Cyclic nucleotide phosphodiesterases (PDEs) are critical in signal transduction pathways since they regulate the cellular concentrations of cyclic nucleotides. The intracellular concentration of cAMP, for example, is controlled by both its synthesis by adenylate cyclase and its hydrolysis by PDEs. Elevated levels of cAMP in platelets and neutrophils block all activation pathways including platelet shape change, aggregation, adhesion, and secretion of granule contents. PDEs are widely distributed in tissues and cells. Nearly all mammalian cells contain multiple PDEs that re currently classified into seven families. The amino acid sequence available show that all these PDE families contain a related carboxy terminal domain with approximately 30% sequence identity, whereas within each family, there is approximately 90% sequence identity. This domain of approximately 270 amino acids is now believed to be the catalytic domain. The proposal focuses on the putative catalytic domain of mainly two enzymes, cGMP-inhibited PDE (PDE III), the predominant cAMP-PDE in platelets, and cAMP-specific PDE (PDE IV), the major cAMP PDE in neutrophils. By utilizing chemical modification, peptide mapping, spectroscopic, and molecular biological techniques. I will characterize the catalytic domain and reveal the amino acid residue(s) involved in the chemistry of hydrolysis. The role of metal ions will be investigated by kinetic spectroscopic and site-directed mutagenesis methods. The affinity labeled amino acid residue (labeled with 8-[4-brom-2,3- dioxobutyl)thio]adenosine-3',5'-cyclic monophosphate (8-BDB-TcAMP)) will be subjected to site-directed mutagenesis and mutant protein will be expressed, purified, and characterized and then compared with native enzyme. All PDEs have amino acid sequence that show highly conserved histidines and glutamates spaced in a manner indicative of possible presence of metal binding sites. The roles of the conserved histidines and glutamates will be investigated by chemical modification and site-directed mutagenesis. The possibility of these residues acting as metal ligands will also be investigated by metal analysis, metal chelation and substitution in both recombinant PDE IV and mutant proteins. In platelets, cAMP hydrolysis by PDE III is inhibited by cGMP in the low muM range but PDE II is stimulated by approximately muM cCMP in the same tissue.
Specific Aim 5 is designed to identify the binding site of cGMP in both enzymes and describe the regulatory domain influenced by the cyclic nucleotide. Specific roles of ht metal cations in the structure or hydrolytic (catalytic) mechanism PDEs will be revealed by a combination of kinetic and spectroscopic methods described in Specific Aim 6.
| Omburo, G A; Jacobitz, S; Torphy, T J et al. (1998) Critical role of conserved histidine pairs HNXXH and HDXXH in recombinant human phosphodiesterase 4A. Cell Signal 10:491-7 |
| Zhu, W H; Majluf-Cruz, A; Omburo, G A (1998) Cyclic AMP-specific phosphodiesterase inhibitor rolipram and RO-20-1724 promoted apoptosis in HL60 promyelocytic leukemic cells via cyclic AMP-independent mechanism. Life Sci 63:265-74 |
| Omburo, G A; Torphy, T J; Scott, G et al. (1997) Inactivation of recombinant monocyte cAMP-specific phosphodiesterase by cAMP analog, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate. Blood 89:1019-26 |
| Ghazaleh, F A; Omburo, G A; Colman, R W (1996) Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase. Biochem J 317 ( Pt 2):495-501 |
