Drosophila NO-dependent guanylyl cyclase is finely regulated by sequential order of coincidental signaling

We investigate the mechanism of regulation of Drosophila-soluble guanylate cyclase. Multiple putative sites of phosphorylation for the major kinases are present on both subunits of the heterodimer. We show that NO activation after binding to the heme group, is specifically modulated by sequential phosphorylations. PKA increases the NO stimulation at optimum level when both subunits are phosphorylated. Phosphorylation by CK (casein kinase-like) first, inhibits the PKA phosphorylation of the alpha subunit and limits the PKA upregulation of the cyclase activity. However, PKA phosphorylation first didn't prevent CK phosphorylation of the two subunits and the sequence PKA/CK induces higher level of NO activation than CK/PKA. These phosphorylations occur independently of NO binding and the direct inhibitory effect of calcium is observed for all the sCG forms. These data show that the sGC activity is regulated in a complex way, and the well-known asymmetry of the two subunits appears to cause the reading of the sequence of regulatory signals. This qualifies sGC as molecular detector on which converge coincidental and/or sequential neuronal signals. Furthermore, due to the fact that NO induction is huge (more than 600-fold obtained with the mammal counterpart), we might consider that any variation in kinases activation and/or calcium concentration in micro area of neuronal processes, provokes locally significant quantitative difference of cGMP synthesis in presence of diffusing NO.