Characterization, purification and cloning of acAMP-responsive element binding protein (CREB) required for the transcription of the rat somatostatin gene
Abstract
The 5$\sp\prime$-end deletion study indicated that the sequence between position $-$60 and $-$43 of the rat somatostatin gene is critical for its high level expression in CA-77 cells. The CA-77 cell line originated from a rat medullary thyroid carcinoma which is known to express the endogenous somatostain gene. By using a somatostatin promoter fragment ($-$70 to $-$29) as radioactive-labeled probe in a gel retardation assay, three sequence-specific DNA binding activities, designated B1, B2 and B3, were identified from extracts of HeLa cells, rat brain, as well as CA-77 cells. The DNaseI protection and methylation interference studies suggested that the protein(s) bind to the region which contains a cAMP-responsive element (CRE, TGACGTCA). A deletion disrupting the CRE sequence abolished not only formation of the three sequence-specific complexes in vitro but also expression of the somatostatin promoter in vivo. The three DNA binding activities from rat brain cell extract were separated by an KCl gradient on a DEAE-Sepharose column. The protein corresponding to the B2 activity was further purified to apparent homogeneity by two cycles of CRE-specific affinity chromatography. The purified protein, named CREB, showed a single band of 43 kDa on a silver-stained SDS-PAGE gel. Its CRE-binding specificity was confirmed by Southwestern blotting, renaturation, and DNaseI protection analyses. The protein was phosphorylated in vitro on serine residue(s) by cAMP-dependent protein kinase. Furthermore, the purified CREB specifically stimulated transcription from the rat somatostatin promoter in an in vitro transcription/complementation assay. A cDNA clone for the CREB protein was isolated from CA-77 cell RNA by means of the polymerase chain reaction (PCR) technique. The cDNA was sequenced from both directions, demonstrating a single open reading frame of 327 amino acids. The CREB protein was expressed both in the reticulocyte lysate and in E. coli. Its specific DNA-binding activity was demonstrated by gel retardation assays and CRE-affinity chromatography. The bacteria-expressed CREB was purified with streptomycin and ammonia sulfate precipitation followed by CRE-affinity chromatography. The purified CREB is able to transactivate the somatostatin promoter using the in vitro transcription/complementation assay. This represents one of the first studies in which a bacteria-expressed eukaryotic transcription factor can function in an eukaryotic in vitro transcription system.
Degree
Ph.D.
Advisors
Dixon, Purdue University.
Subject Area
Biochemistry|Molecular biology
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