hs
is
that
it
is
the
first
multifunctional
CaM-dependent
protein
kinase
reported
to
exist
as
a
monomer.
ACMPK
also
phos-
phorylates
protein
substrates
at
considerably
slower
rates
than
those
reported
for
mammalian
CaM-PK
I1s.
Few
de-
tailed
kinetic
and
substrate
specificity
studies
have
been
reported
comparing
mammalian
CaM-PK
I1s
to
each
other
(32)
and
none
has
been
reported
comparing
mammalian
CaM-PK
Hs
to
those
from
nonmammalian
sources.
De-
Riemer
et
al.
reported
rates
of
phosphorylation
of
synapsin
I
and
MAP-2
of
0.89
and
0.04
nmol
per
min
per
mg
of
Aplysia
CaM-PK
II,
respectively
(41),
which
are
far
below
those
reported
here
for
ACMPK.
The
isolation
of
a
fungal
multi-
function
Ca2+
/CaM-dependent
protein
kinase
capable
of
recognizing
mammalian
protein
substrates
and
being
regu-
lated
by
mammalian
CaM
indicates
a
high
degree
of
func-
tional
conservation
among
CaM-dependent
multifunctional
protein
kinases
in
eukaryotic
organisms.
The
Mr
of
ACMPK
suggests
that
if
it
is
a
CaM-PK
II
it
may
resemble
the
a
subunit
of
the
rat
brain
enzyme
more
closely
than
the
8
subunit.
Comparison
of
the
amino
acid
sequences
of
the
a
(11)
and
B
(12)
subunits
of
rat
brain
CaM-PK
II,
indicates
>90%
identity
between
the
subunits
for
the
amino
terminal
317
residues
ending
at
the
putative
CaM-binding
sites.
This
is
also
the
region
that
contains
all
the
sequences
homologous
to
other
protein
kinases
(11).
For
the
remaining
160
carboxyl-terminal
residues
of
the
a
subunit
the
level
of
similarity
falls
dramatically.
Lin
et
al.
have
postulated
that
the
carboxyl-terminal
region
of
the
a
subunit
(residues
350-478)
is
the
"association
domain,"
which
could
be
impor-
tant
in
the
assembly
of
subunits
of
the
holoenzyme
(11).
The
monomeric
ACMPK
with
an
estimated
Mr
of
42,000
could
be
a
homolog
of
the
a
(Mr,
54,000)
subunit
lacking
this
domain.
We
express
our
appreciation
to
Drs.
Roger
Colbran,
Charles
Schworer,
and
Tom
Soderling
(Vanderbilt
University)
and
to
Dr.
Mary
Kennedy
(California
Institute
of
Technology)
for
generously
sharing
unpublished
information
as
well
as
supplying
valuable
materials
used
in
this
research.
We
thank
Dr.
N.
Ronald
Morris
for
his
enthusiastic
support
of
the
project.
This
research
was
supported
by
National
Institute
of
General
Medicine
Grant
GM
37288
to
D.C.B.,
National
Institutes
of
Health
Grant
NS
11252
to
D.J.W.,
by
a
predoctoral
fellowship
to
S.F.
from
E.
R.
Squibb
and
Sons,
and
by
National
Institutes
of
General
Medicine
Grant
GM
34711
to
N.
Ronald
Moms.
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