From the
Subcellular localization of
Ca
Increases in cytosolic calcium concentration, either by release
from intracellular stores or by influx from the extracellular space,
mediate the effects of many hormones and neurotransmitters on target
tissues. The primary intracellular receptor for calcium is calmodulin
(CaM),
CaMKII activity is
regulated by Ca
Consistent with its diverse
roles, CaMKII exhibits broad yet localized distribution within neurons,
as demonstrated by immunohistochemistry
(13, 14) . It is
concentrated at postsynaptic densities, where it is ideally located to
regulate postsynaptic events, such as long term potentiation. Discrete
localization of CaMKII may play an important role in its physiological
function in at least three ways. 1) Substrate proteins may have to be
colocalized with the kinase to be phosphorylated. 2) Localized
modulation of the Ca
As a first test of the hypothesis that the subcellular
localization of CaMKII may be determined by association with specific
anchoring protein(s), a gel overlay assay was developed to detect
proteins capable of binding to CaMKII. Discrete patterns of
[
As a first step
toward understanding the possible physiological relevance of these
observations, the affinity for the interaction of
[
CaMKII undergoes slower basal autophosphorylation at Thr
The data comparing competition by different
autophosphorylated forms of mCaMKII
Since binding of CaMKII to p190 appears to
require autophosphorylation at Thr
As a
final test of the binding specificity of p190,
[
It is becoming increasingly clear that subcellular
localization/compartmentation of specific proteins plays a significant
role in the functioning of signal transduction pathways. The
intracellular localization of CaMKII varies in different tissues; in
cerebral cortex, about 88% of CaMKII activity is associated with a
particulate (0.1% Triton X-100 insoluble) fraction
(56) ,
probably containing cytoskeletal elements. Association of CaMKII with
specific subcellular structures is likely to play an important role in
determining specific functions of CaMKII. For example, to play a role
in long term potentiation
(20, 57, 58) , it is
probably important for CaMKII to be localized to dendritic spines and
postsynaptic densities, where Ca
One possible
mechanism that may contribute to differential localization is the
specific interaction of CaMKII with a diverse array of anchoring
proteins. Preliminary reports have suggested that CaMKII can interact
with several cellular proteins besides calmodulin. Using an overlay
assay with
The present report describes
experiments using recombinant mouse CaMKII
The forebrain CaMKII-binding proteins were chosen for further
investigation because more is known about the localization of neuronal
CaMKII, and the
The
[
Competition
experiments suggested that prior autophosphorylation of CaMKII at
Thr
The data reported in this manuscript suggest
that p190 may be a PSD protein that specifically anchors activated
CaMKII. However, its precise identity remains unknown. Association of
CaMKII with p190 following activation/autophosphorylation in response
to dendritic Ca
We express our appreciation to James Bann and Susanne
Kloeker for performing a few of the experiments reported; Drs. Jackie
Corbin, Sharron Francis, Lee Limbird, and Brian Wadzinski for
critically reading an initial version of the manuscript and for
invaluable discussions; and Mary Ann Barban for excellent technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/calmodulin-dependent protein kinase II (CaMKII) by
interaction with specific anchoring proteins may be an important
mechanism contributing to the regulation of CaMKII. Proteins capable of
binding CaMKII were identified by the use of a gel overlay assay with
recombinant mouse CaMKII
(mCaMKII
) or Xenopus CaMKII
(xCaMKII
)
P-autophosphorylated at
Thr
as a probe. Numerous
[
P]CaMKII-binding proteins were identified in
various whole rat tissue extracts, but binding was most prominent to
forebrain proteins of 190 kDa (p190) and 140 kDa (p140). Fractionation
of forebrain extracts localized p190 and p140 to a crude
particulate/cytoskeletal fraction and isolated postsynaptic densities.
[
P]mCaMKII
-bound to p190 with an apparent
K
of 609 nM (subunit
concentration) and a B
of 7.0 pmol of
mCaMKII
subunit bound per mg of P2 protein, as measured using the
overlay assay. Binding of 100 nM
[
P]mCaMKII
to p190 was competed by
nonradioactive mCaMKII
autophosphorylated on Thr
(EC
= 200 nM), but to a much lesser
extent by nonradioactive mCaMKII
autophosphorylated on Thr
(EC
> 2000 nM). In addition,
nonphosphorylated mCaMKII
was a poor competitor for
[
P]mCaMKII
binding to p190. The competition
data indicate that Ca
/CaM-dependent
autophosphorylation at Thr
promotes binding to p190,
whereas, Ca
/CaM-independent autophosphorylation at
Thr
does not enhance binding. Therefore, CaMKII may
become localized to postsynaptic densities by p190 following its
activation by an increase of dendritic Ca
concentration.
(
)
which, when activated by calcium,
interacts with many proteins
(1) . CaM targets include several
serine/threonine protein kinases such as myosin light chain kinase,
phosphorylase kinase, and Ca
/calmodulin-dependent
protein kinases I-IV. Within the
Ca
/calmodulin-dependent protein kinase class, CaMKII
is somewhat unique in that it exhibits broad substrate specificity and
is found in most, if not all, tissues. CaMKII was originally isolated
from rat brain and rabbit liver (reviewed in Ref. 2), and four isoforms
(
,
,
,
) have now been identified using molecular
techniques
(3, 4, 5, 6) . Alternatively
spliced mRNAs encoding
and
are predominantly and
exclusively expressed in the nervous system, whereas mRNAs encoding
and
isoforms are predominant in peripheral tissues
(6, 7, 8, 9, 10, 11) . A
combination of 8-12 of the subtypes make up a CaMKII holoenzyme.
In brain, the
isoform is highly expressed in forebrain while
subtypes are universally expressed
(4, 5, 6, 12) . Most, if not all,
neurons contain CaMKII by immunocytochemistry
(13, 14) .
In some brain regions, such as hippocampus, the
isoform alone
accounts for 1% of total protein
(15) . Among neuronal processes
regulated by CaMKII are neurotransmitter release, catecholamine
synthesis, cytoskeletal protein interactions, gene expression, and
postsynaptic responses such as long term potentiation (reviewed in
Refs. 16 and 17). Transgenic mice lacking the CaMKII
gene are
deficient in spatial learning
(18) and also have an abnormal
fear response and aggressive behavior
(19) . In addition, it is
much more difficult to induce long term potentiation in hippocampal
slices from the transgenic animals
(20) .
/CaM binding and by two modes of
autophosphorylation. Ca
/CaM stimulates
autophosphorylation at Thr
resulting in a
Ca
/CaM-independent form
(21, 22, 23, 24) . In the absence of
Ca
/CaM, CaMKII undergoes autophosphorylation at
Thr
and Ser
, which blocks
Ca
/CaM binding, thereby inactivating the enzyme
(25, 26, 27) . Presumably, protein phosphatases
contribute to the regulation of CaMKII autophosphorylation in vivo by dephosphorylating these sites.
concentration
(31) may
differentially regulate CaMKII in discrete subcellular compartments. 3)
Colocalization with different protein phosphatases may affect its
dephosphorylation. Recently, CaMKII
was shown to
contain a nuclear localization signal sequence which is inserted by
alternative splicing just carboxyl-terminal of the calmodulin binding
domain
(32) . However, mechanism(s) that determine subcellular
localization of other CaMKII isoforms are unknown. Protein kinase A is
localized, at least in part, by association with specific AKAPs
(A-kinase anchoring proteins)
(33, 34, 35, 36, 37) . In
addition, activated protein kinase C is thought to bind to proteins
termed RACKs (receptors for activated C kinase)
(38, 39) . Interaction of multifunctional kinases with
specific anchoring proteins may allow broad specificity kinases to have
localized and specific effects within cells. The present studies were
designed to test the hypothesis that localization of CaMKII may also be
determined, at least in part, by anchoring proteins.
Expression of CaMKII in Baculovirus (AcMNPV)
All
procedures were performed as described
(40) . Recombinant
baculovirus expressing wild type mouse CaMKII (mCaMKII
), a
mutant mCaMKII
(T286A), and wild type Xenopus CaMKII
(xCaMKII
) were gifts from Drs. D. Brickey and T. Soderling (Vollum
Institute)
(41, 42) . Suspension cultures were infected
with virus at a multiplicity of infection of 20 plaque-forming units
per cell and grown for 72 h. Cell pellets were stored at -70
°C and then thawed in homogenization buffer at 4 °C for
purification of the kinase as described
(41) .
Rat Forebrain Fractionation
Forebrains (1 g
each) were rapidly removed from female Sprague-Dawley rats (>50 days
of age) and homogenized using a Polytron (two 15-s bursts separated by
30 s on ice) in 10 ml/forebrain ice-cold homogenization buffer (10
mM Tris-HCl (pH 7.5), 1 mM EGTA, 1 mM EDTA,
1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 20
mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin). The
homogenate was centrifuged at 100,000
g for 60 min,
and the supernatant (S1; cytosolic proteins) was removed. The pellet
was rehomogenized in 10 ml of homogenization buffer plus 1% Triton
X-100 and centrifuged as before. The supernatant (S2; membrane
proteins) was removed, and the pellet was resuspended in 10 ml of
homogenization buffer (P2; cytoskeletal proteins). Aliquots of
fractions were stored at -70 °C until needed. All other rat
tissues were homogenized following the same protocol in order to
generate whole tissue extracts.
Ca
Reactions (typically 0.5 ml) were
performed in HEPES (50 mM, pH 7.5), magnesium acetate (2
mM), CaCl/Calmodulin-dependent
Autophosphorylation
(1.5 mM), DTT (2 mM),
and [
-
P]ATP (10 µM; 20000
cpm/pmol). mCaMKII
or xCaMKII
(in 50 mM HEPES, pH
7.5, 1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, and 10%
(v/v) ethylene glycol) was added to 3-10 µM (subunit
concentration) so that 15% glycerol was also present in the
incubations. Calmodulin was added to 2
the kinase subunit
concentration. Following incubation on ice for 60 s, nonradioactive ATP
(2 mM final) was added, followed by an additional 30-s
incubation on ice. The reaction was stopped by the addition of EDTA (10
mM), NaF (25 mM), NaCl (500 mM), and Tween
20 (0.1% v/v), and incubation continued for 1 h on ice. Controls
established that the autophosphorylation stoichiometry did not
significantly increase during the second (30-s) phase. However, the
second phase enhanced recovery from desalting columns (see below),
ensuring that the eluted material was >95% insoluble in 10%
trichloroacetic acid. Autophosphorylation stoichiometries (typically
0.2-0.6 mol of [
P]phosphate per mol of
mCaMKII
subunit) were determined by spotting aliquots on P81
papers (Whatman) and washing in 75 mM phosphoric acid,
followed by liquid scintillation counting
(43) .
[
P]CaMKII was desalted on a Sephadex G-50 (fine)
column (15 ml) equilibrated with Tris-HCl (50 mM, pH 7.4),
NaCl (500 mM), EGTA (0.1 mM), DTT (1 mM),
and Tween 20 (0.1% v/v), collecting 0.5-ml fractions. Peak radioactive
fractions, as determined by Cerenkov counting, were pooled, and the
concentration of [
P]mCaMKII
in the pool was
determined by liquid scintillation counting of aliquots from the pool.
Generally, 60-100% of applied
[
P]mCaMKII
was recovered in the pool.
Nonradioactive autophosphorylations of mCaMKII
,
T286A-mCaMKII
, and xCaMKII
were performed using identical
conditions, except that [
-
P]ATP was
replaced with ATP, and the reaction was terminated with EDTA (5
mM) alone. Nonphosphorylated samples were incubated under
identical conditions, except ATP was replaced with water.
Nonradioactive and nonphosphorylated samples were not desalted.
Ca
Reactions (typically 0.5 ml) were
performed in HEPES (50 mM, pH 7.5), magnesium acetate (2.5
mM), EGTA (2 mM), bovine serum albumin (1 mg/ml), DTT
(2 mM), and ATP (1 mM). Typically, 5 µM
mCaMKII/Calmodulin-independent (Basal)
Autophosphorylation
subunit (in 50 mM HEPES, pH 7.5, 1 mM
EDTA, 1 mM DTT, 50% (v/v) glycerol, and 10% (v/v) ethylene
glycol) was added, and storage buffer was added to bring the final
glycerol concentration to 15% (v/v). Samples were incubated for 60 min
at 30 °C, and then reactions were stopped with EDTA (5
mM). Autophosphorylation stoichiometries were about 1.2 mol of
[
P]phosphate/mol mCaMKII
subunit,
determined by running similar reactions in the presence of
[
-
P]ATP. Nonphosphorylated mCaMKII
was
incubated under the same conditions except ATP was omitted.
Casein Phosphorylation
Casein was incubated
overnight at 30 °C in Tris-HCl (50 mM, pH 7.5), EDTA (0.1
mM), magnesium acetate (10 mM), casein (5 mg/ml), ATP
(0.2 mM), C-subunit of protein kinase A (8.4 µg/ml), and
-mercaptoethanol (0.1% v/v) in a final volume of 0.5 ml
(44) . The reaction was stopped by the addition of EDTA and
sodium pyrophosphate (10 mM each). Similar reactions in the
presence of [
-
P]ATP indicated a typical
stoichiometry of about 0.1 mol of phosphate per mol of casein.
Phosphorylase b Phosphorylation
Phosphorylase
b was incubated for 60 min at 30 °C in Tris/sodium
glycerol 1-phosphate (100 mM each, pH 8.2), magnesium acetate
(10 mM), ATP (0.2 mM), CaCl(0.1
mM), phosphorylase b (10 mg/ml), and phosphorylase
kinase (0.2 mg/ml) in a final volume of 0.5 ml
(45) . NaF (50
mM) and EDTA (25 mM) were then added, and the
incubation was continued for another 60 min. Similar reactions in the
presence of [
P]ATP indicated a typical
stoichiometry of about 0.5 mol of phosphate per mol of phosphorylase
b. Phosphorylation of CaMK-(281-309)-The peptide (50
µM) was incubated for 30 min at 30 °C in HEPES (50
mM, pH 7.5), magnesium acetate (2.5 mM), CaCl
(1.5 mM), ATP (0.5 mM), calmodulin (150
µM), and mCaMKII
(100 nM) in a final volume
of 0.5 ml
(46) , and the reaction was stopped by addition of
EDTA (5 mM). Typically, 0.5 mol of phosphate was incorporated
per mol of peptide.
Overlay Technique
An overlay procedure
(47) was used to detect binding of P-labeled
mCaMKII
or xCaMKII
to cellular proteins. Protein fractions
were separated by SDS-PAGE (10%; 7-cm or 16-cm gels) and transferred
overnight at 4 °C and 30 V to a PVDF membrane (MilliPore or Gelman)
in Tris/glycine buffer (25 mM and 192 mM,
respectively). Prestained standards were also run to permit estimation
of approximate molecular weights of
[
P]CaMKII-binding proteins. Membranes were
blocked for at least 60 min in Tris-HCl (50 mM)/NaCl (200
mM) (TBS) containing Tween 20 (3% v/v) and non-fat powdered
milk (5% w/v) (blocking buffer). The membrane was washed for 30 min in
TBS containing Tween 20 (0.1% v/v) and non-fat powdered milk (5% w/v)
(rinsing buffer) prior to incubation with
[
P]CaMKII. Membranes were then incubated for 2 h
at room temperature with constant rocking in rinse buffer containing
the indicated concentration of
P-labeled CaMKII, followed
by extensive washing with at least 5 changes of rinse buffer and
autoradiography. For affinity determinations and competition
experiments, preparative SDS-PAGE minigels were loaded with
350-720 µg of P2 fraction protein; the resulting membranes
were cut into 5-mm strips (containing 23-48 µg of total
protein), and incubations were performed in Bio-Rad mini incubation
trays (0.5-ml incubation and 1-ml wash per strip).
Quantitation of Specific Bands
Binding of
P-labeled CaMKII to p190 was quantitated by excising the
band from the PVDF membrane and counting in a liquid scintillation
counter. Blanks for each strip were cut from an area of the membrane
which appeared to have no bands. The amount of kinase bound to p190 in
each strip (picomoles) was normalized for the loading of P2 protein on
the strip (milligrams of protein).
CaMKII Western Blot
PVDF membranes were prepared
as described for the overlay assay. Membranes were blocked for 60 min
in blocking buffer (see above) and then incubated with
affinity-purified goat anti-CaMKII antibody (0.1 µg/ml) in blocking
buffer overnight at 4 °C. The antibody was raised to purified
native rat forebrain CaMKII
(48) by Bethyl Laboratories
(Montgomery, TX) and then affinity-purified on a column of recombinant
mCaMKII coupled to Affi-Gel 15 (Bio-Rad), prepared according to
the manufacturer's directions. Membranes were then incubated with
alkaline phosphatase-conjugated rabbit anti-goat secondary antibody
(Vector Laboratories) (1:1000 dilution) for 60 min and developed with
AP Substrate Kit II (Vector Laboratories) according to the directions.
Miscellaneous
All protein concentrations were
determined by a Bradford
(49) protein assay (Bio-Rad) using
bovine serum albumin as standard. Phosphorylase kinase, phosphorylase
b, casein, and prestained standards were from Sigma. The
peptide CaMK-(281-309) was synthesized and characterized as
described previously
(50) . Calmodulin was purified from bovine
brain
(51) . RII and C-subunit were gifts from Dr. J. D.
Corbin (Vanderbilt University); purified RII
contains about 0.5
mol of phosphate/mol of RII
subunit
(52) . Tubulin
(53) and mixed MAPs
(54) were gifts from Dr. R. C.
Williams, Jr. (Vanderbilt University). Postsynaptic densities
(55) were a gift from Drs. S. E. Tan and T. Soderling (Vollum
Institute).
P]mCaMKII
-binding proteins were detected
in various central nervous system and peripheral tissues (Fig. 1,
top). The most abundant binding of
[
P]mCaMKII
was detected in forebrain
extracts, where [
P]mCaMKII
-binding proteins
of 140 and 190 kDa were particularly prominent, but several other
[
P]mCaMKII
-binding proteins were also
detected. Cerebellum and pons/medulla extracts contained generally
lower [
P]mCaMKII
binding activities, and
the pattern of binding was significantly different. The pattern of
[
P]mCaMKII
binding to proteins in
peripheral tissue extracts was different. For example, kidney extracts
contain very few prominent
[
P]mCaMKII
-binding proteins, whereas ovary,
spleen, and lung contain a major
[
P]mCaMKII
-binding protein of about 65 kDa.
Heart extracts contained several
[
P]mCaMKII
-binding proteins between 40 and
220 kDa. Generally, similar patterns of CaMKII-binding proteins were
detected when [
P]xCaMKII
was used as a
probe (Fig. 1, bottom). However, the 65-kDa
CaMKII-binding protein detected in ovary, lung, and spleen was
apparently specific for the
-isoform. Binding of
P-labeled
and
isoforms to all proteins could
be largely competed by excess nonradioactive autophosphorylated ligand
(see below). The pattern of [
P]CaMKII-binding
proteins did not correspond to the Coomassie Blue staining pattern of
the proteins in any of the tissue extracts (data not shown).
Figure 1:
Survey of
rat tissues for CaMKII-binding proteins. The indicated whole tissue
extracts (100 µg/lane) were separated by SDS-PAGE, transferred to
PVDF membranes, and then probed with either
[P]mCaMKII
( top) or
[
P]xCaMKII
( bottom) as described
under ``Experimental Procedures.'' Approximate molecular
masses (kDa) are shown on the left.
In
order to determine whether the
[P]mCaMKII
-binding proteins might play a
role in localizing rat forebrain CaMKII, extracts were separated into
soluble (S1), Triton X-100 soluble (S2), and Triton X-100 insoluble
(P2) fractions (see ``Experimental Procedures''). The three
fractions were subjected to overlay analysis using
[
P]mCaMKII
or probed with a polyclonal
antibody to CaMKII. The most abundant binding of
[
P]mCaMKII
was detected in the P2 fraction,
where proteins of 140 and 190 kDa (p140 and p190) were most prominent
(Fig. 2, left). The pattern of
[
P]mCaMKII
-binding proteins in P2 was very
similar to the [
P]mCaMKII
-binding protein
pattern detected in whole forebrain extracts (Fig. 1,
top). Consistent with this observation,
[
P]mCaMKII
-binding proteins were barely
detectable in S1 and S2. In addition, the relative abundance of
[
P]mCaMKII
-binding proteins in the
particulate fraction (P2) parallels the localization of endogenous
CaMKII immunoreactivity to this fraction, as determined by Western blot
(Fig. 2, middle).
Figure 2:
Subcellular distribution of rat
forebrain CaMKII-binding proteins. Rat forebrain extracts were
fractionated as described under ``Experimental Procedures.''
Fractions were separated by SDS-PAGE (100 µg of protein/lane),
transferred to PVDF membranes, and then probed with either
[P]mCaMKII
( left) or antibodies to
CaMKII ( middle). Purified PSDs (20 µg), purified tubulin
(5 µg), and a mixed MAPs sample (20 µg) were separated by
SDS-PAGE, transferred to PVDF membrane, and then probed with
[
P]mCaMKII
( right).
The P2 fraction is a crude
cytoskeletal preparation which likely contains postsynaptic densities
(PSDs), tubulin, neurofilaments, various microtubule-associated
proteins (MAPs), and other cytoskeletal proteins. In order to further
characterize the location of the
[P]mCaMKII
-binding proteins, overlay assays
were performed using purified tubulin, a mixed MAP fraction (which
contains tubulin, neurofilament proteins,
, and
microtubule-associated proteins; see Ref. 54), and purified PSDs. The
pattern of [
P]mCaMKII
-binding proteins
detected in PSDs was very similar to the pattern in the P2 fraction and
in whole forebrain extracts (compare Fig. 2, left, with
Fig. 2
, right, and Fig. 1). Much weaker binding of
[
P]mCaMKII
to tubulin and to several MAPs
was also detected (Fig. 2, right).
P]mCaMKII
with its binding proteins was
determined. Strips of PVDF membrane containing rat forebrain P2
proteins were incubated with increasing concentrations of
[
P]mCaMKII
. Binding of
[
P]mCaMKII
to p190 was detectable at 25
nM [
P]mCaMKII
subunit and
increased with the concentration of
[
P]mCaMKII
(Fig. 3, top).
Other bands were detectable in the affinity experiments; however, they
were only apparent at higher concentrations of
[
P]mCaMKII
. The portion of each membrane
corresponding to p190 was cut out, and the amount of
[
P]mCaMKII
bound to p190 was determined in
a scintillation counter (Fig. 3, bottom). The data
appear to follow a generally hyperbolic form approaching saturation at
3 µM [
P]mCaMKII
. However,
almost double the [
P]mCaMKII
was bound at
the 4 µM data point, and there was considerably more
variability in the amount of binding detected, as indicated by the
larger standard error. This might indicate that a second binding
component was being detected at 4 µM
[
P]mCaMKII
, or that nonspecific binding was
significantly increased. However, these alternatives could not be
investigated further because 4 µM was the maximum
concentration of [
P]mCaMKII
possible in
these experiments. Scatchard transformation of the data obtained up to
3 µM [
P]mCaMKII
yielded a
straight line (Fig. 3, bottom, inset),
indicating that [
P]mCaMKII
bound to p190
with a K
of 609 nM mCaMKII
subunit (
61 nM holoenzyme) and a B
of 7.0 pmol of [
P]mCaMKII
subunit
bound per mg of P2 protein. Very similar binding parameters were
obtained when the raw data (up to 3 µM) were fitted using
nonlinear regression techniques (data not shown).
Figure 3:
Determination of the affinity for
[P]mCaMKII
binding to p190. Rat forebrain
P2 fraction was fractionated by preparative SDS-PAGE and then
transferred to PVDF membranes. Strips (5 mm) of the blocked membrane
were incubated with the indicated concentration of
[
P]mCaMKII
in a total volume of 0.5 ml.
Top, representative autoradiograms from a single experiment.
The panel on the left was exposed to film for 4 h,
and the panel on the right was exposed for 14 h.
Bottom, quantitation of [
P]mCaMKII
binding to p190 (see ``Experimental Procedures''). Data were
obtained in 10 experiments, and each point is the mean ± S.E. of
at least 3 determinations. The hyperbola is plotted according to
parameters derived from the Scatchard plot ( inset) of the
binding data (
), excluding the 4 µM data point
(
) (see text). Since the amount of
[
P]mCaMKII
bound to the membranes was less
than 0.5% of the total present at all concentrations, the amount of
free ligand was assumed to be equal to the total
concentration.
In order to
establish the specificity of the interaction between
[P]mCaMKII
and p190, competition
experiments were performed. Strips of membrane containing P2 proteins
were incubated with 100 nM
[
P]mCaMKII
, autophosphorylated in the
presence of Ca
/CaM, in the additional presence of
potential nonradioactive competitors. Fig. 4shows competition by
different autophosphorylated forms of mCaMKII
. Representative
experiments are shown in the top and middle panels,
and quantitative data from three independent experiments are shown
below. Competition by mCaMKII
autophosphorylated with
nonradioactive ATP in the presence of Ca
/CaM (same
conditions used to prepare [
P]mCaMKII
) was
concentration-dependent, exhibiting an EC
of about 200
nM and reducing binding by over 90% at 2 µM.
However, if ATP was omitted from the competitor autophosphorylation
reaction ( i.e. nonphosphorylated competitor), only a 20%
reduction in binding was detected at 2 µM competitor.
Therefore, autophosphorylation of mCaMKII
in the presence of
Ca
/CaM increased the potency of competition by more
than 10-fold.
Figure 4:
Competition for
[P]mCaMKII
binding to p190 by mCaMKII
autophosphorylated at different sites. Membrane strips containing
fractionated P2 proteins were incubated with 100 nM
[
P]mCaMKII
in the additional presence of
nonradioactive mCaMKII
that had previously been incubated in the
presence (
,
) or absence (
,
) of ATP with
either Ca
/CaM (
,
) or EGTA (
,
). Top, autoradiograms from a representative experiment
in which the competitor was incubated with Ca
/CaM.
Middle, autoradiograms from a representative experiment in
which the competitor was incubated with EGTA. Bottom,
quantitation of [
P]mCaMKII
binding to p190
(see ``Experimental Procedures'') in the presence of
competitors. Each point represents the mean ± S.E.
( n > 3).
CaMKII can be autophosphorylated at multiple sites;
with Ca/CaM present, Thr
is the primary
site of autophosphorylation when incubations are performed on ice
(22, 23, 24) as above. Additional competition
experiments were performed using mutant mCaMKII
in which
Thr
was changed to Ala (T286A). T286A-mCaMKII
was a
poor competitor (EC
of approximately 2 µM)
for wild type [
P]mCaMKII
(data not shown),
even following incubation on ice with ATP in the presence of
Ca
/CaM (stoichiometry < 0.05 mol of
[
P]phosphate/mol of T286A-mCaMKII
subunit).
in the absence of Ca
/CaM, and possibly other
sites, but not at Thr
(26, 27) .
Therefore, the effect of basal autophosphorylation (with nonradioactive
ATP) on competition was investigated. Binding of
[
P]mCaMKII
was reduced by only about 45% at
2 µM basal autophosphorylated mCaMKII
(Fig. 4).
Furthermore, the omission of ATP from the basal autophosphorylation
reaction had little effect on competition. Therefore,
autophosphorylation of mCaMKII
at the basal site(s) did not affect
the potency of competition for [
P]mCaMKII
binding.
suggest that potent binding of
mCaMKII
to p190 requires autophosphorylation at Thr
.
Autophosphorylation at Thr
is already known to convert
the kinase to an open/active (Ca
-independent)
conformation
(21, 22, 23, 24) , whereas
nonphosphorylated CaMKII or basal autophosphorylated CaMKII adopts a
closed/inactive conformation. Therefore, it seemed possible that
[
P]mCaMKII
binding to p190 may require the
open/active conformation, perhaps because p190 is a substrate for
CaMKII. However, several observations argue against this possibility.
1) Competition for [
P]mCaMKII
binding by
nonphosphorylated CaMKII is unaffected by the presence of CaCl
(2.5 mM) plus CaM (1 µM), which maintains
the competitor in an open/active conformation (not shown). 2) Binding
of [
P]mCaMKII
is essentially unaffected by
a variety of reagents that bind to, or affect binding of substrates to,
the active site, including ATP (0.5 mM), ADP (0.5
mM), syntide-2 (0.25 mM), magnesium acetate (10
mM), EDTA (1 mM), EGTA (1 mM), CaCl
(2.5 mM), and CaCl
(2.5 mM) plus
CaM (1 µM) (data not shown). These data suggest that the
interaction of CaMKII with p190 is not mediated via the catalytic
domain. Furthermore, no competition for binding of 100 nM
[
P]mCaMKII
is observed using a 100-fold
excess of a CaMKII regulatory domain peptide, CaMK-(281-309),
that had been phosphorylated at Thr
(not shown).
Therefore, the regulatory domain itself is insufficient to compete for
the interaction. However, since heat inactivation of
Thr
-autophosphorylated mCaMKII
(15 min at 65 °C)
prevents competition for [
P]mCaMKII
binding
(not shown), the native conformation of the kinase appears to be
necessary for binding.
, another possibility
is that p190 is a Thr
-specific protein phosphatase.
However, binding of 100 nM
[
P]mCaMKII
is unaffected by the inclusion
of protein phosphatase inhibitors ( e.g. 1 µM
microcystin LR) (not shown). In addition, there are no reported protein
phosphatase catalytic subunits in this molecular weight range.
P]mCaMKII
overlay experiments were
performed in the presence of other phosphorylated competitors.
Essentially no competition was observed using 10-fold molar excesses of
glycogen phosphorylase a, phosphocasein, or type II
regulatory subunit of protein kinase A, or using a 10,000-fold molar
excess of free phosphoserine or phosphothreonine (not shown).
concentrations can
be selectively regulated
(59, 60) and the appropriate
substrates ( e.g. receptors and/or ion channels) may also be
localized ( e.g. Ref. 55). However, the mechanism(s) explaining
the diverse localization of CaMKII are unclear.
I-labeled rat brain CaMKII as a probe, CaMKII
was shown to interact with tubulin and unidentified postsynaptic
density proteins of 115 and 155 kDa
(61) . Binding of CaMKII to
actin has also been reported
(62) . Although these interactions
may localize CaMKII to cytoskeletal components, they were not
characterized in detail. More recently, synaptic vesicle CaMKII was
shown to bind to synapsin I
(63) , although this may be a
mechanism for localizing synapsin I to the vesicles, rather than for
localizing CaMKII to vesicles.
isoform
(
P-autophosphorylated in the presence of
Ca
/calmodulin)
([
P]mCaMKII
) as a probe in a gel overlay
assay. Multiple [
P]mCaMKII
-binding proteins
were present in whole tissue extracts, and the pattern and amount of
[
P]mCaMKII
binding varied between tissues
(Fig. 1, top). Although one could question the
physiological relevance of screening peripheral tissues extracts with a
neuronal isoform of CaMKII, the fact that similar CaMKII binding
patterns were observed with the
isoform (Fig. 1,
bottom) might suggest that a conserved domain is involved in
binding. All four known isoform classes (
,
,
,
)
share highly conserved amino-terminal catalytic and regulatory
(residues 1-315; 90% identical) domains. The major differences
arise from the presence of multiple variable insertions immediately
following the regulatory domain
(6) . The remainder of the
carboxyl-terminal domain is 75% homologous in all isoforms. Little data
are available concerning the function of the insertion and
carboxyl-terminal domains, although the deletion of both domains
results in a monomer, rather than the normal holoenzyme
(64) .
isoform is primarily and highly expressed in
forebrain. Analysis of crude subcellular fractions revealed that the
[
P]mCaMKII
-binding proteins were primarily
present in the P2 (cytoskeletal) fraction (Fig. 2). In addition,
purified PSD preparations exhibited a
[
P]mCaMKII
binding protein pattern similar
to the P2 fraction. Weak binding to tubulin was detected, as previously
observed
(61) , and to several unidentified proteins in the
mixed MAP sample. These observations are consistent with the high
concentration of CaMKII at PSDs
(13, 14, 28, 29, 30) and its
association with microtubules and neurofilaments
(65, 66) .
P]mCaMKII
-binding protein that had the
strongest signal in the forebrain P2 fraction (p190) had a
K
of 609 nM for the CaMKII
subunit (
61 nM holoenzyme) (Fig. 3), 20-fold lower
than the concentration of CaMKII in rat forebrain.
(
)
Therefore, the affinity data are consistent with the
possibility that CaMKII and p190 can interact in intact neurons.
However, one should use care extrapolating affinities from immobilized
ligand assays to physiological conditions. Binding to p190 exhibited a
B
of 7.0 pmol of
[
P]mCaMKII
per mg of P2 protein, suggesting
that p190 may be capable of binding 30-40% of the CaMKII in the
P2 fraction, when saturated.
(
)
was required for binding of CaMKII to p190
(Fig. 4; see ``Results''). Therefore, if p190 is
responsible for localizing CaMKII to PSDs, one would expect CaMKII in
isolated PSDs to be Thr
-autophosphorylated and thus
partially Ca
-independent. Although CaMKII is abundant
in isolated PSDs
(28, 29, 30) , substantial
Ca
-independent CaMKII activity has not been detected
without Ca
/CaM-dependent phosphorylation in vitro (67) . This may be because autophosphorylated CaMKII is
less stable than nonphosphorylated kinase
(68) , and, therefore,
active autophosphorylated CaMKII may not survive the isolation
procedure. In agreement with this possibility, the specific CaMKII
activity in isolated PSDs is about 10-fold lower than expected based on
its abundance
(67) . Another possibility is that
nonphosphorylated CaMKII may be localized to PSDs by other mechanisms,
possibly another class of anchoring proteins. Therefore, activation and
autophosphorylation of PSD CaMKII may result in its
``translocation'' to p190. Indeed, previous studies have
suggested that the subcellular distribution of CaMKII in invertebrate
neurons is regulated by autophosphorylation
(69, 70) .
Recent studies also suggest that the association of the RII
subunit of protein kinase A with AKAPs is regulated by phosphorylation
(71) . Alternatively, it has recently been suggested that CaMKII
translocates to PSDs from a soluble fraction following euthanasia
(72) . Therefore, it is possible that the high concentration of
nonphosphorylated CaMKII in isolated PSDs is artifactual, as suggested
previously
(2) .
mobilization may be an important
point of regulation in the Ca
/CaM-dependent
phosphorylation of PSD proteins, such as glutamate receptors
(55) . These phosphorylations are thought to play a critical
role in the regulation of synaptic plasticity. In addition, the
diversity of CaMKII-binding proteins in other tissues suggests that
there may be a variety of anchoring proteins, analogous to AKAPs and
RACKs, which specifically localize CaMKII to discrete subcellular
compartments. These interactions may be important in determining other
physiological processes regulated by Ca
mobilization.
/calmodulin-dependent protein kinase II; protein
kinase A, cyclic AMP-dependent protein kinase; C-subunit, catalytic
subunit of protein kinase A; DTT, dithiothreitol; MAPs,
microtubule-associated proteins; mCaMKII
, recombinant
baculovirus-expressed mouse
isoform of CaMKII; PAGE,
polyacrylamide gel electrophoresis; PSD, postsynaptic densities; PVDF
membranes, polyvinylidene difluoride; RII
, type II regulatory
subunit of protein kinase A; TBS, Tris-buffered saline; xCaMKII
,
recombinant baculovirus-expressed Xenopus
isoform of
CaMKII.
subunit concentration in whole
rat brain extracts by radioimmunoassay (0.7% of total protein) (15).
Assuming 1 g of tissue has a volume of 1 ml and an average subunit
molecular mass of 54 kDa for CaMKII, the average concentration of
CaMKII subunit in forebrain is about 13 µM.
P]mCaMKII
at B
,
there is about 7.0 pmol of p190 (1.31 µg) per mg of P2. This
estimate for the abundance of p190 is consistent with the fact that no
190-kDa protein can be detected in the P2 fraction by Coomassie Blue
staining of SDS-PAGE gels, but should be considered a minimal estimate,
since it assumes 100% efficiency for both the electrophoretic transfer
and renaturation steps of the protocol. By semiquantitative Western
blotting, there is about 1 µg (approximately 19 pmol) of
CaMKII
per mg of P2 protein (R. J. Colbran, unpublished). Thus,
p190 may be capable of binding about 37% of the CaMKII in the P2
fraction when saturated.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.