(Received for publication, October 25, 1996, and in revised form, January 28, 1997)
From the Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2612
Ca2+/calmodulin-dependent
protein kinase II (CaM kinase II) -subunits were cloned from a
porcine aortic smooth muscle cDNA library resulting in
identification of alternatively spliced CaM kinase II
B-
and
C-subunits and a novel
-subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the
G-subunit. A clone predicted to encode a 62.2-kDa
-subunit, designated as
E, was isolated with a
variable domain structure similar to a
B-subunit but
with a 114-nucleotide insertion in the conserved "association"
domain of CaM kinase II subunits. A full-length
E-subunit construct expressed in COS cells resulted in
multimeric CaM kinase II holoenzymes (470 kDa) with activation and
autoregulatory properties similar to expressed holoenzymes composed of
B-,
C-, or
G-subunits.
Expression of
E and related
-subunit mRNAs
containing the 114-base insertion was documented in porcine tissues by
reverse transcriptase-polymerase chain reaction. CaM kinase II subunits containing the 38-amino acid insert were identified by Western analysis
of partially purified CaM kinase II from carotid arterial smooth muscle
and brain using a sequence-specific anti-peptide antibody.
Immunoprecipitations of tissue homogenates indicated a comparatively
high level of expression of subunits containing the insert in brain and
provided evidence for their co-assembly with other more abundant
subunits into CaM kinase II heteromultimers. Our analyses indicate the
following patterns of
-subunit expression: vascular smooth muscle,
B >
C >
E,G; heart,
B >
E,C >
G; brain,
E and related subunits
A,B,C,G.
Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II)1 mediates cellular responses induced by increases in second messenger Ca2+ and has been implicated in the control of such essential functions as synaptic transmission (1, 2), gene transcription (2, 3), and cell growth (4). CaM kinase II specific activity in smooth muscle (5, 6) is about 1/10 of that in brain (7). A significant fraction of the kinase in smooth muscle associates with a myofibrillar fraction (8) and co-purifies with caldesmon, a putative thin filament regulatory protein (9). CaM kinase II is activated in vascular smooth muscle cells over a physiological range of free intracellular [Ca2+] (10) and has been shown to be involved in cell migration (11) and modulation of smooth muscle myosin light chain kinase sensitivity to activator [Ca2+] (6). CaM kinase II has also been implicated in the control of Ca2+ channels (12) and sarcoplasmic reticulum Ca2+/ATPase activity in smooth (13) and cardiac muscle (14, 15) and as an intermediate in the activation of the mitogen-activated protein kinase signaling cascade induced by Ca2+-mobilizing stimuli (16).2
Endogenous CaM kinase II (7) and recombinant isozymes expressed in
Escherichia coli (17) or mammalian cells (18) are large
multimeric proteins composed of 8-10 individual protein kinase
subunits, which can be products of four separate genes. The CaM kinase
II subunit genes are expressed in a tissue-specific manner in that two
of the isoforms ( and
) appear to be largely restricted to the
brain, while the other two isoforms (
and
) are variably
expressed in brain and peripheral tissues (5, 19, 20). Additional
subunit diversity arises through alternative splicing. While the full
spectrum and functional significance of subunit heterogeneity has not
been established, it has been proposed that it provides a mechanism for
producing CaM kinase II isozymes with differing kinetics, substrate
specificities, or subcellular localization (21, 22).
With the general goal of understanding the relationship between CaM
kinase II structure and cellular localization/function in smooth
muscle, studies were carried out to identify -subunit isoforms
expressed in mammalian vascular smooth muscle. A novel CaM kinase II
-subunit variant (
G-subunit) was discovered, and a
unique class of
-subunit variants with a 38-amino acid insertion in
the association domain was documented for the first time in primary
neuronal and cardiovascular tissues. When expressed in COS cells, these
unique subunits formed multimeric holoenzymes (7-8 subunits) with
autoregulatory properties characteristic of known CaM kinase II
isozymes. Although the functional significance of the CaM kinase II
-subunits with the 38-amino acid insertion is not known, these
subunits were variably expressed as proteins in arterial smooth muscle,
heart, and brain, where they appeared in heteromultimeric complexes
with other more abundant CaM kinase II subunits.
A porcine cultured thoracic aorta cell
cDNA library (Uni-ZAP XR vector; Stratagene) was screened with a
990-bp DNA probe corresponding to the rat CaM kinase II -subunit
catalytic domain. The probe was generated by RT-PCR using rat brain RNA
and
-subunit-specific primers and phosphorylated by nick translation
with [32P]dCTP using a kit from Promega. Hybridization
was at 50 °C followed by four washes in 1-0.1 × SSC, 0.1%
SDS at 50 °C. Positive clones were categorized by restriction
endonuclease mapping and size analysis of PCR-amplified targets using
primers that spanned the variable region of the kinase, including
regions V1-3. By comparison with predicted products, the
clones were classified as probable
B-,
C-, or unknown subunits, and representative clones were
chosen for sequencing. pBluescript phagemids containing the putative
-subunit cDNAs were excised from the Lambda ZAP II vector
according to procedures provided by the manufacturer (ExAssist/SOLR
System, Stratagene). Both strands of clone 28 were sequenced by the
dideoxy chain termination method using [
-35S]dATP and
Sequenase (U.S. Biochemical Corp.) with overlapping synthetic
oligonucleotide primers as depicted in Fig. 1. Other clones were
sequenced fully in at least one direction and in both directions in
regions corresponding to the variable domains.
Expression Vectors and Construction of Full-length
pBluescript plasmids containing
-subunit inserts (clones 28, 29, and 35) were linearized by
digestion with XbaI.
-Subunit cDNAs were excised by
incomplete digestion with ApaI, which was necessary due to
the occurrence of an ApaI site in the conserved 3
coding
region of the
-subunit cDNA. Full-length fragments were gel
purified and ligated into pRc/CMV. Clone 6 was incomplete at the 5
end
but terminated in the conserved catalytic domain at a position
corresponding to base +666 in the other
-subunits. A full-length
sequence was constructed by ligating in a single reaction the following
three gel-purified DNA fragments: the 5
region of full-length clone 35 (including the 5
-untranslated region (UTR)) excised using the
XbaI site in the pBluescript multicloning sequence and the
NsiI site in the
-subunit sequence; the 3
fragment of
clone 6 (including 3
-UTR) produced by digestion with NsiI and ApaI; and linearized pRc/CMV vector digested with
XbaI and ApaI. The resulting construct was
confirmed by partial sequencing and was shown to be translated into a
functional CaM kinase II subunit in transfected COS-7 cells.
Total RNA was isolated from
the brain and left ventricle of 7-day-old male swine using acid
guanidinium/phenol/chloroform extraction (23) and from adult swine
carotid artery medial smooth muscle, and neonatal rat cardiomyocytes
using RNAzol (Biotecx). Integrity of the RNA was confirmed by
formaldehyde-agarose gel electrophoresis, and concentration was
estimated by absorbance at 280/260 nm. RT-PCR amplification reactions
were carried out using a commercial kit (Perkin-Elmer Corp.) and
Taq DNA polymerase (Fisher). RT reactions were carried out
with oligo(dT)16 primers and 2 µg of total RNA. PCR
reactions utilized 500 pmol/ml of each primer. In the "nested" PCR
reactions (Fig. 6) 0.5 µg of control -subunit plasmid DNA or 1 µl of the reaction mix from the primary RT-PCR reactions was used as
template. PCR amplifications were carried out on a PTC-100 Thermocycler
(MJ Research, Inc.) using a cycling protocol (35 cycles) recommended by
the PCR kit manufacturer and the following primers:
5
-CCGTGGTACATAATGC-3
(nucleotides 1100-1115) and
5
-ACATGTCCATGTCATC-3
(reverse complement of 1398-1413 in the rat
A-subunit, accession number J04063[GenBank]) to amplify the
V3 region in rat
-subunits (Fig. 3); nucleotides
987-1001 (5
-GTCAACTGAGAGTTC-3
) and the reverse complement of
1252-1266 (5
-CACGTTCATCTGGTA-3
) in the coding sequence of the
porcine
2-subunit (accession number U73504[GenBank]); nucleotides
103-120 in the porcine
C-subunit
(5
-ACCTCCACGCAGGAATAT-3
) and the reverse complement of
5
-TCTGCTCCTCAGCCA-3
in the 114-base V3 insert in the
E-subunit (primer set A, Fig. 6); 5
-CAGATGCTGACTATA-3
(751-765) and 5
-CACAATGCTACAGATG-3
, the reverse complement of 1012-1027 in the porcine
C-subunit (primer set B, Fig.
6); nucleotides 706-723 (5
-CCAGAGTGGGACACGGTG-3
) and the reverse
complement of 1335-1354 (5
-CATCGCCTATATCCGCCTCA-3
) in the porcine
C-subunit (Fig. 7).
For Southern blots (5), oligonucleotide probes were end-labeled using
[-32P]ATP and T4 polynucleotide kinase and purified by
gel filtration over a Pharmacia Sephadex G-50 Nick column. The
oligonucleotide probes used were as follows: 5
-CCAGAGTGGGACACGGTG-3
(nucleotides 706-723 in
C common to all
-subunits);
5
-TTGGCAGGCAGAGCTCC-3
(947-963 in
G recognizing a
V1 sequence common in
A- and
G-subunits); 5
-CAGTCTCGTAAGCCCAG-3
(1011-1027 in
B recognizing a V2 sequence common to
B-,
G-, and
E-subunits); and
5
-GAATGGCAGCTCGGT-3
(recognizing the
E-subunit
V3 sequence).
COS-7 cells (ATCC
CRL1651) were cultured in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) plus 10% fetal bovine serum (Life Technologies,
Inc.). The cells were transfected with CaM kinase II -subunit
cDNAs in the pRc/CMV expression vector (1 µg of plasmid DNA)
using 10 µl of Lipofectamine (Life Technologies, Inc.) in 1 ml of
OPTI-MEM I medium for 16 h and cultured with growth medium for an
additional 24 h. Cells were lysed (4 °C) in buffer A,
containing 50 mM MOPS (pH 8.6), 3 mM EGTA, 100 mM NaF, 250 mM NaCl, 100 mM sodium
pyrophosphate, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 0.2 units/ml aprotinin, 1% Nonidet
P-40, and 50% Hanks' buffered salt solution.
CaM kinase II activity was assayed as described previously with autocamtide-2 (KKALRRQETVDAL) as a peptide substrate (5). To measure generation of Ca2+/CaM-independent or "autonomous" activity, COS cell lysate CaM kinase II was autophosphorylated by preincubating for 30 s or 5 min in 50 mM MOPS (pH 7.4), 10 mM magnesium acetate, 3 mM EGTA, 4 mM calcium chloride, 400 nM calmodulin, 0.2 mM ATP, 15 mM 2-mercaptoethanol at 30 °C. Controls lacked added Ca2+/CaM and ATP. Reactions were stopped by removing a 25-µl aliquot to tubes (4 °C) containing 5 µl of 90 mM EDTA. 5-µl aliquots were subsequently assayed for "total" and "independent" CaM kinase II activity as described previously (5) but with 6 mM CaCl2 added to the total tubes to compensate for EDTA in the autophosphorylated lysates. Autonomous activity was defined as [(independent/total activity) × 100].
Size Fractionation of Expressed CaM Kinase II HoloenzymesGel filtration chromatography was carried out on a
Superose 12 FPLC column (Pharmacia Biotech Inc.) equilibrated and
eluted with buffer A (4 °C) at a flow rate of 0.25 ml/min,
collecting 0.25-ml fractions. The elution of thyroglobulin (669 kDa),
catalase (250 kDa), and ovalbumin (67 kDa) was used to calibrate the
column. Sucrose gradient centrifugation was carried out as described
previously (18, 24). Lysates from -subunit-overexpressing COS cells (300-1000 µg of total protein) were layered on 5-20% sucrose
gradients (4.5 ml) containing 50 mM MOPS (pH 7.0), 150 mM NaCl, 0.5 mM EDTA, 5% glycerol, 0.5%
Nonidet P-40 and centrifuged at 150,000 × g for
16 h at 5 °C. 150-µl fractions were removed from the top, and
aliquots removed for SDS-PAGE (30 µl) and CaM kinase II assays (7.5 µl). Molecular sizes were estimated relative to the sedimentation of
thyroglobulin, catalase, and ovalbumin standards according to the
following formula (24): R = (Mr (unknown)/Mr (std))2/3,
where R is the quotient of the distance traveled by
the unknown in the sucrose gradient divided by the distance traveled by
the standard. The sizes relative to each standard were averaged to obtain estimated Mr values.
Anti-peptide
antibodies were produced in New Zealand White rabbits as described
previously (5, 25). A peptide corresponding to amino acids 39-69 in
the catalytic domain of the CaM kinase II -subunit sequence, which
is common in all CaM kinase II subunits, was solubilized in
phosphate-buffered saline (10 mg/ml), emulsified with Hunter's
Titermax adjuvant (CytRx Corp.), and used to produce the antibody
designated CK2-CAT. A synthetic peptide (PEGRSSRDRTAPSAGMQPQPSLC) from
the V3 sequence in the
E-subunit was
cross-linked to purified protein derivative of tuberculin (Statens
Seruminstitut, Copenhagen) with gluteraldehyde (25), emulsified with
RIBI adjuvant (RIBI Immunochemical Research Inc.), and used to produce
the antibody designated CK2-V3. Antibodies were purified by peptide
affinity column chromatography using cyanogen bromide-activated
Sepharose 4B (Pharmacia). Fractions were eluted with 0.2 M
glycine, dialyzed, and stored in phosphate-buffered saline containing
0.02% sodium azide.
Western blots utilized standard procedures (5). Immunoreactive bands were visualized using horseradish peroxidase (HRP)-coupled 2° antibody and the enhanced chemiluminescent (ECL) detection method (Amersham Corp.). In the case of the Western blots carried out on proteins immunoprecipitated from tissue homogenates with CK2-V3, 1° antibodies were directly cross-linked to HRP to catalyze the ECL reaction (EZ-link Plus activated peroxidase and Freezyme conjugate purification kit; Pierce).
Fresh brain or frozen pulverized heart and carotid artery were
homogenized in 5 volumes of buffer A and centrifuged at 100,000 × g for 40 min, and the supernatant was assayed for CaM kinase II and total protein. A volume of brain extract containing 115 nmol/min
total CaM kinase II activity and volumes of carotid and heart extracts
containing 30 nmol/min of total activity were immunoprecipitated with
CK2-V3 (2 µg of affinity-purified antibody/mg of extract protein) for
16 h at 4 °C. Immune complexes were collected by the addition
of protein A-coupled agarose beads (Pierce) and centrifugation at
10,000 × g for 15 s. Immunoprecipitated proteins
were washed three times with buffer A and then solubilized in sample
buffer and resolved by SDS-PAGE. Control immunoprecipitations were
carried out in the presence of 25 µM immunizing peptide,
25 µM immunizing peptide coupled to carrier protein
(purified protein derivative), and 25 µM unrelated
peptide (IHFHRSGSPTVPI; near the C terminus of CaM kinase II
-subunit) coupled to carrier protein.
CaM kinase II was partially purified from porcine brain and carotid artery medial smooth muscle by DEAE anion exchange chromatography followed by phosphocellulose cation exchange chromatography as described previously (26) to specific activities of 60 and 90 nmol/min/ml, respectively. Synthetic oligonucleotides were synthesized on an Applied Biosystems model 394 DNA synthesizer and synthetic peptides on an Applied Biosystems 431A peptide synthesizer in the Core Molecular Biology Lab at the Weis Center for Research. Autoradiograms and ECL exposures were scanned and digitized with a Molecular Dynamics personal densitometer and ImageQuant 4.0 software.
Four candidate CaM kinase II -subunit clones were
isolated from a cultured porcine thoracic aortic cell cDNA library
and sequenced as shown in Fig.
1.3 A comparison of clone
28 sequence with published rat and human
-subunit sequences
indicated that it was a novel CaM kinase II
-subunit variant. It was
designated the
G-subunit, consistent with published
nomenclature for alternatively spliced
-subunit variants (18, 27,
28). This
-subunit contained two sequences encoding 21 (V1 region) and 23 (V2 region) amino acids,
each of which has been shown to be individually expressed in some CaM kinase II
-subunit isoforms (18, 27) (Fig. 2). Clone
35 was found to lack the V1 insertion identifying it as a
B-subunit cDNA, while clone 29 lacked inserts in
V1 and V2, identifying it as a
C-subunit. Nucleic acid sequences of the porcine
cDNAs were 95% identical to the human sequence in the coding
region and 3
-UTR but only 29% identical in an overlapping region of 21 nucleotides in the 5
-UTR. Predicted amino acid sequences of the
porcine
B and
C clones were 100%
identical to published human
B- and
C-subunit sequences.
Clone 6 was incomplete, lacking the 5-UTR and first 665 bases of the
-subunit coding sequence. Its sequence was otherwise identical to a
B-subunit but with an additional 114-base insert at a
position corresponding to bases 1216 and 1217 of the
G
sequence (Fig. 3). The insertion resulted in the
substitution of an alanine for a valine followed by 38 amino acids in
the translated sequence in a region considered to be part of the
conserved association domain (Figs. 2 and 3). Three
-subunit
variants containing an identical 114-base insert (referred to here as
V3) and either no V2 insert, the 69-base
V2 insert, or a truncated 27-base V2 insert
were recently identified by RT-PCR in cultured human biliary tumor
cells and designated as putative
F-,
E-,
and
D-subunits (28). Based on this, we designated clone
6 as a partial
E-subunit cDNA.
To investigate the possibility that the V3 sequence was CaM
kinase II subunit- and/or species-specific, RT-PCR was used to amplify
products spanning this domain from neonatal rat cardiomyocyte RNA using
primers specific for the rat -subunit sequence. Two major products
were amplified consistent in size with predicted
-subunit targets
containing or lacking the 114-base V3 insert. Sequencing
the RT-PCR products from rat mRNA confirmed a homologous 114-base
V3 insert, nearly identical to the porcine and human sequences except for two nucleotides, which resulted in two amino acid
substitutions (Fig. 3). Similar experiments using
-subunit-specific PCR primers and swine brain, carotid artery, and cardiomyocyte mRNA
amplified only a single product of the size predicted for a
-subunit
lacking a V3 insertion (not shown).
To assess the biochemical properties of the novel CaM
kinase II G- and
E-subunits, the
cDNAs were subcloned into a mammalian expression vector (pRc/CMV)
and expressed in transiently transfected COS-7 cells. Because the
putative
E-subunit clone was incomplete (clone 6), and
additional full-length clones were not isolated from the library, a
full-length sequence was constructed (see "Experimental
Procedures"). Transfection of the cDNAs in COS cells resulted in
6-27-fold increases in Ca2+/CaM-stimulated autocamtide-2
kinase activity in cell lysates (Fig. 4A).
Western analysis identified CaM kinase II subunits in each of the
overexpressing cell lines consistent in size with those predicted based
on amino acid sequences (Fig. 4B). A distinguishing feature
of CaM kinase II is that upon activation it undergoes a transition to a
Ca2+/calmodulin-independent or "autonomous" kinase
activity, a reaction that is facilitated by the multimeric structure of
the holoenzyme (29). Preincubation in the absence of exogenous
substrate resulted in the rapid and extensive conversion of each
expressed
-subunit CaM kinase II isozyme to its autonomous form
(Fig. 4C).
The position of the V3 insertion within the conserved
association domain raised the possibility that multimeric complexes composed of E-subunits might be disrupted or modified in
size. However, gel filtration analysis of both CaM kinase II
E- and
B-subunits expressed in COS cells
(Fig. 5A) and Western analysis of the column
fractions with CK2-CAT (Fig. 5B) indicated that most of the
kinase nearly co-eluted with a thyroglobulin (669 kDa) standard.
Sucrose gradient centrifugation confirmed expression of
B,E,G-subunits as multimers in COS cells with estimated
sizes as follows:
B, 448 kDa;
E, 471 kDa;
G, 425 kDa. These data suggest that the
-subunits
form multimers of 7 or 8 subunits, a finding consistent with
previous results describing the human
B-subunit (18). A
variable fraction of Ca2+/CaM-dependent kinase
activity, consistent in size with subunit monomers, was also resolved
by both gel filtration (Fig. 5A) and sucrose density
centrifugation. However, only weakly immunoreactive 35-40-kDa bands
could be detected in these fractions (Fig. 5B), suggesting
that the source of kinase activity was proteolytic fragments of the
expressed subunits.
Structural Characterization of Expressed CaM Kinase II
To better define the
-subunit structures that contain the V3 insertion
in vivo, RT-PCR was carried out using mRNA from porcine brain, carotid artery, and cardiomyocytes such that only
-subunit-derived targets that contained the 114-base V3
insert and spanned the V1 domain were amplified (Fig.
6). A second PCR amplification was carried out using
templates amplified from the primary RT-PCR reactions and nested
primers spanning V1 and V2. The predominant products obtained from this reaction were identical in size to the
product amplified from the control
B-subunit clone. This experiment confirmed that
-subunits containing the V3
insert are in fact expressed at the mRNA level in primary porcine
tissues and indicated that the insert occurred mainly in combination
with the 69-base V2 insert (consistent with the sequence of
clone 6 or the predicted
E-subunit) or possibly a
63-base V1 insert, which would represent yet another
-subunit variant. Other minor products were amplified with the
nested primers, some of which were similar in size to the products
amplified from control
C and
G subunit
clones, suggesting low level in vivo expression of
alternative
-subunit isoforms containing the V3
insert.
To
provide an estimate of the relative expression of -subunits
containing the V3 insert compared with other
-subunits,
RT-PCR experiments were carried out using primers that spanned all
three variable domains. Products amplified from porcine brain, arterial smooth muscle, and cardiomyocyte mRNA and control plasmid DNA (Fig.
7A) were Southern blotted with
32P-labeled oligonucleotide probes that hybridized
specifically to the 63-base V1, 69-base V2, or
114-base V3 sequence or to a sequence common to all
-subunits (Fig. 7B). Hybridization with the common probe
produced an autoradiogram with a pattern essentially identical to the
ethidium bromide-stained gel (Fig. 7A). In smooth muscle,
the most abundant PCR product hybridized with the
B-specific V2 probe and was consistent in
size with that amplified from the control
B-subunit
cDNA. Less abundant were products that were consistent in size with
that expected from the
C-subunit and hybridized only
with the common probe (not shown). Minor amounts of a 781-base product
that hybridized with both the V1- and
V2-specific probes were amplified from smooth muscle
mRNA, consistent with low level expression of the
G-subunit. Hybridization of the arterial smooth muscle
PCR products with the V3 probe was weak, suggesting that
-subunit transcripts containing the 114-base V3 insert
must be a minor fraction of total
-subunit mRNA in this
tissue.
The pattern of PCR products amplified from cardiomyocyte RNA was
similar to that in smooth muscle, but in this case products consistent
in size with the E-subunits and hybridizing with the V3-specific probe were more abundant. In contrast, the
largest and most abundant
-subunit products amplified from porcine
brain RNA included the 114-base V3 insertion. Probes
specific for both the V2 and V1 inserts also
hybridized with these targets, implying the expression of both the
E-subunit and another isoform predicted to have the
63-base V1 insert in combination with the 114-base V3 insert. Only minor amounts of a 718-base product, which
would reflect expression of the
B-subunit, or a 649-base
product, consistent with the
C-subunit, were amplified
from brain RNA. A minor product slightly smaller than the
G-subunit and hybridizing with the V1-specific primer was amplified from brain RNA, probably
reflecting expression of the
A-subunit, which was the
original
-subunit isoform cloned from brain (27).
To confirm expression of CaM
kinase II -subunits containing the 114-base V3 insert at
the protein level in primary tissues and to gain insight into its
assembly into multimeric CaM kinase II isozymes in vivo, an
anti-peptide antibody was produced (CK2-V3) against a portion of the
predicted 38-amino acid insert. The specificity of CK2-V3 was
established by Western analysis of lysates from COS cells
overexpressing recombinant
-subunits. In a mixture of such lysates a
62-kDa band was identified, consistent with the predicted size of the
E-subunit (Fig. 8A). A 52-kDa
band in the lysates also cross-reacted with CK2-V3 but not with CK2-CAT (Fig. 8B), indicating a protein unrelated to CaM kinase II.
A consistent observation was a relatively weaker immunoreactivity of
the expressed
C- and
E-subunits with
CK2-CAT compared with
B- and
G-subunits,
which is apparent in mixtures of COS cell lysates containing
approximately equal activities of each of the expressed subunits (Fig.
8B, lane 1).
Analysis of partially purified fractions of CaM kinase II from carotid
artery smooth muscle with CK2-CAT indicated CaM kinase II subunits with
apparent sizes in the range of 54-58 kDa (Fig. 8B). These
subunits were not cross-reactive with CaM kinase II - or
-subunit-specific antibodies (not shown) and therefore probably
represent mixtures of
- and
-subunits. Similar fractions from
porcine brain contained primarily 50- and 58-60-kDa subunits, which
can be attributed to expression of CaM kinase II
- and
-subunits
as previously established for this tissue (7). Bands consistent in size
with the
E-subunit (62 kDa) were barely detectable in
either brain or carotid fractions immunoblotted with CK2-CAT. When the
same membrane was probed with CK2-V3, a 62-kDa band that co-migrated
with recombinant
E-subunit standards was detected in the
partially purified CaM kinase II fractions from both tissues, indicating expression of the
E-subunit or a similarly
sized CaM kinase II subunit containing the V3 insert (Fig.
8A). Smooth muscle CaM kinase II preparations also contained
CK2-V3 cross-reactive bands, which were estimated to be 56 and 64 kDa,
consistent in size with
C- and
G-like
subunits containing the 38-amino acid V3 insert. The
preparation from brain also contained minor amounts of unidentified
faster and slower migrating subunits cross-reactive with the CK2-V3
antibody. To determine the composition of native CaM kinase II isozymes
containing
G- and related subunits, aliquots of porcine
brain, carotid artery medial smooth muscle, and cardiac tissue
homogenates were immunoprecipitated with CK2-V3. The immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with CK2-V3 (Fig.
9A) and CK2-CAT (Fig. 9B), which
had been directly conjugated to HRP. This avoided the need for
secondary HRP-coupled anti-rabbit IgG antibodies in the detection
protocol that would have also detected the IgG subunits in the
immunoprecipitates and obscured signals in the 50-kDa range. CK2-V3
immunoprecipitates from all three primary tissues contained a primary
62-kDa band, which was immunoblotted with the same antibody and
co-migrated with the
E-subunit immunoprecipitated from
overexpressing COS cells (Fig. 9A). The addition of a
180-fold molar excess of the immunizing peptide (relative to estimated
antibody binding sites), either free or coupled to the carrier protein,
prevented immunoprecipitation of
E- (Fig. 9A)
and co-immunoprecipitating subunits (Fig. 9B). An unrelated
peptide had no effect, establishing the specificity of the
immunoprecipitations. Co-immunoprecipitation of other CaM kinase II
subunits from all three primary tissues, as detected with CK2-CAT (Fig.
9B), indicated the formation of CaM kinase II
heteromultimers in vivo. Compared with carotid or heart
extracts, brain extracts contained higher concentrations of
-subunits immunoprecipitable with CK2-V3, consistent with the
relatively high level of mRNA expression for these subunits
(Fig. 7B).
Compared with CaM kinase II isozymes from brain, which are
composed of - and/or
-subunits, relatively little work has been carried out on the properties and function of CaM kinase II isozymes expressed in peripheral tissues, which are composed primarily of
-
and/or
-subunits. Porcine models have been widely used in both
in vivo cardiovascular studies (30) and in vitro,
where biophysical studies on isolated arterial tissues and biochemical studies of purified proteins have contributed significantly to our
current knowledge concerning the regulation of smooth muscle contractility (31). Our preliminary biochemical characterization of
porcine carotid artery CaM kinase II (8) provided a rationale for the
present studies, which were initiated to gain a better understanding of
smooth muscle CaM kinase II structure.
Attention was focused on CaM kinase II -subunits, which appear to be
the principal isoform expressed in this differentiated tissue
(32).4 Analysis of
-subunits cloned from
the cultured porcine aortic vascular smooth muscle library resulted in
the identification of two novel variants designated
G
and
E, bringing the total number of reported
-subunits to seven (18, 28). The 21-amino acid V1
sequence in the
G-subunit is common only to the original
-subunit (
A) cloned from brain and is homologous to a
similar insert in the CaM kinase II
-subunit (33). The
G-subunit was shown in the present study to be expressed
in carotid and heart, but it appears to be a relatively small fraction
of the total
-subunit pool. The 114-base
-subunit V3
insert, which we identified in clone 6, was also identified by RT-PCR
in human biliary tumor cells and reported while the current study was
in progress (28). Although that analysis did not include the
V1 region of the
-subunit shown in Fig. 2, a partial
sequence containing both the 69-base V2 insert and 114-base
V3 insert was designated as a
E-subunit. To
avoid confusion, we chose to retain this nomenclature and designated clone 6 as a partial
E-subunit. As shown here, this
114-base V3 insert is well conserved across mammalian
species with only a few differences in nucleotide and primary sequence
between the rat and porcine or human homologues.
This study provides the first direct evidence that
E-subunit and related subunit mRNAs that contain the
38-amino acid V3 insert are expressed and translated
in vivo in primary tissues. CaM kinase II subunits
containing this sequence were found to be variably expressed and were
most abundant in brain, where they were the principal
-subunit
variant. While clearly detectable as protein in partially purified
fractions of CaM kinase II from carotid artery smooth muscle, based on
the RT-PCR analysis of
-subunit RNA and immunoprecipitation
experiments with the V3 insert specific antibody,
E-like subunits appear to represent a relatively small
fraction of the total
-subunit pool in this tissue and also in
heart. It should be noted that the
E-subunit was cloned
from a cDNA library from cultured aortic smooth muscle cells, which
are known to revert to a dedifferentiated phenotype similar to smooth
muscle cells found in the developing animal (34). In the present study,
neonatal animals were the source of the porcine brain and hearts, while
carotid arteries were from mature swine. It is possible that
E- and related subunits may be developmentally regulated
and expressed at higher levels in tissues from immature animals.
It is likely that other -subunit variants containing both the
23-amino acid V1 and 38-amino acid V3 inserts
exist. Indirect evidence for this included the hybridization of both
V1- and V3-specific probes to
-subunit
RT-PCR products amplified from brain RNA (Fig. 7) and multiple bands in
the Western blots using the V3 sequence-specific antibody
(Fig. 8). A novel CaM kinase II
-subunit cDNA, designated
3, cloned from neonatal rat islet cells (35) contains a
longer nonhomologous sequence (258 bases encoding 86 amino acids)
inserted at the same position in the association domain as the
-subunit V3 insert. In the
-subunit gene, this is at
the boundary between exons IV and V (33). The Drosophila CaM
kinase II gene is also alternatively spliced at this site, producing
four CaM kinase II subunit variants (36) indicating evolutionary
conservation of variants with modifications in this region. However,
this class of splice variants may be subunit-specific, since similar
-subunit variants were not identified in the cDNA library or by
RT-PCR using brain, smooth muscle, or heart mRNA- and
-subunit-specific primers.
BLAST searches of the peptide sequence data bases failed to detect
peptide sequences strongly homologous to the 38-amino acid -subunit
that might provide clues as to the possible function of the insert.
Expression of this sequence in the
-subunits does not grossly alter
holoenzyme structure or autoregulatory properties, as evidenced by the
close similarity between
B- and
E-subunits expressed in COS cells with
respect to holoenzyme size and kinetics of
autophosphorylation-dependent generation of autonomous
kinase activity (Fig. 4). At this point we can only speculate that the V3 sequence may be important in targeting CaM kinase II
isozymes to specific subcellular compartments as has been shown for a
specific V2 sequence found in other CaM kinase II subunits,
which targets isozymes to the nucleus (21, 22).
In general, not much is known specifically about the subunit
composition of CaM kinase II holoenzymes. Therefore, it is significant that multiple CaM kinase II subunits were found to co-immunoprecipitate from tissue homogenates with subunits containing the 38-amino acid
V3 sequence using the V3 sequence-specific
antibody. While the co-immunoprecipitating subunits have not been
identified, their sizes in brain are consistent with - (50 kDa) and
-subunits (58-60 kDa). In the case of smooth muscle, which contains
primarily 54- and 58-kDa CaM kinase II subunits, the 62-kDa
E-subunits appear to differentially co-precipitate
(co-assemble) with the 58-kDa subunits. In heart, the primary
co-precipitating subunit(s) has an apparent size of 50-52 kDa and may
represent a
2- and/or
3-subunit that has
been described in this tissue (5, 20). This experiment indicates that
in vivo
E-subunits form heteromultimeric holoenzymes with other more abundant CaM kinase II subunits. Similar evidence of heteromultimeric CaM kinase II holoenzymes in brain and
carotid smooth muscle was obtained by immunoprecipitation with an
antibody specific for the unique C terminus of the
2-subunit and related subunits (8).
A smooth muscle CaM kinase II activity has been purified from chicken
gizzard and reported to have a tetrameric structure, slow
autophosphorylation and autoactivation responses, and unique autophosphorylation sites (32). These properties were suggested to be
due to a smooth muscle B-subunit. However, in the
present and previous studies (18), CaM kinase II holoenzymes composed of
B-subunits expressed in COS cells were found to have
properties typical of most other reported CaM kinase II holoenzymes,
including a multimeric structure of 6-9 subunits and rapid kinetics of
autophosphorylation under optimal conditions (Fig. 4). It is possible
that the specific V2 sequence in the avian
B-subunit (32) accounts for the unusual properties of
the kinase. With respect to our original goal of defining CaM kinase II
structure in mammalian vascular smooth muscle, we conclude that at
least four
-subunit variants (
B,
C,
E, and
G) are expressed in carotid
arterial smooth muscle. Of these, the
B- and
C-subunit mRNAs, which are translated into proteins
of 58 and 56 kDa, respectively, are most abundant. Recombinant
B- and
C-subunits comigrate with, and
could account for, the 58- and 54-kDa CaM kinase II subunits purified
from carotid artery (Fig. 8). Additional studies aimed at defining the
expression and properties of CaM kinase II holoenzymes containing
-subunit variants are required to complete the characterization of
the arterial smooth muscle CaM kinase II.
We gratefully acknowledge the technical assistance of Charla Sweeley and David Cooney.