Immunofluorescent localization of cyclic nucleotide-dependent protein kinases on the mitotic apparatus of cultured cells

Cyclic nucleotides and cyclic nucleotide-dependent protein kinases have been implicated in the regulation of cell motility and division, processes that depend on the cell cytoskeleton. To determine whether cyclic nucleotides or their kinases are physically associated with the cytoskeleton during cell division, fluorescently labeled antibodies directed against cyclic AMP, cyclic GMP, and the cyclic nucleotide- dpendent protein kinases were used to localize these molecules in mitotic PtK1 cells. Both the cyclic GMP-dependent protein kinase and the type II regulatory subunit of the cyclic AMP-dependent protein kinase were localized on the mitotic spindle. Throughout mitosis, their distribution closely resembled that of tubulin. Antibodies to cyclic AMP, cyclic GMP, and the type I regulatory and catalytic subunits of the cyclic AMP-dependent protein kinase did not label the mitotic apparatus. The association between specific components of the cyclic neucleotide system and the mitotic spindle suggests that cyclic nucleotide-dependent phosphorylation of spindle proteins, such as those of microtubules, may play a fundamental role in the regulation of spindle assembly and chromosome motion

Phosphodiesterase 8A, cAMP-specific

Phosphodiesterase 8A (PDE8A) is a 3′,5′-cyclic-nucleotide phosphodiesterase that specifically catalyses the hydrolysis of cAMP to AMP. PDE8A is one of the two isoenzymes of the PDE8 family, the other being PDE8B. These two highly similar proteins have several common features that distinguish them from other cAMP-specific PDEs: they have very high affinity for the substrate cAMP; they are insensitive to the non-specific PDE inhibitor IBMX. They contain a PAS (Per, Arnt and Sim) and a REC (receiver) domain, both of which are observed in many signal transduction proteins. The possible function(s) of the PAS domain in PDE8 is still unknown. PDE8A mRNA is not expressed in all tissues but has been detected in several and is highest in testis, spleen, small intestine, heart, ovary, colon and kidney. Until early 2009, the lack of specific small-molecule inhibitors slowed the study of the physiological relevance of PDE8A; thus to date many aspects of this PDE's functions (that is, whether it is activated or regulated during certain cellular process) remain in large part unknown. So far, functions for PDE8A have been described in Leydig cells, where lack of PDE8A causes increased testosterone production in response to luteinizing hormone; T cells, where it seems to be induced during T cell activation and may control chemotaxis; and cardiocytes, where it seems to regulate calcium handling

GAF domains: two-billion-year-old molecular switches that bind cyclic nucleotides

GAF domains represent one of the largest families of small-molecule binding units present in nature. The first mammalian GAF domains discovered were the cGMP-binding regulatory domains of several cyclic nucleotide phosphodiesterases (PDEs). The crystal structure of the PDE2A GAF domains has provided our first look at the architecture of the binding site for the second messenger cGMP. The topology of this site differs greatly from all other previously determined cyclic nucleotide binding sites. In PDE2A, cGMP binds to a well-defined pocket in one of the two GAF domains that is analogous to the ligand-binding pocket of the distantly related PAS domains of photoactive yellow protein and FixL. The consensus cGMP-binding motif suggests strongly that only certain GAF domains will bind cGMP. Although the detailed mechanism for how cGMP binding to the GAF domain regulates catalysis remains to be determined, recent data from a GAF domain-containing cAMP-stimulated adenylyl cyclase from Anabaena suggest a mechanism conserved across two billion years of evolution. Because of their unique ligand-binding topologies, the GAF domains of PDEs are likely to offer good new targets for rational drug design.status: publishe

Cyclic nucleotide-binding GAF domains in phosphodiesterases and adenylyl cyclases

edition: 2ndstatus: publishe