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 isola ted bovine heart calpastatin cDNAs and 5'-regions of the calpastatin g ene. Analysis of 5'-cDNA sequence identified a new translation initiat ion site that is in frame and 204 nucleotides upstream of the previous ly designated start site, Conceptual translation from this upstream AU G 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 k nown protein, Immunoblot studies demonstrated that heart and liver con tain a calpastatin protein of 145 kDa on SDS-polyacrylamide gel electr ophoresis that comigrates with full-length bacterially expressed calpa statin and calpastatin produced by coupled in vitro transcription-tran slation from the upstream AUG, An antibody raised against the XL regio n 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 with in 272 nucleotides upstream of transcription initiation of the calpast atin gene is sufficient to direct moderate level transcription. Promot er sequences further upstream act to inhibit or stimulate transcriptio nal activity. Exposure of transfected cells to dibutyryl cAMP resulted in a 7-20-fold increase in promoter activity for constructs containin g at least 272 nucleotides of upstream promoter sequence. Deletion ana lysis indicates that at least one cAMP-responsive element resides with in 102 nucleotides of transcription initiation.