The Bovine Calpastatin Gene Promoter and a New N-terminal Region of the Protein Are Targets for cAMP-dependent Protein Kinase Activity*

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.

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.
Calpastatin is a proteinase inhibitor that is specific for calpains, a family of calcium-activated neutral proteases that regulate a wide range of Ca 2ϩ -dependent cellular processes (1)(2)(3)(4)(5)(6). Calpastatin binds to and inhibits the calpains in the presence of Ca 2ϩ , 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)(14)(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 K i for inhibition of m-calpain than for inhibition of -calpain, whereas an unphosphorylated form preferentially inhibited -calpain (3,19). This relative specificity forversus mcalpain 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 5Ј-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.
Isolation of Bovine Calpastatin cDNA and Genomic Sequences-Approximately 1.7 ϫ 10 5 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 ϫ SSC at room temperature for 30 min) using a 152-bp 5Ј-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 doublestranded DNA pBluescript plasmids containing cDNA inserts.
To isolate bovine genomic sequence in the 5Ј-region of the calpastatin gene, 1.1 ϫ 10 6 recombinant phage from a bovine heart genomic library (Stratagene) were screened using a 139-bp PCR product from the 5Ј-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 5Ј-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; 5Ј-AUGGCGACGAUGGAUGT-GTTCC-3Ј) near the 5Ј-end of the calpastatin cDNA was labeled with [␥-32 P]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 g of total RNA from bovine heart tissue, 10 pmol of [␥-32 P]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 MgCl 2 , 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 ϫ 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 ϫ TTBS and then incubated in 1 ϫ 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 5Ј-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 5Ј-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 MgCl 2 , 1 mM dithiothreitol, 1 g/ml PKA. The phosphorylation reaction was initiated by adding 26.25 Ci of [␥-32 P]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 ϫ 10 5 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 g/ml Gentamicin, 1:100 antibiotic-antimycotic. The following day, cells were transfected with 2 g 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 l 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 l 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 l of cell extract per reaction was incubated at 37°C for 1 h with 1 l of [ 14 C]chloramphenicol (25 Ci/ml, Amersham Corp.), 8 l of 40 mM acetyl-CoA (Pharmacia Biotech Inc.), and 100 l 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 l of ethyl acetate. Unacetylated and acetylated [ 14 C]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.

Isolation of Full-length Bovine
Calpastatin cDNAs-To identify 5Ј-bovine calpastatin mRNA sequence, 5Ј-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 5Ј-RACE was used to screen a bovine heart cDNA library. Three independent cDNAs were obtained that differed in the amount of 3Ј-untranslated region and in their 5Ј-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).
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 5Ј 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 (AGU-AUGA) 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.   FIG. 1. A, structure of three calpastatin cDNAs obtained from a bovine heart cDNA library. cDNAs differ in the amount of 3Јuntranslated region and in their 5Ј-termination point. cDNAs 2 and 3 extend 5Ј 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.
FIG. 2. cDNA and deduced amino acid sequence of bovine heart calpastatin. cDNA sequence was derived by combining cDNA 1, which extends farthest 3Ј, with the 5Ј sequence of cDNA 2. Extreme 5Ј-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.
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).
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 fulllength 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)(28)(29).
Analysis of the deduced amino acid sequence of the fulllength 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 5Ј-Region of the Bovine Calpastatin Gene-Using 5Ј-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 5Ј-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  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). located in exon 4. mRNA sequence further 5Ј is encoded by three exons separated by large introns spanning at least 60 kb.
To define the transcription initiation site, primer extension was performed using heart muscle RNA as template and a 22-nt primer located just 5Ј 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 5Ј terminus of the 130-nt primer extension product is contiguous within the genomic sequence, indicating that it is encoded by a single exon.
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 Ϫ162 and Ϫ41 (relative to the most 5Ј initiation of transcription identified by primer extension) and a putative CAAT box between nt Ϫ125 and Ϫ122 (Fig. 7). To determine whether this upstream genomic region possesses promoter activity, DNA fragments extending from nt Ϫ1667, Ϫ1242, Ϫ944, Ϫ671, Ϫ272, Ϫ102, or Ϫ31 to nt ϩ130 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.
Transient transfection studies show that the 5Ј-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 Ϫ1667 and Ϫ944, for example, increased promoter activity more than 2-fold, while deletion to nt Ϫ671 reduced CAT activity more than 7-fold. Truncation at nt Ϫ102, which deleted a potential CAAT box element at nt Ϫ125, reduced CAT expression to barely detectable levels. Further deletion to nt Ϫ31 abolished promoter activity. These results demonstrate that DNA elements located within nt Ϫ1667 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.
␤-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 5Ј-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)(32)(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 ϩ129 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 5Ј 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 5Ј 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 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. 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 5Ј-flanking region of the calpastatin gene can direct moderate level transcription of a heterologous cDNA and that sequence elements between nt Ϫ1667 and Ϫ272 can act to both stimulate and inhibit gene expression. Deletion to nt Ϫ102, 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 cAMPresponsive 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 Ϫ76 just downstream of an Sp1 site, and a second (TGAC) located at nt Ϫ20. 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 aminoterminal region is a target of PKA phosphorylation, its removal from the calpain inhibitory domains may serve a regulatory function.