The ultimate goal of this project is to provide unequivocal demonstration of the existence of a nuclear factor(s) which presumably binds to and regulates the transcription of genes known to be subject to control by cAMP in eukaryotic cells. It is postulated that such a protein ought to exist that is likely to be a substrate for the cAMP-dependent protein kinase (PK) which exhibits low affinity for the relevant specific DNA sequences until it has been phosphjorylated. We have preliminary evidence that such a factor is, in fact, present in cAMP-treated but not control nuclei. A variety of approaches will be taken with the goals of clearly demonstrating the existence of such a protein and developing methods for its purification. Nitrocellulose filter binding of various specific restriction fragments isolated from two cAMP-inducible genes (PEPCK and prolactin) will be conducted with salt extracts of nuclei from control and cAMP-treated cells. PAGE analysis of possible complexes will be run+/- competng DNA and footprinting will be done with Exonuclease III. The ability of various fragments to exhibit factor binding will be compared with their ability to confer cAMP-inducibility upon a heterologous gene (CAT) after injection into Xenopus oocytes or transfecion into mammalian cells. Attempts will be made to render control extracts (nuclear and cytosolic) capable of specific DNA binding by addition of the pure C subunit of PK an ATP. Transient expression of plasmids constructed with various fragements of the two inducble genes inserted upstream of the E. coli CAT gene in a pSV2 derived plasmid which contains the SV40 enhancer will be sought by injectin into oocytes and by transfection. cAMP inducibility will be scored in oocytes after injecion of pure PK subunit, PK subunit antibodies or addition of progesterone. Deletion and reorientation mutants should allow location of the critical regulatory elements (s) and to determine if it acts as an enhancer. A combination of the in vitro binding and in vivo expression systems will be used to assist purificaion of the putative trans acting protein subject to regulation by PK. The use of two gnes from different cells and species should allow us to determine whether or not the putative protein is a ubiquitous mediator of cAMP contol of eukaroytic gene expression and to discover what is the basis of its presumed interaction with specific regions of genes under control.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031538-04
Application #
3279595
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1983-09-15
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Tennessee Knoxville
Department
Type
Schools of Arts and Sciences
DUNS #
City
Knoxville
State
TN
Country
United States
Zip Code
37996
Lu, G; Schlichter, D; Wicks, W D (1992) Characterization of rat liver nuclear proteins which recognize the cAMP responsive element. Int J Biochem 24:1763-71
Lu, G H; Schlichter, D; Wicks, W D (1992) Interaction of a nuclear factor 1-like protein with a cAMP response element-binding protein in rat liver. Int J Biochem 24:455-64
Huang, Z M; Thewke, D; Gong, Q Q et al. (1991) Functional recognition of the neuronal tyrosine hydroxylase cAMP regulatory element in different cell types. Brain Res Mol Brain Res 11:309-19
Spielholz, C; Schlichter, D; Wicks, W D (1988) Cyclic adenonosine monophosphate does not affect the stability of the messenger ribonucleic acid for tyrosine aminotransferase in cultured hepatoma cells. Mol Endocrinol 2:344-9
Williams, J A; Schlichter, D; Wicks, W D (1988) Progesterone decreases DNA binding factor activity and the expression in Xenopus oocytes of a cAMP responsive gene from rat liver. Second Messengers Phosphoproteins 12:261-70
Lee, C Q; Miller, H A; Schlichter, D et al. (1988) Evidence for a cAMP-dependent nuclear factor capable of interacting with a specific region of a eukaryotic gene. Proc Natl Acad Sci U S A 85:4223-7
Schlichter, D; Miller, H; Wicks, W D (1986) On the role of protein kinase subunits in the control of eukaryotic gene expression. J Cyclic Nucleotide Protein Phosphor Res 11:149-54