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Vol. 273, Issue 1, 660-666, January 2, 1998
From the Departments of To investigate the regulation of calpastatin gene
expression, we isolated bovine heart calpastatin cDNAs and
5
The Bovine Calpastatin Gene Promoter and a New N-terminal Region
of the Protein Are Targets for cAMP-dependent Protein
Kinase Activity
,§, and Biochemistry,
§ Nutritional Sciences, and ¶ Cell Biology and Anatomy,
University of Arizona, Tucson, Arizona 85721
-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.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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