Novel Ca2+/Calmodulin-dependent Protein Kinase II gamma -Subunit Variants Expressed in Vascular Smooth Muscle, Brain, and Cardiomyocytes*

(Received for publication, October 25, 1996, and in revised form, January 28, 1997)

Harold A. Singer Dagger , Holly A. Benscoter and Charles M. Schworer

From the Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2612

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) gamma -subunits were cloned from a porcine aortic smooth muscle cDNA library resulting in identification of alternatively spliced CaM kinase II gamma B- and gamma C-subunits and a novel gamma -subunit variant predicted to encode a 60.2-kDa polypeptide, which was designated the gamma G-subunit. A clone predicted to encode a 62.2-kDa gamma -subunit, designated as gamma E, was isolated with a variable domain structure similar to a gamma B-subunit but with a 114-nucleotide insertion in the conserved "association" domain of CaM kinase II subunits. A full-length gamma 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 gamma B-, gamma C-, or gamma G-subunits. Expression of gamma E and related gamma -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 gamma -subunit expression: vascular smooth muscle, gamma B > gamma C > gamma E,G; heart, gamma B > gamma E,C > gamma G; brain, gamma E and related subunits >>  gamma A,B,C,G.


INTRODUCTION

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 (alpha  and beta ) appear to be largely restricted to the brain, while the other two isoforms (delta  and gamma ) 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 gamma -subunit isoforms expressed in mammalian vascular smooth muscle. A novel CaM kinase II gamma -subunit variant (gamma G-subunit) was discovered, and a unique class of gamma -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 gamma -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.


EXPERIMENTAL PROCEDURES

Cloning and Sequencing of CaM Kinase II gamma -Subunit cDNA

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 gamma -subunit catalytic domain. The probe was generated by RT-PCR using rat brain RNA and gamma -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 gamma B-, gamma C-, or unknown subunits, and representative clones were chosen for sequencing. pBluescript phagemids containing the putative gamma -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 [alpha -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.


Fig. 1. Sequencing strategy of CaM kinase II gamma -subunit clones. Clones were isolated from a cultured porcine thoracic aortic cell cDNA library. Solid bars indicate the translated region, and the thin lines represent the UTR. The number of bases in the clone before and after the initiation site are indicated. Shaded regions indicate the approximate position and length of variably expressed sequences relative to clone 28. Gaps indicate absence of sequence. Both strands of cDNA were sequenced with a set of overlapping primers (approximate length and position indicated by arrows) as indicated for clone 28. The approximate position of an NsiI restriction site used to generate a fusion product between clone 6 (C-terminal region) and clone 28 (N-terminal region) is indicated.
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Expression Vectors and Construction of Full-length gamma E-Subunit

pBluescript plasmids containing gamma -subunit inserts (clones 28, 29, and 35) were linearized by digestion with XbaI. gamma -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 gamma -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 gamma -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 gamma -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.

RT-PCR and Southern Analysis

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 gamma -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 gamma A-subunit, accession number J04063[GenBank]) to amplify the V3 region in rat gamma -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 delta 2-subunit (accession number U73504[GenBank]); nucleotides 103-120 in the porcine gamma C-subunit (5'-ACCTCCACGCAGGAATAT-3') and the reverse complement of 5'-TCTGCTCCTCAGCCA-3' in the 114-base V3 insert in the gamma 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 gamma 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 gamma C-subunit (Fig. 7).


Fig. 6. Expression of gamma -subunit transcripts containing the 114-base V3 insertion. RNA from porcine brain, carotid artery vascular smooth muscle (VSM), and heart was analyzed by RT-PCR. The approximate position of PCR primers (`A' Primers) and the region amplified in the primary reaction is depicted at the top. The downstream primer was complementary to sequence at the 3' end of the V3 insert. The resulting ethidium bromide-stained products are shown in the bottom left panel. An aliquot from each of the primary RT-PCR reactions was amplified a second time using nested primers (`B' Primers), which spanned the V1 and V2 regions. The products were compared in size with products amplified using the same primers and control plasmid DNA as templates (bottom right panel).
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Fig. 3. CaM kinase II gamma -subunit V3 domain. Nucleotide and deduced amino acid sequences of the V3 domain and some flanking conserved sequence are from porcine gamma G- and gamma E-subunit clones and a rat cardiomyocyte RT-PCR product amplified using rat gamma -subunit specific primers, which flanked this region. The porcine sequence differs from human gamma -subunit sequences (18) by a single nucleotide (t in porcine, c in human) shown in boldface type and marked with an asterisk. Differences in nucleotide and deduced amino acid sequence between porcine and rat sequence in this region are in boldface type.
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Fig. 7. Analysis of gamma -subunits expressed in brain, carotid artery vascular smooth muscle, and heart. Total RNA from the tissues was analyzed by RT-PCR and Southern blotting. The PCR primers spanned all three variable regions, providing an analysis of the entire pool of gamma -subunit variant mRNAs. A, ethidium bromide-stained agarose gel of size-fractionated RT-PCR products compared with controls amplified from a mixture of plasmid DNAs containing gamma -subunit sequences. B, Southern analysis of the same gel using end-labeled 32P-oligonucleotides (Var1, Var2, and Var3) complementary to sequences specific for each of the variable domain sequences (V1, V2, and V3, respectively). The same blot was sequentially hybridized and stripped of each probe.
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For Southern blots (5), oligonucleotide probes were end-labeled using [gamma -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 gamma C common to all gamma -subunits); 5'-TTGGCAGGCAGAGCTCC-3' (947-963 in gamma G recognizing a V1 sequence common in gamma A- and gamma G-subunits); 5'-CAGTCTCGTAAGCCCAG-3' (1011-1027 in gamma B recognizing a V2 sequence common to gamma B-, gamma G-, and gamma E-subunits); and 5'-GAATGGCAGCTCGGT-3' (recognizing the gamma E-subunit V3 sequence).

Expression of gamma -Subunits in COS-7 Cells

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 gamma -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 Assay and Autophosphorylation

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 Holoenzymes

Gel 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 gamma -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.

Immunoprecipitation and Western Blotting

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 alpha -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 gamma 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 delta -subunit) coupled to carrier protein.

Miscellaneous Methods

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.


RESULTS

Cloning and Identification of CaM Kinase II gamma -Subunit Variants

Four candidate CaM kinase II gamma -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 gamma -subunit sequences indicated that it was a novel CaM kinase II gamma -subunit variant. It was designated the gamma G-subunit, consistent with published nomenclature for alternatively spliced gamma -subunit variants (18, 27, 28). This gamma -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 gamma -subunit isoforms (18, 27) (Fig. 2). Clone 35 was found to lack the V1 insertion identifying it as a gamma B-subunit cDNA, while clone 29 lacked inserts in V1 and V2, identifying it as a gamma 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 gamma B and gamma C clones were 100% identical to published human gamma B- and gamma C-subunit sequences.


Fig. 2. Structure of cloned CaM kinase II gamma -subunits. Conserved regions (top) are indicated by open rectangles, and variably expressed sequences are shown by bars. Conserved regions of amino acid sequence (bottom) are shaded. The variable regions are labeled as V1-3. Clone 28 represents a novel combination of variable sequences and is designated as a gamma G-subunit. Clone 6 encodes a variable domain sequence that includes a 38-amino acid V3 insert, consistent with the predicted sequence of a human gamma -subunit previously designated as gamma E (28). The conserved domain separating the V2 and V3 regions is divided by a dotted line, because the sequence in the first half of this domain (EPQ ... GIK) is variably expressed in other CaM kinase II subunits.
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Clone 6 was incomplete, lacking the 5'-UTR and first 665 bases of the gamma -subunit coding sequence. Its sequence was otherwise identical to a gamma B-subunit but with an additional 114-base insert at a position corresponding to bases 1216 and 1217 of the gamma 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 gamma -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 gamma F-, gamma E-, and gamma D-subunits (28). Based on this, we designated clone 6 as a partial gamma 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 gamma -subunit sequence. Two major products were amplified consistent in size with predicted gamma -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 delta -subunit-specific PCR primers and swine brain, carotid artery, and cardiomyocyte mRNA amplified only a single product of the size predicted for a delta -subunit lacking a V3 insertion (not shown).

Properties of gamma -Subunit Variants Expressed in COS-7 Cells

To assess the biochemical properties of the novel CaM kinase II gamma G- and gamma E-subunits, the cDNAs were subcloned into a mammalian expression vector (pRc/CMV) and expressed in transiently transfected COS-7 cells. Because the putative gamma 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 gamma -subunit CaM kinase II isozyme to its autonomous form (Fig. 4C).


Fig. 4. Properties of CaM kinase II gamma -subunits expressed in COS cells. A, CaM kinase II specific activities in COS cell lysates from cells transiently transfected with CMV vector (CMV) or CMV vector containing gamma -subunit DNAs. Activities were measured using autocamtide-2 as a peptide substrate. Total reflects activities measured with saturating concentrations of Ca2+ and calmodulin activators, and Independent reflects activities from lysates measured in the absence of added activators (n = 4-6). B, immunoblots of lysates from control and gamma -subunit-overexpressing COS cells using an anti-peptide antibody (CK2-CAT), which recognizes a sequence in the N-terminal region common in all known CaM kinase II subunits. The arrows indicated the mobility and size of molecular mass standards. The predicted sizes of gamma -subunits based on amino acid composition are as follows: gamma B, 58,365; gamma C, 55,960; gamma E, 62,164; and gamma G, 60,201 Da. C, generation of autonomous activity in COS cell lysates from cells overexpressing gamma -subunit DNA after a 30-s or 5-min preincubation in the absence of exogenous substrate and either in the absence (solid symbols) or presence (open symbols) of activators to regulate autophosphorylation and conversion to an activator-independent or "autonomous" form. The autonomous activities in these samples were subsequently assayed using exogenous substrate in the absence of free Ca2+. Total activities in the autophosphorylated samples were not different from their respective nonautophosphoryated controls (n = 2-3).
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The position of the V3 insertion within the conserved association domain raised the possibility that multimeric complexes composed of gamma E-subunits might be disrupted or modified in size. However, gel filtration analysis of both CaM kinase II gamma E- and gamma 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 gamma B,E,G-subunits as multimers in COS cells with estimated sizes as follows: gamma B, 448 kDa; gamma E, 471 kDa; gamma G, 425 kDa. These data suggest that the gamma -subunits form multimers of 7 or 8 subunits, a finding consistent with previous results describing the human gamma 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.


Fig. 5. Size fractionation of recombinant CaM kinase II gamma -subunit holoenzymes. A, gel filtration analysis of autocamtide-2 kinase activities in COS cell lysates from cells overexpressing gamma B- and gamma E-subunit cDNA using a Superose 12 FPLC column. Shown are Ca2+/CaM-dependent activities. Activator-independent activities and autocamtide-2 kinase activities in control "empty" CMV vector-transfected cells were negligible in these experiments. Fractions containing eluted peaks of thyroglobulin (Thy.; 669 kDa), catalase (Cat.; 250 kDa), and ovalbumin (Ova.; 66 kDa) standards are indicated. B, immunoblots of Superose 12 column fractions using CK2-CAT, an antibody that recognizes a sequence in the N-terminal region common in all known CaM kinase II subunits.
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Structural Characterization of Expressed CaM Kinase II gamma -Subunits Containing the V3 Insert

To better define the gamma -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 gamma -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 gamma B-subunit clone. This experiment confirmed that gamma -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 gamma E-subunit) or possibly a 63-base V1 insert, which would represent yet another gamma -subunit variant. Other minor products were amplified with the nested primers, some of which were similar in size to the products amplified from control gamma C and gamma G subunit clones, suggesting low level in vivo expression of alternative gamma -subunit isoforms containing the V3 insert.

Relative Expression of gamma -Subunit Variants in Vivo

To provide an estimate of the relative expression of gamma -subunits containing the V3 insert compared with other gamma -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 gamma -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 gamma B-specific V2 probe and was consistent in size with that amplified from the control gamma B-subunit cDNA. Less abundant were products that were consistent in size with that expected from the gamma 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 gamma G-subunit. Hybridization of the arterial smooth muscle PCR products with the V3 probe was weak, suggesting that gamma -subunit transcripts containing the 114-base V3 insert must be a minor fraction of total gamma -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 gamma E-subunits and hybridizing with the V3-specific probe were more abundant. In contrast, the largest and most abundant gamma -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 gamma 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 gamma B-subunit, or a 649-base product, consistent with the gamma C-subunit, were amplified from brain RNA. A minor product slightly smaller than the gamma G-subunit and hybridizing with the V1-specific primer was amplified from brain RNA, probably reflecting expression of the gamma A-subunit, which was the original gamma -subunit isoform cloned from brain (27).

Identification of gamma -Subunits with the V3 Insert in Brain and Arterial Smooth Muscle

To confirm expression of CaM kinase II gamma -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 gamma -subunits. In a mixture of such lysates a 62-kDa band was identified, consistent with the predicted size of the gamma 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 gamma C- and gamma E-subunits with CK2-CAT compared with gamma B- and gamma 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).


Fig. 8. Expression of gamma E-subunit protein in brain and carotid artery. Partially purified fractions of CaM kinase II from brain and carotid artery were probed with a primary antibody raised against the unique 38-amino acid V3 sequence found in the gamma E-subunit (panel A, CK2-V3) or with an antibody common to all CaM kinase II subunits (panel B, CK2-CAT). A mixture of recombinant standards (gamma -subunit overexpressing COS cell lysates) is included as a control in the first lane. The mobility of size standards is indicated at the right of each panel.
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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 alpha - or beta -subunit-specific antibodies (not shown) and therefore probably represent mixtures of gamma - and delta -subunits. Similar fractions from porcine brain contained primarily 50- and 58-60-kDa subunits, which can be attributed to expression of CaM kinase II alpha - and beta -subunits as previously established for this tissue (7). Bands consistent in size with the gamma 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 gamma E-subunit standards was detected in the partially purified CaM kinase II fractions from both tissues, indicating expression of the gamma 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 gamma C- and gamma 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 gamma 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 gamma 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 gamma 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 gamma -subunits immunoprecipitable with CK2-V3, consistent with the relatively high level of mRNA expression for these subunits (Fig. 7B).


Fig. 9. Immunoprecipitation of CaM kinase II holoenzymes with CK2-V3. Brain, carotid artery, heart extracts, and control COS cell lysates from cells expressing the gamma B- and gamma E-subunits were immunoprecipitated using the antibody that specifically recognizes CaM kinase II subunits containing the unique 38-amino acid V3 insert (IP: CK2-V3). The precipitated proteins were then resolved by SDS-PAGE and immunoblotted using the same antibody (CK2-V3*) that had been directly conjugated to HRP to provide a catalyst for chemiluminescent detection (panel A). The blots were then stripped and reprobed with HRP-conjugated CK2-CAT (CK2-CAT*) (panel B), a subunit-nonselective antibody, to detect other CaM kinase II subunits that co-precipitated in multimeric complexes with gamma E-like subunits. The lanes containing carotid and heart samples are shown at an exposure 3 times longer than the control and brain samples to partially compensate for differences in the relative signal strengths. Brain immunoprecipitation (IP) and control lanes are as follows. Lane 1, IP; lane 2, IP and 25 µM V3 immunizing peptide; lane 3, IP and 25 µM V3 peptide coupled to carrier; lane 4, IP and 25 µM unrelated peptide coupled to carrier.
[View Larger Version of this Image (54K GIF file)]



DISCUSSION

Compared with CaM kinase II isozymes from brain, which are composed of alpha - and/or beta -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 delta - and/or gamma -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 gamma -subunits, which appear to be the principal isoform expressed in this differentiated tissue (32).4 Analysis of gamma -subunits cloned from the cultured porcine aortic vascular smooth muscle library resulted in the identification of two novel variants designated gamma G and gamma E, bringing the total number of reported gamma -subunits to seven (18, 28). The 21-amino acid V1 sequence in the gamma G-subunit is common only to the original gamma -subunit (gamma A) cloned from brain and is homologous to a similar insert in the CaM kinase II beta -subunit (33). The gamma 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 gamma -subunit pool. The 114-base gamma -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 gamma -subunit shown in Fig. 2, a partial sequence containing both the 69-base V2 insert and 114-base V3 insert was designated as a gamma E-subunit. To avoid confusion, we chose to retain this nomenclature and designated clone 6 as a partial gamma 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 gamma 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 gamma -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 gamma -subunit RNA and immunoprecipitation experiments with the V3 insert specific antibody, gamma E-like subunits appear to represent a relatively small fraction of the total gamma -subunit pool in this tissue and also in heart. It should be noted that the gamma 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 gamma E- and related subunits may be developmentally regulated and expressed at higher levels in tissues from immature animals.

It is likely that other gamma -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 gamma -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 beta -subunit cDNA, designated beta 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 gamma -subunit V3 insert. In the beta -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 delta -subunit variants were not identified in the cDNA library or by RT-PCR using brain, smooth muscle, or heart mRNA- and delta -subunit-specific primers.

BLAST searches of the peptide sequence data bases failed to detect peptide sequences strongly homologous to the 38-amino acid gamma -subunit that might provide clues as to the possible function of the insert. Expression of this sequence in the gamma -subunits does not grossly alter holoenzyme structure or autoregulatory properties, as evidenced by the close similarity between gamma B- and gamma 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 alpha - (50 kDa) and beta -subunits (58-60 kDa). In the case of smooth muscle, which contains primarily 54- and 58-kDa CaM kinase II subunits, the 62-kDa gamma 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 delta 2- and/or delta 3-subunit that has been described in this tissue (5, 20). This experiment indicates that in vivo gamma 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 delta 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 gamma B-subunit. However, in the present and previous studies (18), CaM kinase II holoenzymes composed of gamma 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 gamma 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 gamma -subunit variants (gamma B, gamma C, gamma E, and gamma G) are expressed in carotid arterial smooth muscle. Of these, the gamma B- and gamma C-subunit mRNAs, which are translated into proteins of 58 and 56 kDa, respectively, are most abundant. Recombinant gamma B- and gamma 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 delta -subunit variants are required to complete the characterization of the arterial smooth muscle CaM kinase II.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL49426 (to H. A. S.) and a fellowship from the American Heart Association, Pennsylvania Affiliate (to S. T. A.).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.
Dagger    To whom correspondence should be addressed: Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave., Danville, PA 17822-2612. Tel.: 717-271-8669; Fax: 717-271-6701; E-mail: has @smtp.geisinger.edu.
1   The abbreviations used are: CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; PCR, polymerase chain reaction; RT, reverse transcriptase; PAGE, polyacrylamide gel electrophoresis; UTR, untranslated region; MOPS, 4-morpholinepropanesulfonic acid; FPLC, fast protein liquid chromatography; CMV, cytomegalovirus.
2   S. T. Abraham, H. A. Benscoter, and H. A. Singer, submitted for publication.
3   Complete cDNA sequences and deduced amino acid sequences for the four porcine clones shown in Fig. 1 have been entered in GenBankTM and assigned the following accession numbers: gamma B (clone 35), U72970[GenBank]; gamma C (clone 28), U72071[GenBank]; gamma E (clone 6), U72972[GenBank]; gamma G (clone 29), U72973[GenBank]. The sequence of the rat gamma -subunit RT-PCR product spanning the V3 domain shown in Fig. 3 has been assigned accession number U73503[GenBank].
4   H. A. Singer, unpublished observations.

ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Charla Sweeley and David Cooney.


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