CYCLIC AMP AND PROTEIN KINASES IN CELL REGULATION

A central goal of this research program is to determine how eukaryotic
cells control the localization and expression of cAMP-dependent protein
kinases (cA-PKs). The predominant cA-PK in the mammalian central
nervous system (CNS) contains an R subunit isoform (RIIBeta) that
appears to adapt the holoenzyme for specialized functions in brain. A
striking characteristic of RIIBeta is its ability to bind tightly to
intracellular structures in neurons. Thus, signals carried by cAMP in
the CNS might be targeted to specialized intraneuronal compartments by
high-affinity RIIBeta binding proteins. We have discovered two novel
particulate proteins, bovine brain P75 and rat brain P150, that bind
RIIBeta with high-affinity. The putative P75 and P150 anchor proteins
are expressed almost exclusively in brain. We will now clone and
characterize a full-length cDNA for P75. The RIIBeta binding domain of
P75 will be characterized by in vitro mutagenesis, expression and
functional analysis. The regulation of the expression of RIIBeta and
P150 by cAMP and steroid hormones will be examined in the neuronal cell
line E5. Regulatory elements in the P75 and RIIBeta genes that govern
cell-specific expression will be determined. The regional, cellular and
intraneuronal localization of P150 and RIIBeta in brain will be
determined by immunocytochemistry in combination with light and electron
microscopy. The RIIBeta binding domain of P75 will be used to explore
the intracellular localization of RII subunits in nonneuronal cells. We
recently identified Caenorhabditis elegans as an excellent system for
studying (a) the regulation of R and C (catalytic subunit) expression
and (b) the functions of cA-PKs in development and differentiation. We
have cloned and characterized the C. elegans Rce, Cce and
metallothionein (MT) genes and flanking regions. We will now
investigate temporal, cell-specific and developmentally-controlled
aspects of Rce and Cce promoter activation (gene expression) by using
their promoter/enhancer regions to drive expression of the
Beta-galactosidase reporter gene in developing, transgenic C. elegans.
Finally, we will target the overexpression of the Cce subunit of cA-PK
to intestinal cells via the gut-specific MT promoter/enhancer. High
level gene expression will be obtained by adding cadmium to the medium.
cDNAs corresponding to cell-specific genes regulated by cA-PK will be
cloned and characterized.