To investigate the regulation of calpastatin gene
expression, we isolated bovine heart calpastatin cDNAs and
5
-regions of the calpastatin gene. Analysis of 5
-cDNA sequence
identified a new translation initiation site that is in frame and 204 nucleotides upstream of the previously designated start site.
Conceptual translation from this upstream AUG produces a protein
containing 68 additional N-terminal amino acids. This "XL" region
contains three potential PKA phosphorylation sites but shares no
homology with other regions of calpastatin or with any known protein.
Immunoblot studies demonstrated that heart and liver contain a
calpastatin protein of 145 kDa on SDS-polyacrylamide gel
electrophoresis that comigrates with full-length bacterially
expressed calpastatin and calpastatin produced by coupled in
vitro transcription-translation from the upstream AUG. An
antibody raised against the XL region recognized the 145-kDa band,
demonstrating that the upstream AUG is utilized and that the 145-kDa
band represents full-length calpastatin in vivo. Transient
transfection assays demonstrated that sequence within 272 nucleotides
upstream of transcription initiation of the calpastatin gene is
sufficient to direct moderate level transcription. Promoter sequences
further upstream act to inhibit or stimulate transcriptional activity.
Exposure of transfected cells to dibutyryl cAMP resulted in a
7-20-fold increase in promoter activity for constructs containing at
least 272 nucleotides of upstream promoter sequence. Deletion analysis
indicates that at least one cAMP-responsive element resides within 102 nucleotides of transcription initiation.
 |
INTRODUCTION |
Calpastatin is a proteinase inhibitor that is specific for
calpains, a family of calcium-activated neutral proteases that regulate
a wide range of Ca2+-dependent cellular
processes (1-6). Calpastatin binds to and inhibits the calpains in the
presence of Ca2+, although how this interaction is
regulated remains largely unknown.
Calpastatin is encoded by a single gene in birds and mammals that
produce a number of closely related protein isoforms via alternative
splicing (7, 8). Partial calpastatin cDNAs have been cloned from
several species, including cattle, pig, rat, rabbit, and human (9-12).
Conceptual translation using an initiation codon first identified in
the rabbit cDNA yields a protein of 718 amino acids with a
predicted molecular mass of 76 kDa (9). The protein contains four
repeating domains, each of which is capable of binding to calpain and
inhibiting proteolytic activity such that a single calpastastin
molecule can inhibit several calpain molecules in vitro
(13-15). An additional N-terminal L domain shares no homology with the
inhibitory domains and is of unknown function.
Although mechanisms controlling calpastatin inhibitory activity are not
yet understood, studies in vitro and in vivo
suggest that calpastatin is regulated both at the level of protein
abundance and via postranslational modification. Following
administration of
-agonists to steers, calpastatin mRNA and
protein levels and calpain inhibitory activity increase 2-fold, and
this increased calpastatin activity has been linked to decreased
protein degradation during muscle hypertrophy (16-18).
-Agonists
bind to the
2-adrenergic receptor and activate signaling
cascades that involve cAMP-dependent protein kinase
(PKA),1 raising the
possibility that calpastatin gene transcription is responsive to a
PKA-dependent signaling pathway. Calpastastin is also
phosphorylated by PKA (3, 19), and some evidence suggests that
phosphorylation can alter calpastatin function. A phosphorylated form
of calpastatin isolated from rat skeletal muscle has been reported to
have a lower Ki for inhibition of m-calpain than for
inhibition of µ-calpain, whereas an unphosphorylated form
preferentially inhibited µ-calpain (3, 19). This relative specificity
for µ- versus m-calpain can be shifted in vitro
by PKA-dependent phosphorylation or alkaline phosphatase
treatment.
These findings indicate that PKA may be involved in regulating both
calpastatin gene transcription and the extent of calpastatin protein
phosphorylation. Relatively little is known about the regulation of
calpastatin gene expression, however, since no calpastatin gene
promoter has been characterized in any species. Furthermore, analysis
of the known calpastatin amino acid sequence has identified only a
single potential PKA phosphorylation site within domain 1. As each of
four domains within calpastatin shows potent calpain inhibitory
activity, it has not been understood how phosphorylation at a single
site might modulate calpastatin activity.
Here we report the cloning of 5
-regions of calpastatin cDNAs and
transcriptional regulatory regions of the bovine calpastatin gene.
Analysis of the cDNA sequence identified a previously unreported upstream translation initiation site that yields a calpastatin protein
with 68 additional N-terminal amino acids. This "XL" region is
present on calpastastin protein in heart and liver, and is phosphorylated by PKA. Transfection experiments show that the calpastatin gene promoter is up-regulated by dibutyryl cAMP, indicating that both the calpastatin promoter and protein are targets for PKA
activity.
 |
EXPERIMENTAL PROCEDURES |
5
-Rapid Amplification of Complementary DNA 5
-Ends
(RACE)--
5
-RACE was performed as described (20) using total RNA
from adult bovine heart and primers derived from a published partial calpastatin cDNA sequence (12). Three nested calpastatin-specific primers, CPB8 (5
-CCTGTATCTGAUGAGTGCTTGGG-3
), CPB3
(5
-GGTAGGCTTTTTGGCTCTGTGTG-3
), and CPB1
(5
-GTTCCTTTGGCTTTACTTCTTGGG-3
) were used. Single-stranded cDNA
template was generated using 5 µg of bovine heart total RNA and the
CPB8 primer according to standard protocols. Following removal of
excess primers using Centricon-100 spin filters (Amicon Corp.), poly(A)
was added to 5
-cDNA ends by incubation in a solution containing 10 units of terminal deoxytransferase (Promega), 0.2 mM dATP, 100 mM cacodylate buffer (pH 6.8), 1 mM CoCl2, and 0.1 mM dithiothreitol
at 37 °C for 5 min, followed by heating to 65 °C for 5 min to
stop the reaction. Second strand cDNA was synthesized using the
52-nt hybrid primer QT
(5
-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT-3
), and two
rounds of PCR were then performed using the primer QO (5
-CCAGTGAGCAGAGTGACG-3
) and the calpastatin-specific primer CPB3,
followed by the primer QI (5
-GAGGACTCGAGCTCAAGC-3
) and CPB1. PCR reaction products were separated on a 1% agarose gel, excised, and sequenced.
Isolation of Bovine Calpastatin cDNA and Genomic
Sequences--
Approximately 1.7 × 105 recombinant
phage from a bovine heart Uni-ZAP XR cDNA library (Stratagene) were
plated and screened at moderate stringency (final wash: 0.1% SDS,
0.1 × SSC at room temperature for 30 min) using a 152-bp 5
-RACE
PCR product. Clones giving positive signals were plaque-purified and
rescreened two additional times to eliminate false positives. The
plaque-purified cDNA phage clones were converted into respective
pBluescript plasmid counterparts by in vivo excision
according to the manufacturer's protocols to produce double-stranded
DNA pBluescript plasmids containing cDNA inserts.
To isolate bovine genomic sequence in the 5
-region of the calpastatin
gene, 1.1 × 106 recombinant phage from a bovine heart
genomic library (Stratagene) were screened using a 139-bp PCR product
from the 5
-end of the calpastatin cDNA as probe. Clones giving
positive signals were plaque-purified, and genomic inserts were
subjected to restriction enzyme analysis. Fragments hybridizing to an
oligonucleotide from the extreme 5
-end of the calpastatin cDNA
were subcloned into pBluescript for additional characterization.
Northern Blot and Primer Extension--
Total RNA was isolated
from adult bovine heart according to the method of Chomczynski and
Sacchi (21). Northern analysis was performed essentially as described
(22) using a 413-nt PCR product from the middle of the cDNA. For
primer extension, an oligonucleotide (CPB522;
5
-AUGGCGACGAUGGAUGTGTTCC-3
) near the 5
-end of the calpastatin
cDNA was labeled with [
-32P]dATP (7000 mCi/mmol,
ICN). The annealing reaction was performed at 85 °C for 5 min
followed by incubation at 55 °C for 90 min in a solution containing
10 µg of total RNA from bovine heart tissue, 10 pmol of
[
-32P]dATP-labeled CPB522, 250 mM KCl, 250 mM Tris-HCl (pH 8.3 at 45 °C). Reverse transcription was
carried out at 45 °C for 45 min in a reaction containing 1 mM dNTP, 12.5 units of avian myeloblastosis virus reverse
transcriptase, 50 mM Tris-HCl (pH 8.3), 50 mM
KCl, 10 mM MgCl2, 0.5 mM
spermidine, and 10 mM dithiothreitol. Primer extension
products were separated on a 6% polyacrylamide-urea gel and visualized
by autoradiography.
Western Blot Analysis--
Western blots were performed as
described by Towbin et al. (23). Whole tissue extracts from
bovine heart and liver, a crude ammonium sulfate fraction of whole
heart homogenates, or recombinant proteins representing full-length
calpastatin or domains 1-4, were electrophoresed on 10% SDS-PAGE and
transferred to nitrocellulose. Membranes were incubated in 5% nonfat
dry milk in 1 × TTBS (0.1% Tween 20, 100 mM
Tris-HCl, pH 7.5, and 0.9% NaCl) buffer at room temperature for 1 h, washed in 1 × TTBS and then incubated in 1 × TTBS, 1%
BSA plus 1F7, a monoclonal antibody that recognizes an epitope in the
C-terminal portion of bovine calpastatin, or a chicken antiserum
prepared against amino acids 17-33 of the XL region. Following washes
and incubation with horseradish peroxidase-conjugated goat anti-mouse
or anti-chicken IgG, peroxidase was visualized using SuperSignal
substrate (Pierce).
Construction of Recombinant Plasmids for Promoter Analysis
and Expression of Calpastatin cDNA--
A genomic
HindIII fragment containing 1667 bp of 5
-flanking region,
158 bp of exon 1, and approximately 2400 bp of intron 1 cloned into
pBluescript KS served as template for constructing pCS-1667. PCR
amplification was performed using the forward primer KS located within
pBluescript vector and a reverse primer CPB36, which is located in exon
1 and contains an additional XbaI site at its 5
-end. The
PCR product was restricted with HindIII plus XbaI
and cloned into the pCAT-BASIC reporter vector (Promega). The resulting
plasmid clone, pCS-1667, contains 1667 nt upstream of transcription
initiation linked to the CAT reporter gene. pCS-1667 was used as a
template to generate pCS-1242, pCS-944, pCS-671, pCS-272, pCS-102, and
pCS-31.
Production of Recombinant Calpastatin Proteins and PKA
Phosphorylation Assay--
Calpastatin cDNAs representing
full-length calpastatin, domain L plus region XL, or domains 1-4 were
cloned into the pCAL-n vector (Stratagene) to generate fusion proteins
containing an N-terminal 26-amino acid calmodulin binding unit.
Recombinant plasmids expressing full-length, domains 1-4, or domain L
and region XL were designated pCalp786, pCalp569, and pCalp217,
respectively. Recombinant proteins were isolated using a calmodulin
affinity column according to the manufacturer's protocols
(Stratagene).
PKA was a generous gift from Dr. Mike Walsh (University of Calgary).
0.1 mg/ml of the partially purified recombinant protein pCalp786,
pCalp569, or pCalp217 was added to a reaction solution containing 20 mM NaHEPES (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 1 µg/ml PKA. The phosphorylation
reaction was initiated by adding 26.25 µCi of
[
-32P]ATP and 0.25 mM ATP. Following
incubation at 30 °C for 30 min, reactions were boiled for 5 min at
95 °C and loaded onto a 7.5-20% gradient SDS-PAGE gel.
Transient Transfection and CAT Assay--
One day prior to
transfection, 5 × 105 NIH3T3 cells were plated onto a
35-mm tissue culture plate in medium consisting of Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum, 50 µg/ml Gentamicin, 1:100 antibiotic-antimycotic. The
following day, cells were transfected with 2 µg of plasmid DNA using
LipofectAMINE (Life Technologies). For some experiments, dibutyryl cAMP
(Sigma) was added to culture medium at a final concentration of 1 mM 24 h after transfection. 48 h following transfection, cultures were rinsed with 2 ml of chilled TBS (50 mM Tris-HCl, 90 mM NaCl, pH 7.5). 750 µl of
chilled STE (40 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.4) was then added to detach cells from the
plate. Cells were pelleted, brought up in 200 µl of lysis buffer
(0.25 M Tris-HCl, pH 7.8, 0.5% Triton X-100), placed on
ice for 10 min, and sonicated. Protein concentrations of extracts were
determined using the BCA protein assay kit (Pierce).
CAT activities were measured essentially by the method previously
described (24). Briefly, 70 µl of cell extract per reaction was
incubated at 37 °C for 1 h with 1 µl of
[14C]chloramphenicol (25 µCi/ml, Amersham Corp.), 8 µl of 40 mM acetyl-CoA (Pharmacia Biotech Inc.), and 100 µl of 250 mM Tris-HCl (pH 7.5). Reactions were terminated
by extraction with 1 ml of ethyl acetate. Ethyl acetate was transferred
to a separate tube and dried, and extracted chloramphenicol was
dissolved in 20 µl of ethyl acetate. Unacetylated and acetylated
[14C]chloramphenicol were resolved by thin layer
chromatography (TLC) in 90% chloroform, 10% methanol and quantitated
using an Instant Imager (Packard). CAT activities were normalized to
protein content within extracts, and each construct was assayed in at
least three independent transfection experiments.
 |
RESULTS |
Isolation of Full-length Bovine Calpastatin cDNAs--
To
identify 5
-bovine calpastatin mRNA sequence, 5
-RACE (20) was
performed using mRNA from adult bovine heart and primers derived
from a published partial calpastatin cDNA (12). A 152-bp cDNA
fragment containing additional upstream sequence identified by 5
-RACE
was used to screen a bovine heart cDNA library. Three independent
cDNAs were obtained that differed in the amount of 3
-untranslated
region and in their 5
-termination point (Fig. 1A). Overlapping regions of
all clones were identical except for a 66-nt deletion within cDNA 3 that probably represents an alternatively spliced exon (8). The size of
these cDNAs (4296, 3430, and 2544 nt) corresponded approximately to
the size of three mRNAs identified by Northern blot using heart
muscle RNA (Fig. 1B; see also Ref. 9).

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Fig. 1.
A, structure of three calpastatin
cDNAs obtained from a bovine heart cDNA library.
cDNAs differ in the amount of 3 -untranslated region and in their
5 -termination point. cDNAs 2 and 3 extend 5 beyond an AUG that is
in frame and 204 nucleotides upstream of the previously designated
initiation of translation. B, Northern blot of bovine heart
RNA showing three bands obtained using calpastatin cDNA as probe.
The size of these three bands corresponds to the size of cDNAs
1-3.
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|
Published characterizations of partial calpastatin cDNAs from
human, rabbit, pig, and cattle have proposed a translation initiation site that yields a predicted protein of 639-718 amino acids, depending on species (9-12, 16). Designation of this translation start site
derived from the rabbit cDNA (9), in which a stop codon was
identified 135 nt upstream of the proposed AUG. This upstream stop
codon is not present in any of the bovine cDNAs that we isolated. Additionally, cDNAs 2 and 3 extend 5
to a potential translation initiation site that is in frame and 204 nt upstream of the previously designated start site (Fig. 1A). This upstream AUG is in
excellent "Kozak consensus" context for translation initiation
(GCCAUGG), whereas the previously designated downstream
site (AGUAUGA) is a poor translation initiation site
(37). Conceptual translation from the upstream AUG produces a protein
containing 68 additional N-terminal amino acids (region XL). The XL
region shares no homology with other regions of calpastatin or with any
known protein. Full-length calpastatin cDNA sequence and conceptual
translation are shown in Fig. 2.

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Fig. 2.
cDNA and deduced amino acid sequence of
bovine heart calpastatin. cDNA sequence was derived by
combining cDNA 1, which extends farthest 3 , with the 5 sequence
of cDNA 2. Extreme 5 -mRNA sequence and the transcription
initiation site were determined by primer extension and comparison with
genomic sequence spanning this region. Domain boundaries are indicated;
amino acid sequence of the XL region is shown in boldface
type. The dashed underlined region denotes the sequence
absent from cDNA 3. The single underlined sequence
denotes regions that differ from a previously published bovine skeletal
muscle calpastatin cDNA (12). Asterisks indicate polyadenylation sites.
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|
To verify that the upstream translation initiation site is functional,
coupled in vitro transcription-translation experiments were
performed using calpastatin cDNA 1, which contains only the downstream ATG, or cDNA 2, which contains both the upstream and downstream ATGs (Fig. 3A). The
largest translation product arising from the upstream ATG of cDNA 2 runs on SDS-PAGE at an apparent molecular mass of 145 kDa (Fig.
3B), almost twice the predicted molecular mass of 84 kDa.
cDNA 1 produced a protein of 135 kDa, reflecting initiation at the
downstream AUG. The anomalous migration of calpastatin on
SDS-PAGE has been previously reported (10, 14, 25).

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Fig. 3.
A, cDNA clones used for coupled
in vitro transcription-translation. cDNA 1 contains a
single downstream ATG, while cDNA 2 contains an additional ATG 204 nt further upstream. B, autoradiograph showing SDS-PAGE
analysis of coupled in vitro transcription-translation reactions, containing no DNA (lane 1), cDNA 1 (lane 2), or cDNA 2 (lane 3) as template.
While the largest translation product produced from cDNA 1 migrated
at 135 kDa, cDNA 2 produced a predominant product of 145 kDa.
C, immunoblot analysis of bacterially expressed calpastatin
or whole adult heart or liver lysates. mAb 1F7, produced against a
C-terminal calpastatin epitope, recognized a protein of approximately
150 kDa, representing bacterially expressed calpastatin protein
produced from cDNA clone 2 containing the upstream AUG plus a 4-kDa
calmodulin binding domain (lane 1). mAb 1F7 recognized a
band of 145 kDa in whole heart and liver homogenates (lanes 2 and 3). A smaller band of 135 kDa was also detected
in liver. Three bands were recognized by mAb 1F7 in a crude ammonium
sulfate fraction of whole heart homogenate (lane 4), one
band migrating at 145 kDa and probably representing full-length
calpastatin, plus two smaller bands of approximately 120 and 110 kDa.
In contrast, an antiserum raised against a peptide from the N-terminal
XL region recognized only the 145-kDa band in the ammonium sulfate
fraction (lane 5).
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To determine whether the upstream AUG is utilized in vivo,
Western analyses were performed using whole cell extracts, an ammonium sulfate fraction of crude bovine heart homogenate, or bacterially expressed full-length calpastatin. Monoclonal antibody (mAb) 1F7, which
recognizes an epitope in the C-terminal region of calpastatin, identified a 145-kDa protein in whole heart extracts that runs slightly
faster than bacterially expressed full-length calpastatin protein
(pCalp 786, Fig. 4) containing an
additional 4-kDa calmodulin binding domain, and comigrates with the
largest in vitro translation product (Fig. 3C,
lanes 1 and 2). A 145-kDa band that comigrates with the full-length in vitro translation product plus a
second band of 135 kDa were detected in whole liver extracts (Fig.
3C, lane 3). This lower band could represent
translation initiation at the downstream AUG or a proteolytic cleavage
product of the full-length protein. mAb 1F7 recognized a band of 145 kDa plus two smaller bands in an ammonium sulfate fraction of crude
heart muscle homogenate (Fig. 3C, lane 4). These
lower bands migrate more rapidly than calpastatin protein initiated at
the downstream AUG, and their origin is unclear; they may arise from
proteolysis (26), or they may be products of alternative splicing. The
110- and 120-kDa polypeptides represent the calpastatin protein
isolated biochemically and studied extensively by several laboratories (27-29).

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Fig. 4.
PKA kinase assay of bacterially expressed
calpastastin proteins. A, constructs used to express
full-length or truncated calpastatin proteins in E. coli.
pCalp786 represents full-length calpastatin; pCalp569 is truncated just
N-terminal to domain 1; pCalp217 contains regions XL and L. Asterisks indicate predicted PKA phosphorylation sites.
Gray boxes indicate a 4-kDa calmodulin binding domain
present to facilitate protein purification. B, SDS-PAGE
autoradiograph showing pCalp786 (lane 1), pCalp569
(lane 2), and pCalp217 (lane 3). All three
protein products were phosphorylated by PKA.
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To confirm that calpastatin contains the XL region in vivo,
a polyclonal antisera was prepared against a 17-amino acid peptide from
the XL region (amino acids 17-33). This antisera recognized only the
145-kDa band in the crude ammonium sulfate fraction (Fig. 3C, lane 5). Taken together, these results
demonstrate that the upstream AUG is utilized and that the XL region is
present within calpastatin protein in vivo.
Analysis of the deduced amino acid sequence of the full-length
calpastatin protein identified three potential PKA phosphorylation sites clustered within the N-terminal XL region and a fourth site within domain 1 (Fig. 4). To determine whether the XL domain is a
target of cAMP-dependent phosphorylation, full-length and
truncated calpastatin proteins were expressed in E. coli,
partially purified, and then challenged in a kinase assay using
purified PKA. As shown in Fig. 4, both an N-terminal partial
calpastatin protein containing regions XL and L, and a C-terminal
partial protein consisting of domains 1-4, are phosphorylated by
PKA.
Structure of the 5
-Region of the Bovine Calpastatin
Gene--
Using 5
-sequence from the calpastatin cDNA as probe,
four clones were isolated from a bovine genomic library. Extensive
subcloning and sequencing has defined the structure of the 5
-region of
the calpastatin gene (Fig. 5). Comparison
between the genomic and heart cDNA sequences revealed that the
previously proposed (downstream) translation initiation site is located
in exon 4. mRNA sequence further 5
is encoded by three exons
separated by large introns spanning at least 60 kb.

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Fig. 5.
Genomic organization and phage clones
spanning the 5 -region of the bovine calpastatin gene. Intron and
exon structure is shown along with the location of some restriction
enzyme recognition sites HindIII (H),
XbaI (X), SmaI (S), and
NdeI (N). The upstream and putative downstream
translation sites in exons 1 and 4, respectively, are shown.
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To define the transcription initiation site, primer extension was
performed using heart muscle RNA as template and a 22-nt primer located
just 5
to the upstream AUG (Fig. 6).
Three major extension products were obtained of 118, 124, and 130 nt.
Each band may reflect a distinct transcription initiation site, since multiple start sites are common for genes that lack TATA boxes and that
are GC-rich in their proximal promoter regions (see below). The region
between the upstream AUG and the 5
terminus of the 130-nt primer
extension product is contiguous within the genomic sequence, indicating
that it is encoded by a single exon.

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Fig. 6.
Primer extension analysis of calpastatin
mRNA. Bovine heart poly(A)+ mRNA was annealed
to a primer located just 5 of the upstream AUG. Lane 1 is a
G sequencing ladder used as a size marker. Radiolabeled primer
extension products are shown in lane 2. Three major bands were obtained of 118, 124, and 130 nt.
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PKA Responsiveness of the Calpastatin Gene Promoter--
Sequence
immediately upstream of exon 1 is GC-rich and contains four potential
SP-1 binding sites between nt
162 and
41 (relative to the most 5
initiation of transcription identified by primer extension) and a
putative CAAT box between nt
125 and
122 (Fig.
7). To determine whether this upstream
genomic region possesses promoter activity, DNA fragments extending
from nt
1667,
1242,
944,
671,
272,
102, or
31 to nt +130
were cloned immediately upstream of the CAT reporter gene and
transfected into NIH3T3 cells. Promoter activity was assessed 48 h
later by enzymatic CAT assay.

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Fig. 7.
Sequence of the bovine calpastatin gene
5 -flanking region. The sequence extends 1667 nt upstream of the
initiation of transcription, which is designated as nt +1. Truncation
points for promoter-CAT constructs are indicated. Sp1 and GC boxes are located in two clusters within the proximal promoter region and upstream of nt 750. Double underlines indicate sequences
that are potentially important for cAMP responsiveness. The
single underline denotes a putative CAAT box.
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Transient transfection studies show that the 5
-flanking region of the
calpastatin gene possesses moderate transcriptional activity (Fig.
8, Table
I). CAT expression from pCS-1667CAT was approximately 0.8% of pCATControl, which contains the CAT gene under
control of the strong SV40 viral promoter. Progressive deletion of
promoter sequence identified regions that both stimulate and suppress
transcriptional activity. Deletion of sequence between nt
1667 and
944, for example, increased promoter activity more than 2-fold, while
deletion to nt
671 reduced CAT activity more than 7-fold. Truncation
at nt
102, which deleted a potential CAAT box element at nt
125,
reduced CAT expression to barely detectable levels. Further deletion to
nt
31 abolished promoter activity. These results demonstrate that DNA
elements located within nt
1667 of initiation of transcription of the
calpastatin gene can direct moderate level expression of a heterologous
cDNA and that cis elements within this promoter region can both
stimulate and inhibit transcriptional activity.

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Fig. 8.
cAMP responsiveness of the bovine calpastatin
promoter. Calpastatin-CAT reporter constructs containing varying
amounts of promoter sequence were transfected into NIH3T3 cells.
24 h following transfection, dibutyryl cAMP (1 mM) was
added to some cultures. All cultures were sacrificed 48 h
following transfection. The graph shows CAT activity relative to
pCATControl, which contains the SV40 promoter driving CAT. Data
represent the average of at least three independent experiments.
Representative CAT assays are shown.
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Table I
Effect of dibutryl cAMP on calpastatin promoter activity
CAT activities are relative to pCATControl, which contains the CAT gene
driven by the SV40 viral promoter.
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-Agonist-induced muscle hypertrophy is accompanied by increased
steady-state levels of calpastatin mRNA (18). Since
-agonists act through the cAMP-dependent protein kinase signaling
pathway, we asked whether transcription of the calpastatin promoter is enhanced following administration of dibutyryl cAMP, a constitutive activator of PKA. As shown in Fig. 8 and Table I, the addition of
dibutyryl cAMP to culture medium resulted in a 5-17-fold increase in
calpastatin promoter activity for constructs containing at least 272 nt
of promoter sequence. Transcriptional activity of pCAT-Control was not
increased by dibutyryl cAMP treatment. Interestingly, pCS-102, which
showed extremely low basal promoter activity, was induced to moderate
levels (approximately equivalent to basal levels of pCS-1667) following
administration of dibutyryl cAMP. At least one cAMP-responsive element
is therefore likely to reside within 102 nt of transcription
initiation.
 |
DISCUSSION |
A New N-Terminal Region of Bovine Calpastatin--
We have cloned
calpastatin cDNAs from bovine heart and identified a new upstream
translation initiation site that yields 68 previously unidentified
amino acids on the N terminus of the protein. This XL region is present
in calpastatin in vivo and is a substrate for
phosphorylation by PKA. We have also cloned the 5
-region of the
calpastatin gene and defined an active promoter region, the
transcriptional activity of which is enhanced by dibutyryl cAMP, a
constitutive activator of PKA. Thus, both the calpastastin gene
promoter and the XL region of the protein are targets of PKA
activity.
A wide range of molecular weights have been reported for calpastatin
isolated from cells and tissues, with estimates ranging from 17 to 172 kDa (1). Some of this variability may be due to protein degradation,
while some is likely to result from alternative splicing or different
translation initiation sites (7-9, 11, 30). Autolysis may represent an
additional mechanism for regulating calpain activity (31-33), and
proteolytic processing may also be involved in regulating calpastatin
activity (34). Further confusion has arisen from the widespread
observation that calpastastin runs anomalously on SDS-PAGE (10, 14,
25). The largest reported calpastastin protein, isolated from bovine
heart muscle, has an apparent molecular mass of 145 kDa (35). A
calpastatin protein of similar apparent molecular mass has also been
reported in skeletal muscle (36).
Several observations argue strongly that the upstream AUG we have
identified in bovine heart calpastatin mRNA is the predominant translation initiation site. First, prior designation of a downstream initiation of translation was based upon a partial rabbit calpastastin cDNA containing a stop codon 135 nt upstream of the proposed AUG (9). Bovine calpastatin cDNAs do not contain a stop codon at this
site. Second, the upstream AUG located at nt +129 is the first
potential start site downstream of transcription initiation. An open
reading frame extends from this AUG through to the C terminus of the
protein that is in frame with calpastatin amino acid sequence derived
from peptide sequencing (9, 10, 27). Third, the downstream AUG is a
poor potential start site, whereas the upstream AUG shows excellent
consensus with other translation initiation sites (37). Fourth,
calpastastin produced from the upstream AUG by coupled in
vitro transcription-translation runs at an apparent molecular mass
of 145 kDa, comigrating with calpastatin identified by Western blot in
whole cell extracts from heart and liver. Finally, antibodies prepared
against a peptide from the predicted amino acid sequence of the XL
region recognizes this 145-kDa protein. The protein running at 145-kDa
therefore represents full-length calpastastin, contains the XL region,
and has a predicted molecular mass of 84 kDa.
Since our results clearly show that the upstream AUG is utilized
in vivo, the question arises as to whether the downstream AUG is functional. For rabbit, the presence of a stop codon 108 nt 5
of the downstream AUG precludes the use of any potential upstream start
site. Calpastatin cDNA sequence presently available from other
species does not extend far enough 5
to determine whether upstream
start sites are present. In bovine, Western blots of whole liver
extracts detected calpastatin bands of 145 and 135 kDa (Fig.
3C). The lower band comigrates with an in vitro translation product initiated at the downstream AUG, raising the possibility that both start sites can be utilized. Alternatively, the
135-kDa calpastatin protein might arise through proteolysis. We have
provided evidence indicating that PKA phosphorylates sites within the
XL region, which are present in full-length (145-kDa) calpastatin but
are absent from smaller calpastatin proteins. Regardless of whether the
135-kDa calpastastin arises through alternative start site selection or
through proteolysis, removal of the XL region might play a regulatory
role by altering phosphorylation patterns on the protein.
PKA Responsiveness of the Calpastatin Gene Promoter--
The
proximal promoter region of the bovine calpastatin gene is GC-rich and
lacks a TATA box, characteristics of genes that are widely expressed in
different cell types. Transfection analyses show that the 5
-flanking
region of the calpastatin gene can direct moderate level transcription
of a heterologous cDNA and that sequence elements between nt
1667
and
272 can act to both stimulate and inhibit gene expression.
Deletion to nt
102, which eliminates a putative CAAT box, almost
completely abolishes promoter activity.
The finding that dibutyryl cAMP increases calpastatin promoter activity
between 5- and 20-fold demonstrates that transcription of the
calpastatin gene can be up-regulated by PKA. Binding of
-agonists to
the
2-adrenergic receptor activates a
PKA-dependent signaling pathway and leads to an increase in
calpastatin protein levels (18). Our results suggest that this occurs
through an increase in gene transcription. Some cAMP-responsive genes
contain a consensus cAMP-responsive cis element (CRE) consisting of the palindromic sequence (TGACGTCA) (38). Variations of the canonical CRE
have also been reported, including CRE half-sites that can bind
proteins such as CREB, ATF-2, and Jun, albeit at reduced affinity
compared with the intact palindromic CRE (for a review see Ref. 39).
pCS-102, which shows no basal promoter function, exhibits moderate
transcriptional activity following exposure of transfected cells to
dibutyryl cAMP, indicating that at least one cAMP-responsive element is
located within 102 nt of transcription initiation. This proximal
promoter region contains two potential CRE half-sites, one (GGTCA) at
nt
76 just downstream of an Sp1 site, and a second (TGAC) located at
nt
20. Detailed deletion and mutation studies are presently under way
to determine whether these or other non-CRE cis elements confer cAMP
responsiveness to the calpastatin promoter.
Taken together, our results indicate that activation of a
cAMP-dependent signaling pathway increases both calpastatin
gene transcription and calpastastin protein phosphorylation. Modulating protein activity through changes in gene transcription, however, represents a relatively slow regulatory mechanism; one possibility is
that newly synthesized calpastatin is phosphorylated in the XL region,
rendering it inactive pending a subsequent regulatory step. Calpastatin
is phosphorylated in vivo (30, 40) and also appears highly
susceptible to proteolysis, which has been shown to cleave the L
domain (and therefore also the XL region) from domains 1-4. Since this
amino-terminal region is a target of PKA phosphorylation, its
removal from the calpain inhibitory domains may serve a
regulatory function.