From the Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translocation of protein kinases with broad substrate specificities between different subcellular compartments by activation of signaling pathways is an established mechanism to direct the activity of these enzymes toward particular substrates. Recently, we identified two isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), which are targeted to the nucleus by an alternatively spliced nuclear localization signal (NLS). Here we report that cotransfection with constitutively active mutants of CaM kinase I or CaM kinase IV specifically blocks nuclear targeting of CaM kinase II as a result of phosphorylation of a Ser immediately adjacent to the NLS of CaM kinase II. Both CaM kinase I and CaM kinase IV are able to phosphorylate this Ser residue in vitro, and mutagenesis studies suggest that this phosphorylation is both necessary and sufficient to block nuclear targeting. Furthermore, we provide experimental evidence that introduction of a negatively charged residue at this phosphorylation site reduces binding of the kinase to an NLS receptor in vitro, thus providing a mechanism that may explain the blockade of nuclear targeting that we have observed in situ.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphorylation and dephosphorylation reactions control a myriad of signal transduction processes within the cell including cell growth and differentiation, metabolic pathways, and gene expression. The specificity of some kinases mediating these reactions is attained by a strict substrate specificity that limits the action of these dedicated kinases to a single or limited number of potential targets. Other kinases, however, are able to phosphorylate a large number of proteins in vitro, so the in vivo specificity of these kinases must occur through a different mechanism. Examples of these multifunctional or general protein kinases include protein kinase A, protein kinase C, and the Ca2+/calmodulin-dependent protein kinase (CaM kinase)1 family consisting of CaM kinase I, CaM kinase II, and CaM kinase IV (reviewed in Refs. 1 and 2). All of these kinases are able to phosphorylate nuclear transcription factors in vitro at sites that either activate or repress gene expression and so all of these kinases have the capacity, at least theoretically, to alter cellular phenotype through changes in protein expression. Over the past decade, the evidence that this actually occurs in vivo is becoming increasingly strong; for example, there is now abundant evidence that protein kinase A mediates activation of the cyclic AMP response element-binding protein through phosphorylation of a key Ser residue (3, 4).
Nuclear localization of a kinase is necessary for phosphorylation of nuclear proteins such as transcription factors, although there are examples of transcription factors that are activated in the cytoplasm and then translocate to the nucleus (5, 6). The ability of the catalytic subunit of protein kinase A to be released from cytoplasmic tethering and then passively diffuse into the nucleus, where it can phosphorylate nuclear proteins such as cyclic AMP response element-binding protein, has been described (7). More recently, active export of protein kinase A out of the nucleus under certain conditions based on a nuclear export signal has also been reported (8). Regulation of protein kinase C localization appears to occur, at least in part, through isoform-specific targeting to anchoring proteins that are presumably located near its substrates (9). CaM kinase I has been localized predominantly to the cell cytoplasm, while CaM kinase IV is predominantly nuclear (10, 11), although the mechanisms governing the subcellular localization of these two kinases have not yet been elucidated.
Isoform-specific targeting of CaM kinase II to the nucleus based on an
alternatively spliced nuclear localization sequence (NLS) has been
described (12, 13). CaM kinase II is composed of a multigene family
derived from four related genes: ,
,
, and
(reviewed in
Refs. 1 and 2). The
B,
B, and
A isoforms of CaM kinase II all share a common core NLS
amino acid sequence, KKRK, which is homologous to the NLS of the SV40
large T antigen (12). Nuclear localization of the
B and
B isoforms of CaM kinase II both in situ and
in vivo has been reported (12, 13);
A is also
presumably a nuclear isoform, although this has not been investigated.
Other isoforms of CaM kinase II do not contain any known NLS sequence
and localize to the cytoplasm. Interestingly, CaM kinase II forms
multimers of 8-12 subunits in situ and in vivo,
and the final localization of the multimer to the nucleus or cytoplasm
appears to be based on whether it is composed of predominantly nuclear
or cytoplasmic isoforms (12). These multimers are of sufficient size
(400-600 kDa) that any transport of intact holoenzyme across the
nuclear membrane is likely to require an active, nuclear export signal
or NLS-dependent process. Immunostaining of brain sections
has demonstrated nuclear localization of CaM kinase II in regions with
high expression levels of nuclear forms and cytoplasmic localization of
the kinase in regions expressing predominantly cytoplasmic forms (13).
Thus, like the other multifunctional protein kinases, CaM kinase II
does appear to have access to the cell nucleus, but such access depends
on the type and ratio of isoforms expressed.
Many different nuclear events have been ascribed to CaM kinase II. This
kinase is capable of regulating nuclear envelope breakdown in sea
urchin eggs (14), releasing Xenopus oocytes from meiotic metaphase arrest (15), promoting the maturation of oocytes (16), and
blocking the cell cycle at the G2 to M transition (17). CaM
kinase II has also been shown to activate the CCAAT enhancer element-binding protein (C/EBP
) (18) and recently has been shown
to block activity of the transcription factor cyclic AMP response
element-binding protein and either block or activate the related
transcription factor ATF-1 through phosphorylation of both positive and
negative regulatory sites (19-21). Many of these studies utilized a
truncated mutant of CaM kinase II, which does not associate into
holoenzymes and is able to diffuse freely between the nucleus and
cytoplasm as a result of its small size. Presumably, nuclear
localization of CaM kinase II would be required in order to mediate
processes involving phosphorylation of nuclear transcription factors. A
recent study demonstrates that, among three transfected isoforms of CaM
kinase II, only the nuclear-targeted isoform is able to activate an
atrial natriuretic factor promoter construct in cultured ventricular
myocytes, while the two cytoplasmic isoforms of CaM kinase II do not
affect expression of this construct (22).
Recently, autophosphorylation of cytoplasmic CaM kinase II was shown to
cause translocation of the kinase to the postsynaptic density in
cultured hippocampal neurons (23). We are interested in whether there
are mechanisms other than isoform-specific expression that can affect
the abililty of CaM kinase II to localize to the nucleus and, by doing
so, be available to translate Ca2+ elevations into the
processes described above. The capability of many NLS-containing
proteins to translocate to the nucleus is affected either positively or
negatively by phosphorylation (reviewed in Ref. 24). We have studied
the ability of the other multifunctional kinases, protein kinase A,
protein kinase C, CaM kinase I, and CaM kinase IV, to alter nuclear
targeting of CaM kinase II through phosphorylation. We report here that
CaM kinase I and CaM kinase IV are capable of phosphorylating a Ser
residue immediately adjacent to the NLS of B- and
B-CaM kinase II and that this phosphorylation causes
reduced binding to an NLS receptor and blocks targeting of CaM kinase
II to the cell nucleus.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Construction of CaM Kinase II Mutants--
The cloning of the
,
B, and
B isoforms of CaM kinase II
into the SR
eukaryotic expression vector has been described
previously (12, 13, 25). Mutants of
B-CaM kinase II
incorporating the following underlined Ser to Ala and Ser to Asp
mutations adjacent to the NLS, KKRKAAAA (S332-335A),
KKRKDDDD (S332-335D), KKRKSSSA (S335A) and
KKRKSSAS (S334A) were generated by
oligonucleotide-mediated, site-directed PCR mutagenesis of wild-type
B-CaM kinase II with subcloning of the amplified, mutant
fragment back into its corresponding position in
B-CaM
kinase II. The method for PCR site-directed mutagenesis has been
described (26). Mutants of
B-CaM kinase II incorporating
the following underlined Ser to Ala, Ser to Asp, and Ser to Glu mutants
adjacent to the NLS, KKRKASSS (S332A), KKRKSASS
(S333A), KKRKDSSS (S332D), KKRKESSS (S332E),
KKRKSDSS (S333D), and KKRKSESS (S333E), were
derived through oligonucleotide site-directed mutagenesis of
B-CaM kinase II according to the manufacturer's
protocols (CLONTECH Transformer kit). All mutants described above code for proteins identical to full-length, wild-type
B-CaM kinase II except for the underlined mutations. A
mutant incorporating the NLS of
B-CaM kinase II into the
5'-end of
-CaM kinase II was generated by annealing two
49-mers, 5'-GATGAATGATGGCGTGAAGAAAAGAAAGTCCAGTTCCAGCGTTGGAGCT-3' and
5'-CCAACGCTGGAACTGGACTTTCTTTTCTTCACGCCATCATTCATCTGCA-3', and then
cloning this annealed multimer into the PstI and
SacI sites of hemagglutinin-tagged
-CaM kinase II (27)
(the tagged version of
-CaM kinase II was used because it contains
5'-cloning sites not present in untagged
-CaM kinase II). This
produces a mutant that is identical to full-length
-CaM kinase II
except for replacement of amino acid residues 2 and 3 from
-CaM
kinase II with 13 amino acids encompassing residue numbers 324-336 of
B-CaM kinase II and four residues retained from the
hemagglutinin tag. The 5'-sequence of this construct beginning with
residue 1 is as follows:
MNDGVKKRKSSSSVGAQLIT ... The underlined
portion denotes the region derived from
B-CaM kinase II
including the NLS, the portion in italics denotes the retained region
from the hemagglutinin tag, and the portion in normal lettering
following the italics resumes with residue 4 of
-CaM kinase II and
continues uninterrupted through the remainder of the protein (not
shown). All mutants were screened initially by restriction analysis and
then verified by dideoxynucleotide sequencing. Restriction enzymes and
oligonucleotides were obtained from Life Technologies, Inc.
Construction of CaM Kinase I, CaM Kinase IV, and Calmodulin
Expression Constructs--
Human CaM kinase I clones, including 1) a
full-length, wild-type clone, 2) a
Ca2+/calmodulin-independent, truncated clone coding for
residues 1-294, and 3) these same two constructs with an activating
T177D mutation were a generous gift from A. Means (Duke University)
(28). In order to clone these constructs into the SR expression
vector, the PstI site of SR
was cleaved and blunted with
Klenow fragment, and an oligonucleotide linker containing a
BglII site (Life Technologies, Inc.) was ligated into this
site by standard techniques. The CaM kinase I clones were then cut with
BamHI and EcoRI, producing fragments encoding the
entire kinase reading frame, which was then cloned into the
EcoRI and BglII sites of SR
. Rat CaM kinase IV, including a full-length, wild-type clone as well as a
Ca2+-independent, truncated clone coding for residues
1-313 cloned into a pCMV expression vector was also a gift of A. Means
(29). The full reading frame for CaM kinase IV was cloned out of this vector by cutting at the 5'-end of the coding region with
NotI, blunting this site with T4 DNA polymerase, and then
cutting the 3'-end with ApaI. This fragment was then cloned
into the EcoRV and ApaI sites of SR
. A clone
containing the entire reading frame of human calmodulin was a generous
gift of P. Yaswen. SR
-calmodulin was created by cutting the 5'-end
of this clone with NcoI, blunting with Klenow fragment,
cutting the 3'-end with KpnI, and then cloning this fragment
into the EcoRV and KpnI sites of SR
. All
subcloning procedures were performed according to standard techniques,
and all enzymes and buffers were obtained from Life Technologies and New England Biolabs Inc. SR
-protein kinase A was a gift of M. Muramatsu (DNAX, Palo Alto, CA). An expression vector for CaM kinase
kinase was a generous gift of Thomas Soderling (30). SR
-protein
kinase C and SR
-calcineurin have been described (31).
Cell Culture and Transfection--
COS-7 cells were maintained
in Dulbecco's modified Eagle's medium with 10% supplemented calf
serum (Hyclone Labs) in a 10% CO2 incubator as described
previously (25). DNA was purified using the Qiagen and Merlin (Bio 101, Vista, CA) systems according to the manufacturer's protocols. Cells
were plated onto 35-mm cell culture dishes (Falcon) on the day prior to
transfection at a density such that they were approximately 70%
confluent on the day of transfection. For each transfection, 1.5-1.75
µg of total DNA was combined with 5 µl of Lipofectamine (Life
Technologies, Inc.), and transfection was carried out according to the
manufacturer's recommendations. The transfection medium was replaced
with Dulbecco's modified Eagle's medium plus 10% supplemented calf
serum 5 h post-transfection, and the cells were then left in the
incubator for a total of approximately 48 h post-transfection
prior to fixation or harvest. For immunofluorescence studies, 0.25 µg
of CaM kinase II was cotransfected with 1.25 µg of empty SR
vector, SR
-CaM kinase I, or SR
-CaM kinase IV. For cotransfections
including calmodulin and/or CaM kinase kinase, 0.25 µg of CaM kinase
II was cotransfected with 0.5 µg of CaM kinase I or IV with or
without 0.5 µg of SR
-calmodulin with or without 0.5 µg of CaM
kinase kinase. For NLS binding studies, 1.5 µg of CaM kinase II was
used for transfection.
Immunocytofluorescence--
Fixation and staining of COS-7 cells
were performed on 35-mm tissue culture dishes as described previously
(12). Immunodetection of -CaM kinase II and
B-CaM
kinase II was performed with a monoclonal antibody that recognizes both
isoforms of CaM kinase II. Immunodetection of hemagglutinin-tagged CaM
kinase II was performed with a monoclonal antibody to this tag
(Boehringer Mannheim). Secondary antibody detection with a
rhodamine-linked goat anti-mouse antibody and fluorescence microscopy
and photography were performed as described (12). In order to
quantitate nuclear localization of the kinase, 100 transfected cells
per plate were scored in a blinded fashion as to whether the kinase was
predominantly nuclear or predominantly cytoplasmic. The identity of the
nucleus was verified by comparative phase-contrast microscopy. Cells in
which definitive localization of CaM kinase II staining could not be
established (a small fraction of the total) were omitted from
analysis.
Peptide Phosphorylation--
Purified CaM kinase I was a
generous gift of A. Nairn (Rockefeller University), and purified CaM
kinase IV was a generous gift of A. Means. Peptides with the amino acid
sequences CGVKKRKSSSSVQMME and CGVKKRKASSSVQMME
were generated on an automated peptide synthesizer. Phosphorylation of
these peptides by CaM kinase I and CaM kinase IV was carried out for 5 min at 30 °C in a 50-µl reaction containing 50 mM
PIPES, pH 7.5, 10 mM MgCl2, 0.5 mM
CaCl2, 10 µg/ml calmodulin, 0.1 mg/ml bovine serum
albumin, 20 µM [-32P]ATP (2.5 Ci/mmol),
and 50 µM peptide. Reactions were initiated by the
addition of kinase and terminated by the addition of 10 µl of 30%
trichloroacetic acid. Reaction mixes were then spotted onto P-81
phosphocellulose paper (Whatman), washed extensively with water, and
then counted for Cerenkov radiation.
Protein Phosphorylation--
B-CaM kinase II,
made kinase-inactive with a K42M point mutation (referred to as
B-CaM kinase IIi) as well as
B-CaM kinase IIi with the S332A point
mutation were expressed in COS-7 cells and purified as described (25,
31, 32). Phosphorylation of these two inactive mutants of
B-CaM kinase II by purified CaM kinase I was performed
at 30 °C for 5 min in a 50-µl reaction mix containing 50 mM PIPES, pH 7.5, 1 mM dithiothreitol, 10 mM MgCl2, 50 µM
[
-32P]ATP (2.5 Ci/mmol), 1 µM
calmodulin, 1.2 mM CaCl2, and 0.1 mg/ml bovine
serum albumin. Phosphorylation of these CaM kinase II mutants by
purified CaM kinase IV was performed under similar conditions, except
that bovine serum albumin was used at 1 mg/ml and the specific activity
of [
-32P]ATP was 1.0 Ci/mmol. Equal amounts of
purified
B-CaM kinase IIi with or without
S332A were used in each reaction mix as verified by SDS-polyacrylamide
gel electrophoresis and Coomassie staining of the CaM kinase II samples
(data not shown). The concentration of CaM kinase II used between
experiments ranged from 0.01 to 0.02 mg/ml. The phosphorylation
reactions were initiated by adding either CaM kinase I or CaM kinase IV
at a concentration of 5 µg/ml to the reaction mix and terminated by
the addition of 5× Laemmli sample buffer. The reactions were then
analyzed by SDS-polyacrylamide gel electrophoresis followed by
autoradiography. Scanning densitometry was performed on an Arcus AGFA
II scanner using NIH Image for data analysis. Phosphorylation of
B-CaM kinase IIi by either CaM kinase I or
CaM kinase IV was quantified by densitometry and assigned the value of
100%; phosphorylation of
B-CaM kinase IIi S332A was divided by this value to determine its corresponding percentage of phosphorylation.
NLS Receptor Binding Experiments--
The NLS receptor (NLS-R)
m-pendulin fused with a His6 tag within the bacterial
expression vector pET-30a (Novagen) was a generous gift of S. Adam
(Northwestern University, Chicago, IL) (33). The NLS-R construct was
transformed into BL21 bacteria (Novagen) and grown in 200 ml of LB
medium containing 50 µg/ml of kanamycin and amplified with 1 mM isopropyl -D-thiogalactopyranoside as described (33). The bacteria were pelleted, and the pellet was resuspended in 10 ml of 10% glycerol, 0.2% Nonidet P-40, 0.1 mM Tris (carboxyethyl)phosphine, 1 µg/ml leupeptin, 1 µg/ml aprotonin, 0.2 mM phenylmethylsulfonyl fluoride,
0.2 mM sodium metabisulfite and 1 µg/ml pepstatin A. The bacterial suspension was frozen with liquid nitrogen, thawed, and
then sonicated for a total of 3 min on ice at a setting of 50 W. The
lysate was then centrifuged for 30 min at 14,000 × g
at 4 °C. The resulting supernatant was then filtered through a
0.45-µm filter, aliquoted, quick frozen with liquid N2,
and then stored at
80 °C. The His6 NLS-R was complexed to Ni2+-containing agarose resin (Qiagen) at a ratio of 6 mg of NLS-R-containing lysate to 50 µl of resin for 60 min at 4 °C
with tumbling in 1 ml of 20 mM Tris, pH 8.0, 500 mM NaCl, and 5 mM imidazole. The beads were
then spun down and washed once in the same buffer and then three times
in interaction buffer, a modification of a buffer used by Rexach and
Blobel for NLS/NLS-R interaction (34) containing 40 mM
Hepes, pH 7.0, 150 mM KOAc, 2 mM MgOAc, 20 mM imidazole, 0.1 mM TCEP, 0.1% Tween 20, 0.1% casaminoacids (Difco), 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and
1 µg/ml pepstatin A. COS-7 cells transfected with
-CaM kinase II wild type,
B-CaM kinase II wild type, and mutants of
B-CaM kinase II containing the individual point
mutations S332A, S332D, and S332E were lysed by sonication and
centrifuged, and kinase activity in the soluble fraction was assayed as
described (12). Kinase lysates were diluted in interaction buffer to
produce equal concentrations of kinase based on the kinase activity
assay, and these were then added to equal aliquots of the
Ni2+-resin·NLS-R complexes (10 µl of resin per sample
in 250 µl of interaction buffer) and tumbled at 4 °C for 60 min.
After tumbling, the resin was spun down at 3,000 × g
for 1 min and then washed four times over 15 min in interaction buffer.
The resin pellets were then stripped of protein in 20 µl of buffer
containing 40 mM Tris, pH 8.0, 100 mM EDTA, and
500 mM NaCl. The supernatants stripped from the beads were
then run on a 9% denaturing polyacrylamide gel electrophoresis gel and
transferred to nitrocellulose, and the resulting blots were
subjected to Western blotting for
-isoforms of CaM kinase II as
described previously (14). Each sample of kinase to be used for NLS-R
binding was also subjected to Western blotting in parallel with the
NLS-R-bound samples to ensure that kinase concentration as determined
by kinase activity was equal to kinase level detected by Western
blot. Densitometry and image analysis were performed as described above
for phosphorylation experiments. For all quantitative analysis, the
amount of each kinase that bound to the NLS-R·Ni2+-resin
complex was divided by the amount of that kinase in the prebinding
samples, and this ratio was compared for each kinase isoform and mutant
analyzed, with the binding of wild type
B-CaM kinase II
assigned the value of 100%. All chemicals used were obtained from
Sigma except as indicated. For other NLS receptor constructs,
His6-NPI-1 was a gift of S. Adam (33), GST-NPI-1 and
GST-NPI-3 were gifts of R. O'Neil and P. Palese (35), and GST-karyopherin
was a gift of M. Rexach and G. Blobel (Rockefeller University) (34).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cotransfection with CaM Kinase I or IV Blocks Nuclear Targeting of
B- and
B-CaM Kinase II--
As we
previously reported (12, 13),
-CaM kinase II, which lacks a
functional NLS, localizes exclusively to the cytoplasm (Fig.
1A) when transfected into
COS-7 cells, while
B-CaM kinase II, which contains an
NLS, localizes almost entirely to the cell nucleus (Fig. 1B
and Table I). We next sought to determine
whether the activation of any known signal transduction pathway could alter the localization of
B-CaM kinase II to the
nucleus. In order to do this,
B-CaM kinase II was
cotransfected into COS-7 cells with cDNA constructs encoding
several different protein kinases: the catalytic subunit of protein
kinase A and constitutively active truncation mutants of CaM kinase I,
CaM kinase IV, protein kinase C
, and the protein phosphatase
calcineurin. After 48 h, the localization of CaM kinase II was
determined by immunofluorescence using a monoclonal antibody that
recognizes
-CaM kinase II isoforms. No effect on the nuclear
localization of
B-CaM kinase II was noted when
cotransfected with protein kinase A, protein kinase C, or calcineurin
(data not shown). However, when cotransfected with constitutive,
Ca2+/calmodulin-independent mutants of CaM kinase I or CaM
kinase IV, the localization of
B-CaM kinase II was
almost completely cytoplasmic (Fig. 1, C and D,
and Table I). In order to determine whether this effect was cell
type-specific, we performed similar experiments in PC12 cells (a rat
pheochromocytoma cell line) and HEK-293 cells (a human kidney cell
line) and found a similar blockade of
B-CaM kinase II
nuclear targeting by CaM kinase I and CaM kinase IV (data not shown).
COS-7 cells transfected only with CaM kinase I or CaM kinase IV did not
show any detectable immunofluorescence when stained with the CaM kinase
II antibody, ruling out the possibility that antibody cross-reactivity
with CaM kinase I or CaM kinase IV was the basis for this result (data
not shown).
|
|
The Blockade of CaM Kinase II Nuclear Targeting Is
Specific--
In order to determine whether CaM kinase I and CaM
kinase IV exert a specific block on nuclear targeting of CaM kinase II or instead block nuclear import in a general fashion, we cotransfected constitutive CaM kinase I or CaM kinase IV into COS-7 cells with fos-LacZ, a fusion of the transcription factor
fos with LacZ (-galactosidase) that localizes to the
nucleus as a result of a bipartite NLS derived from the fos
protein (36). We found that nuclear localization of the
fos-LacZ fusion protein was preserved when cotransfected into COS-7 cells under conditions identical to those in the experiments involving blockade of CaM kinase II nuclear targeting (data not shown).
This suggests that CaM kinase I and CaM kinase IV exert a specific
blockade on nuclear localization of CaM kinase II rather than a
generalized blockade of nuclear import.
The First Ser following the NLS Is Required for Blockade of Nuclear
Targeting--
Blockade of nuclear import by phosphorylation of
residues adjacent to an NLS has been seen for some but not all nuclear
targeted proteins (Table II; reviewed in
Ref. 24). We therefore sought to determine whether nuclear targeted
isoforms of CaM kinase II have any possible phosphorylation sites
adjacent to the NLS. As can be seen in Fig.
2, both B-CaM kinase II
and
B-CaM kinase II, which have been shown to be
targeted to the nucleus, as well as
A-CaM kinase II,
which has an identical NLS, all have a string of four Ser residues
immediately following the core NLS sequence KKRK. These four Ser
residues correspond to residues 332-335 in both
B-CaM
kinase II and
B-CaM kinase II and to residues 355-358 in
A-CaM kinase II. In order to determine whether
phosphorylation of one or more of these Ser residues is responsible for
blockade in nuclear targeting by CaM kinase I and CaM kinase IV, we
mutated all four Ser residues to Ala (S332-335A) in
B-CaM kinase II. Like wild-type
B-CaM
kinase II, this mutant localizes almost entirely to the nucleus when
transfected into COS-7 cells. Unlike
B-CaM kinase II,
however, nuclear targeting of this quadruple Ser to Ala mutant is not
blocked by cotransfection with constitutive CaM kinase I or CaM kinase
IV (Fig. 3 and Table I). To determine if
only one Ser is responsible for this effect, we next mutated each of
the four Ser residues individually to Ala. The mutant in which the
first Ser following the NLS was mutated to Ala (S332A) shows nuclear
targeting that is not affected by coexpression with CaM kinase I or CaM
kinase IV and is indistinguishable from the targeting of the mutant
with all four Ser residues mutated to Ala. In contrast, the other three
mutants with single Ser to Ala mutations two, three, and four residues
following the NLS (S333A, S334A, and S335A, respectively) behave like
wild-type
B-CaM kinase II and are blocked from entering
the nucleus by CaM kinase I and CaM kinase IV. Immunostaining of the
B-CaM kinase II S332A and S333A mutants with and without
CaM kinase I or CaM kinase IV is shown in Fig. 3, and compiled data for
all mutants are displayed in Table I.
|
|
|
A Negative Charge following the NLS Is Sufficient for Blockade of
Nuclear Targeting--
The preceding experiments clearly show that the
Ser immediately following the CaM kinase II NLS is essential for
blockade of nuclear targeting by CaM kinase I and CaM kinase IV and
might be a target for phosphorylation by these kinases. To determine if
a negative charge adjacent to the NLS of CaM kinase II is sufficient to
block nuclear targeting of the kinase, we generated a mutant of
B-CaM kinase II in which all four Ser resides following
the NLS are mutated to Asp (S332-335D) to mimic the negative charge that phosphorylation of these residues would produce. When transfected into COS-7 cells, this mutant localized to the cytoplasm, consistent with the hypothesis that phosphorylation of one or more of these residues could block nuclear targeting of CaM kinase II (data not
shown). In order to further examine this effect, we individually mutated Ser332 and Ser333 to both Asp and Glu.
Mutation of Ser332 to either Asp or Glu caused the kinase
to be localized entirely to the cytoplasm when transfected into COS-7
cells (Fig. 4, A and
B), while mutation of Ser333 to Asp or Glu
resulted in mutants with entirely normal nuclear localization (Fig. 4,
C and D). 100 cells from each mutant were scored
blindly for nuclear or cytoplasmic localization of the kinase as
described for the experiments in Table I, and all 100 of the cells
transfected with the S332D or S332E mutant of
B-CaM kinase II were scored as having cytoplasmic localization of the kinase,
while all 100 of the cells transfected with the S333D or S333E mutant
were scored as having nuclear localized kinase. Thus, a negatively
charged residue replacing the first Ser following the NLS abolishes
nuclear targeting of
B-CaMK II, while a negative charge
at the second position does not appreciably alter nuclear targeting.
|
CaM Kinases I and IV Can Phosphorylate the Ser Adjacent to the NLS
of CaM Kinase II in Vitro--
A study by Miyano et al.
(37), which did not address nuclear targeting, demonstrated that a
peptide encompassing the NLS and four adjacent Ser residues of
A-CaM kinase II was a substrate for CaM kinase IV and
that this phosphorylation was on the first Ser. In order to determine
if CaM kinase I could also phosphorylate this site, we synthesized
peptides derived from
B-CaM kinase II with the amino
acid sequence CGVKKRKSSSSVQMME (the S peptide) as well as
an otherwise identical peptide with the first Ser mutated to Ala,
CGVKKRKASSSVQMME (the A peptide). These peptides correspond to residues 326-340 of
B-CaM kinase II; the initial Cys
on each peptide was engineered for coupling purposes unrelated to these experiments. In agreement with the previous study, we found that purified wild type CaM kinase IV did phosphorylate the Ser-containing peptide in the presence of Ca2+ and calmodulin and that
phosphorylation of the A peptide was more than 7-fold reduced compared
with the S peptide. Additionally, we found that purified wild type CaM
kinase I was also capable of phosphorylating the S peptide in the
presence of Ca2+ and calmodulin and that phosphorylation of
the A peptide was reduced by more than 4-fold compared with the S
peptide (data not shown).
|
Blockade of Nuclear Targeting Is Not Dependent on the Position of
the NLS within CaM Kinase II--
We sought to determine whether
moving the NLS of CaM kinase II from the central region of the protein
(based on primary structure) to the N terminus would alter the ability
of CaM kinase I or CaM kinase IV to block nuclear targeting of the
kinase. To accomplish this, we generated a mutant of -CaM kinase II
containing an N-terminal fusion of 13 amino acids derived from the NLS
region of
B-CaM kinase II encompassing the KKRK sequence
as well as the four adjacent serines and neighboring residues that
complete the CaM kinase I and CaM kinase IV consensus phosphorylation
sequences (Figs. 5A and
6A). When transfected into
COS-7 cells, the NLS was able to function at the N terminus of normally
cytoplasmic
-CaM kinase II, efficiently directing this protein to
the nucleus. When cotransfected with constitutive CaM kinase I or CaM
kinase IV, nuclear targeting of this protein was blocked as effectively
as was found for wild type
B-CaM kinase II (Fig.
6B).
|
A Negative Charge Adjacent to the NLS of CaM Kinase II Reduces
Binding to an NLS Receptor--
Does a negatively charged phosphate
immediately adjacent to the positively charged NLS inhibit binding of
this sequence to the NLS-R? To address this question, we developed an
assay to assess binding of CaM kinase II to the NLS-R m-pendulin
coupled via a His6 tag to Ni2+-complexed
agarose resin. Other putative NLS receptor proteins including
His6-tagged NPI-1, GST-tagged NPI-1, GST-tagged NPI-3, and
GST-tagged karyopherin were used in similar resin binding experiments, but none bound to
B-CaM kinase II nearly as
well as m-pendulin (data not shown), and so His6 m-pendulin
was used for all subsequent NLS-R binding experiments. The NLS-R
effectively bound NLS-containing
B-CaM kinase II
wild-type but showed almost no binding to NLS-deficient
-CaM kinase
II wild-type, thus demonstrating the specificity of the assay for
NLS-containing proteins (Fig. 7,
A and B). In addition, a previously described
mutant of
B-CaM kinase II in which the first two Lys
residues of the NLS sequence were mutated to Asn (KKRK
mutated to NNRK), disrupting nuclear targeting of the
kinase (12), was similarly unable to bind to the NLS-R in this assay,
while wild type
B-CaM kinase II bound as well to the
NLS-R as did
B-CaM kinase II (data not shown). Mutation
of S332A in
B-CaM kinase II did not inhibit binding to
the NLS-R, but the S332D and S332E mutants demonstrated substantially reduced binding to the NLS-R (Fig. 7A). The S333D mutant of
B-CaM kinase II did not show reduced binding to the
NLS-R compared with wild-type
B-CaM kinase II (data not
shown). Sufficient experimental trials were performed to allow
statistical analysis comparing the binding to the NLS-R of wild-type
B-CaM kinase II (standardized to 100% binding)
versus that of
-CaM kinase II wild type and of
B-CaM kinase II mutants S332A and S332D. There was
negligible binding (4.2 ± 4.6%) of
-CaM kinase II to the
NLS-R, while the binding of the S332D mutant of
B-CaM
kinase II was 51.9 ± 5.7% of
B-CaM kinase II wild
type, and the binding of the S333A mutant was not significantly
different (115 ± 16%) from
B-CaM kinase II wild
type (Fig. 7B). These results demonstrate that a negatively charged residue immediately downstream from the NLS of
B-CaM kinase II reduces binding of this protein to the
NLS-R compared with wild-type
B-CaM kinase II.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This paper demonstrates that phosphorylation by CaM kinase I or CaM kinase IV of a Ser adjacent to the NLS of nuclear targeted isoforms of CaM kinase II can block its nuclear targeting. CaM kinases I and IV have widespread tissue distribution and have been detected in such locations as the thalamus, hypothalamus, and heart, where nuclear targeted isoforms of CaM kinase II are expressed (10, 13, 38, 39). Thus, the co-localization of CaM kinase I or IV with nuclear targeted CaM kinase II that we have produced in situ through transfection is likely to occur in vivo. We have also examined the effects on nuclear targeting of CaM kinase II when coexpressed with protein kinase A or protein kinase C, two other multifunctional protein kinases, as well as the protein phosphatase calcineurin, all three of which have been shown to alter targeting of certain other NLS-containing proteins (reviewed in Ref. 24). None of these three signal transduction mediators was able to appreciably alter nuclear targeting of CaM kinase II. CaM kinase I and CaM kinase IV are both capable of modifying the function of many proteins through phosphorylation, and this study adds another important potential function to the cellular effects ascribed to these kinases.
The block of nuclear targeting with constitutively active truncation
mutants of CaM kinases I and IV summarized above was not observed with
full-length, Ca2+/calmodulin-dependent CaM
kinase I or IV, even when the CaM kinase I T177D mutant was used or
when CaM kinases I and IV were cotransfected with calmodulin and/or CaM
kinase kinase and combined with varying lengths of stimulation with
Ca2+-ionophore (data not shown). Wild type CaM kinases I
and IV stimulated with Ca2+/calmodulin were able to
phosphorylate this key site in vitro (Fig. 5), however.
Furthermore, phosphorylation of this site is likely to be sufficient to
block nuclear targeting of CaM kinase II as mutant B-CaM
kinase II S332D and S332E are both completely cytoplasmic (Fig. 4).
Activity-dependent modulation of CaM kinase II localization
by phosphorylation in vivo may occur rather slowly during
synthesis of nascent kinase or during cell division when the nuclear
membrane is disrupted rather than as a rapid
Ca2+-dependent shuttle for the kinase in and
out of the nucleus. It may be difficult to recreate in situ
the conditions that govern regulation of CaM kinase II nuclear
targeting in vivo.
There are a number of possible factors that may hinder the ability of wild type CaM kinases I and IV coupled with Ca2+-elevating stimuli to effectively phosphorylate this site on CaM kinase II and block its nuclear targeting in the transfection system that we have used. The nature of our transfection experiments may require sustained phosphorylation of this site for as long as 48 h in order to see a block in nuclear targeting of the kinase. This is likely to occur with constitutive CaM kinase I or IV but may be difficult to achieve with wild type kinase. Activation of wild type CaM kinases I and IV in cultured cells is likely to be transient if it is like activation of CaM kinase II in these cells. In the case of CaM kinase II, we find that the in situ activation levels are low in the basal state and can be activated to higher levels only transiently. For example, the basal level of CaM kinase II autonomous activity (a measure of the activation state of the enzyme) is approximately 2% in PC12 cells (40, 41), and a similarly low basal level of CaM kinase II autonomy has been measured in COS-7 cells.2 In contrast, primary cultures of hippocampal neurons have a much higher basal level of CaM kinase II autonomy of 15.5% despite having a low resting [Ca2+]i of 15-43 nM (42), while homogenates of various brain regions have basal CaM kinase II autonomy values ranging from 4 to 10.5% (42, 43) and homogenates from cardiac muscle also have a relatively high basal level of CaM kinase II autonomy of approximately 8%.3 CaM kinase II autonomy can be increased by application of Ca2+-elevating agents to cultured cells but then returns to basal levels over several minutes despite continued application of stimulus. Although equivalent activation experiments for CaM kinases I and IV have not been performed, these enzymes may also be less active in cultured cell lines than in primary neuronal or cardiac cells. This may make it difficult to maintain phosphorylation of the Ser adjacent to the CaM kinase II NLS for the 48 h required in our transfection experiments. Furthermore, activation of CaM kinases I and IV requires a complex series of phosphorylation events including both autophosphorylation and phosphorylation by an additional CaM kinase I/IV kinase (28, 44-46), and it is possible that we are not able to fully activate wild type CaM kinase I and IV in our in situ cell system. The fact that neither cotransfection with CaM kinase kinase with or without calmodulin nor the use of mutant CaM kinase I T177D allowed full-length CaM kinases I or IV to block nuclear targeting of CaM kinase II makes this somewhat less likely, however.
It is also possible that a kinase other than CaM kinase I or IV may
actually phosphorylate this key site on CaM kinase II in
vivo. The study by Miyano et al. (37) examining
phosphorylation of this site on the peptide derived from the NLS of
A-CaM kinase II found that extracts from all brain
regions examined could phosphorylate this Ser in a
Ca2+/calmodulin-dependent manner. It was not
established whether this activity was the result of CaM kinase IV, CaM
kinase I, or another kinase, although chromatography suggested that CaM
kinase IV is the predominant
Ca2+/calmodulin-dependent kinase with this
activity in cerebellum. Kinetic analysis demonstrated that this peptide
is in fact only a moderately good substrate for CaM kinase IV, with
Km = 8.0 µM versus
Km = 2.6 µM for synapsin I and a
similar Vmax for both peptides (37). In
vitro and in situ studies indicate the importance of
the first Ser following the NLS for the regulation of nuclear targeting
of CaM kinase II, and depending on the cell type, it may be CaM kinase
I or IV or another as yet unidentified Ser/Thr kinase with similar
specificity that phosphorylates this site in vivo.
Most NLS sequences can be moved within a protein and even fused onto
completely different proteins and still retain their nuclear targeting
function, although in some cases the position of an NLS relative to
surrounding residues can have a profound impact on NLS function (47).
We chose to move the NLS of B-CaM kinase II from the
central region of the protein's primary structure to its extreme N
terminus. Based on the "flower and petal" structure which has been
proposed by Kanaseki et al. (48) to describe the quaternary
holoenzyme structure of CaM kinase II, this would move the NLS a
substantial distance from the central globular core to the peripheral
catalytic region of the holoenzyme. We found the NLS to be able in this
position to efficiently target the kinase to the nucleus. This
N-terminal NLS mutant was designed to contain all of the CaM kinase I
and CaM kinase IV consensus requirements contained within
B-CaM kinase II, and we found that both of these kinases
were also capable of blocking nuclear localization of this mutant. This
suggests that the effect of CaM kinases I and IV on nuclear targeting
of CaM kinase II is a local effect on the region surrounding the NLS
and probably does not depend on any particular three-dimensional
structure of the NLS within the kinase, i.e. phosphorylation
causing the kinase to fold in such a way as to obscure the NLS.
Alteration in nuclear targeting of proteins by phosphorylation is not a new phenomenon; there are many examples of phosphorylation either enhancing or inhibiting nuclear translocation of a variety of NLS-containing proteins (reviewed in Ref. 24). One mechanism for inhibition of NLS function is phosphorylation-induced NLS "masking," which refers to the hypothesis that a phosphate adjacent to the NLS inhibits binding to the NLS-R as a result of its charge or a conformational effect (24). Given that negatively charged regions of the NLS-R are thought to bind to the positively charged NLS of nuclear targeted proteins for nuclear import to occur (49), it is not surprising that a negative charge immediately adjacent to this site may inhibit this interaction. Although this theory has been advanced to explain inhibition of nuclear targeting by phosphorylation of several NLS-containing proteins (Table II), this has never been demonstrated directly.
Using an NLS-R binding assay, we present evidence that the
alternatively spliced 13-amino acid sequence containing the NLS consensus sequence KKRK is critical for binding of B-CaM
kinase II to the NLS-R m-pendulin. Additionally, we demonstrate that a
negative charge adjacent to this NLS reduces binding to the NLS-R
by approximately 50%, while mutation of this site to a neutral Ala
does not significantly alter binding to the NLS-R. A recent study
examining the enhancement of nuclear targeting of the SV40 large T
antigen (a protein with multiple phosphorylation sites, some of
which enhance while others inhibit nuclear targeting) by
phosphorylation gave the first evidence that phosphorylation can affect
binding of an NLS-containing protein to an NLS-R (50). In this study,
the authors report that phosphorylation of SV40-LacZ fusions by casein
kinase II, which causes a large increase in nuclear import rate, may be
mediated by an observed 40% increase in binding of this protein to the
NLS-R. Thus, the 50% decrease in NLS-R binding that we have observed
with a negatively charged residue adjacent to the NLS appears to be in
the range that can have a profound impact on nuclear targeting. Perhaps
a doubly charged phosphate adjacent to the NLS will lead to an even
larger decrease in NLS-R binding than we report here, but the
stoichiometric phosphorylation of CaM kinase II by CaM kinase I or CaM
kinase IV that will be required to examine this may be difficult to
attain in vitro. Regardless, our mutational analysis
demonstrates that a single negatively charged residue is able to
completely block nuclear targeting; whether this is wholly or only
partly mediated by the 50% decrease in binding to the NLS-R that we
have observed has not been established.
The possibility that there is cross-talk within the family of
multifunctional Ca2+/calmodulin-dependent
protein kinases raises some very intriguing questions. It is known that
different frequencies of Ca2+ stimuli can lead to
completely opposite effects on cellular phenotype; for example, high
frequency stimulation of neurons leads to long term synaptic
potentiation, while the same stimulus delivered at lower frequencies
causes long term synaptic depression (51). Furthermore, several studies
have implicated CaM kinase II in the generation of long term synaptic
potentiation (52, 53), which also requires new gene transcription in
order to be maintained (54). The finding that CaM kinase II is capable
of repressing activity of the transcription factors cyclic AMP response
element-binding protein and ATF-1 while CaM kinase I and CaM kinase IV
both activate these transcription factors (20) makes this long term
synaptic potentiation/long term synaptic depression consideration
especially provocative. Could different profiles of Ca2+
stimuli lead to altered nuclear targeting of CaM kinase II via Ca2+-dependent phosphorylation? The observation
that nuclear isoforms of CaM kinase II have been found predominantly
within the brain (B) (13) and heart (
B)
(55), where alterations in cellular physiology are strongly tied to the
frequency and amplitude of Ca2+ oscillations, lends support
to the idea that the regulation of CaM kinase II nuclear targeting in
this manner could have physiologic relevance. The fact that the three
nuclear isoforms of CaM kinase II, each derived from a different gene,
all contain this adjacent string of four Ser residues that has not been
noted in any other nuclear targeted protein suggests that these are
likely to be of biologic importance. The data presented here
demonstrate that phosphorylation of the first of these Ser residues
mediated by CaM kinases I and IV can decrease binding to the NLS-R and
block access of CaM kinase II to the nucleus, where control of gene expression takes place. Through mechanisms such as this, cells may be
able focus the action of multifunctional kinases to discrete periods of
time and space.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Means for providing us with
CaM kinase IV protein and CaM kinases I and IV expression constructs,
Dr. A. Nairn for CaM kinase I protein, Dr. M. Muramatsu for the protein
kinase A expression construct, Dr. T. Soderling for the CaM kinase
kinase expression construct, Dr. P. Yaswen for the calmodulin
construct, Drs. R. O'Neil and P. Palese for the NPI-1 and NPI-3
expression constructs, Drs. M. Rexach and G. Blobel for the
karyopherin- expression construct, and Dr. S. Adam for the
m-pendulin expression construct as well as useful discussion on nuclear
import.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health (NIH) Grants GM40600 and GM 30179.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ Recipient of NIGMS, NIH, Grant 5T32 GM07365.
¶ To whom correspondence should be addressed. Tel.: 650-723-7668; Fax: 650-725-3958; E-mail: schulman{at}cmgm.stanford.edu.
1 The abbreviations used are: CaM kinase, Ca2+/calmodulin-dependent protein kinase II; NLS, nuclear localization signal; NLS-R, NLS receptor; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase.
2 E. K. Heist and H. Schulman, unpublished observations.
3 M. E. Anderson, A. P. Braun, T. Lu, Y. Wu, H. Schulman, and R. J. Sung, unpublished observations.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|