Characterization of a factor which binds to the cAMP response element of the neuronal tyrosine hydroxylase gene

Author

Douglas P. Thewke

Date of Award

12-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Wesley D. Wicks

Committee Members

John Koontz, Dan Roberts, Dave Brian

Abstract

Tyrosine hydroxylase (TH) is expressed in sympathetic neurons and is the rate limiting enzyme in catecholamine biosynthesis. TH is subject to both short-term and long-term regulation depending upon the nature of nerve stimulation. Short-term regulation occurs at the level of its catalytic activity, principally by phosphorylation by protein kinase A, protein kinase C and Ca²⁺ /calmodulin kinase. Long-term regulation is the result of an induction of TH synthesis and appears to involve the same signal transduction pathways. cAMP treatment of pheochromocytoma cells with agonists that raise intracellular cAMP levels activates protein kinase A and stimulates TH activity as well as increases TH gene transcription. The cAMP responsive element (CRE) responsible for cAMP induction of TH transcription has been mapped by deletion transfection analysis to a region between -41 and -60 base pairs in the TH promoter relative to the start site of transcription (Huang et al., (1991)Mol. Brain Res. 11, 309- 319). Within this region is the palindromic TGACGTCA motif found in the CREs of other cAMP inducible promoters. Transient transfection of reporter constructs bearing a short synthetic oligonucleotide derived from this region was found to be capable of conferring cAMP responsiveness onto a heterologous promoter.

The goal of this study was to identify and characterize the CRE binding protein (CREB) involved in regulating transcription of TH in response to elevated cAMP levels. By using synthetic oligonucleotides containing the TH CRE as probes for in vitro binding assays, a TH CRE binding protein (CREB^TH) was detected in nuclear extracts obtained from pheochromocytoma cells and brain tissue which are known to express TH and in human placenta choriocarcinoma cells which do not express TH but demonstrate cAMP inducibility of transfected reporter constructs bearing the TH CRE. This factor was demonstrated to specifically bind CRE-containing oligonucleotides and not to several mutant oligonucleotides. The specificity displayed by this factor for these oligonucleotides correlated with the ability of the oligonucleotides to confer cAMP responsiveness in transient transfection analysis. CREB^TH was determined to have an apparent subunit molecular weight of 47,000 daltons and was highly purified from pig brain nuclear extract by DNA affinity chromatography. Furthermore, CREB^TH was phosphorylated in vitro by the catalytic subunit of protein kinase A consistent with its proposed role in the regulation of TH transcription by cAMP.

In order to determine the relatedness of CREB^TH to several other CRE binding proteins, its in vitro binding characteristics were compared to those of bacterially expressed CREB-1, CRE-BP1 and ATFa. CREB^TH displayed a different molecular weight on southwestern blots and a different relative mobility in gel mobility shift assays suggesting that CREB^TH might be a novel factor. However, immunological data indicated that CREB^TH might be related to CREB-1 since it reacted with antibodies directed to the protein kinase A phosphorylation site of CREB-1. The results from phosphopeptide mapping of CREB-1 and CREB^TH reveal that they have similar A kinase phosphorylation domains and peptide mapping of ¹²⁵I-labeled CREB-1 and CREB^TH indicates that these factors are highly related if not identical.

Recommended Citation

Thewke, Douglas P., "Characterization of a factor which binds to the cAMP response element of the neuronal tyrosine hydroxylase gene. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10789