Cyclic nucleotides as affinity tools: phosphorothioate cAMP analogues address specific PKA subproteomes.

cAMP (adenosine-3',5'-cyclic monophosphate) is a general second messenger controlling distinct targets in eukaryotic cells. In a (sub)proteomic approach, two classes of phosphorothioate cAMP affinity tools were used to isolate and to identify signalling complexes of the main cAMP target, cAMP dependent protein kinase (PKA). Agonist analogues (here: Sp-cAMPS) bind to the regulatory subunits of PKA (PKA-R), together with their interaction partners, and cause dissociation of a holoenzyme complex comprising PKA-R and catalytic subunits of PKA (PKA-C).

Chemical tools selectively target components of the PKA system.

Background: In the eukaryotic cell the cAMP-dependent protein kinase (PKA) is a key enzyme in signal transduction and represents the main target of the second messenger cAMP. Here we describe the design, synthesis and characterisation of specifically tailored cAMP analogs which can be utilised as a tool for affinity enrichment and purification as well as for proteomics based analyses of cAMP binding proteins.

Results: Two sets of chemical binders were developed based on the phosphorothioate derivatives of cAMP, Sp-cAMPS and Rp-cAMPS acting as cAMP-agonists and -antagonists, respectively.

P elements inserted in the vicinity of or within the Drosophila snRNP SmD3 gene nested in the first intron of the Ornithine Decarboxylase Antizyme gene affect only the expression of SmD3.

The Drosophila gene for snRNP SmD3 (SmD3) is contained in reverse orientation within the first intron of the Ornithine Decarboxylase Antizyme (AZ) gene. Previous studies show that two closely linked P elements cause the gutfeeling phenotype characterized by embryonic lethality and aberrant neuronal and muscle cell differentiation. However, the exact nature of the gene(s) affected in the gutfeeling phenotype remained unknown.