Abstract

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.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022792-16
Application #
3271338
Study Section
Biochemistry Study Section (BIO)
Project Start
1976-03-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
16
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461

  Publications

Han, Ping; Sonati, Pushpalatha; Rubin, Charles et al. (2006) PDE7A1, a cAMP-specific phosphodiesterase, inhibits cAMP-dependent protein kinase by a direct interaction with C. J Biol Chem 281:15050-7
Zhang, L; Wu, S L; Rubin, C S (2001) Structural properties and mechanisms that govern association of C kinase adapter 1 with protein kinase C3 and the cell periphery. J Biol Chem 276:10476-84
Zhang, L; Wu, S L; Rubin, C S (2001) A novel adapter protein employs a phosphotyrosine binding domain and exceptionally basic N-terminal domains to capture and localize an atypical protein kinase C: characterization of Caenorhabditis elegans C kinase adapter 1, a protein that avidly binds pr J Biol Chem 276:10463-75
Rossi, E A; Li, Z; Feng, H et al. (1999) Characterization of the targeting, binding, and phosphorylation site domains of an A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog that are encoded by a single gene. J Biol Chem 274:27201-10
Davare, M A; Dong, F; Rubin, C S et al. (1999) The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons. J Biol Chem 274:30280-7
Feliciello, A; Cardone, L; Garbi, C et al. (1999) Yotiao protein, a ligand for the NMDA receptor, binds and targets cAMP-dependent protein kinase II(1). FEBS Lett 464:174-8
Li, Z; Rossi, E A; Hoheisel, J D et al. (1999) Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene. J Biol Chem 274:27191-200
Angelo, R; Rubin, C S (1998) Molecular characterization of an anchor protein (AKAPCE) that binds the RI subunit (RCE) of type I protein kinase A from Caenorhabditis elegans. J Biol Chem 273:14633-43
Feliciello, A; Rubin, C S; Avvedimento, E V et al. (1998) Expression of a kinase anchor protein 121 is regulated by hormones in thyroid and testicular germ cells. J Biol Chem 273:23361-6
Dong, F; Feldmesser, M; Casadevall, A et al. (1998) Molecular characterization of a cDNA that encodes six isoforms of a novel murine A kinase anchor protein. J Biol Chem 273:6533-41

Showing the most recent 10 out of 36 publications