To investigate the regulation of calpastatin gene expression, bovine heart calpastatin cDNAs and 5' regions of the calpastatin gene were isolated. 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 PAGE 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 protein in vivo. The organization of the calpastatin 5' genomic region was determined by comparing calpastatin cDNA and genomic sequences. The region encompassing exon 1-4 contains large introns and spans at least 60 kb. Calpastatin promoter sequence analysis revealed that it belong to the family of "house keeping" genes which lack TATA box and are GC rich at the proximal promoter regions. 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 and stimulate transcriptional activity. Exposure of transfected cells to dibutryl cAMP resulted in a seven to twenty fold increase in calpastatin promoter activity for constructs containing at least 272 nucleotides of upstream promoter sequence. Deletion and mutation analyses identified a cAMP responsive element at nt-76. These findings demonstrate that calpastatin gene and protein are both targets for cAMP-dependent kinase activity. beta-Agonist treatment can induce both calpastatin gene transcription and protein phosphorylation.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/288832 |
| Date | January 1998 |
| Creators | Cong, Mei, 1966- |
| Contributors | Antin, Parker B., Goll, Darrel E. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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