The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP-dependent protein kinase activity

Mei Cong, Valery F. Thompson, Darrel E. Goll, Parker B Antin

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)660-666
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number1
DOIs
StatePublished - Jan 2 1998

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Cyclic AMP-Dependent Protein Kinases
Genes
Transcription
Proteins
Nucleotides
Complementary DNA
calpastatin
Phosphorylation
Gene Expression Regulation
Electrophoresis
Gene expression
Liver
Transfection
Polyacrylamide Gel Electrophoresis
Assays
Amino Acids
Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

The bovine calpastatin gene promoter and a new N-terminal region of the protein are targets for cAMP-dependent protein kinase activity. / Cong, Mei; Thompson, Valery F.; Goll, Darrel E.; Antin, Parker B.

In: Journal of Biological Chemistry, Vol. 273, No. 1, 02.01.1998, p. 660-666.

Research output: Contribution to journalArticle

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