Title

 

Authors

R. Corradino

Doctorate School in Pharmacology
Dept. of Pharmacobiological Sciences, University “Magna Græcia” of Catanzaro, Campus “S. Venuta”, 88100 Catanzaro (Italy)

 

Abstract

Mitochondria are highly dynamic organelles that continuously change their morphology through events of fusion and fission. Fusion process is regulated by the combined action of mitofusin 1, mitofusin 2 and OPA1, otherwise fission is modulated by the GTPase dynamin-related protein-1 (Drp1) and its mitochondrial docking protein Fis1. Drp1 is a cytosolic protein which is able to translocate to mitochondria after fission stimulation. This ability is tightly controlled through different mechanisms, such as phosphorylation or nitrosylation.  A recent paper showed a specific inhibitory phosphorylation on Ser637 mediated by PKA and the opposite effect of calcineurin, that dephosphorylates Drp1 triggering fission and translocation of the protein to mitochondria (Cereghetti et al.,2008). These data accounted for a switch between phosphorylated and dephosphorylated form which is able to modulate Drp1 function In a previous work we  demonstrated the new role of nitric oxide in the regulation of mitochondrial dynamics. In particular we showed that NO inhibited Drp1 and maintained fission at low level, leading to the formation of long interconnetted mitochondrial network during muscle differentiation. NO acted through cGMP production and its effect was due to G-kinase activation, as clearly demonstrated by the inhibitory role of KT5823. Our hypothesis was the specific phosphorylation of Drp1 at PKG-dependent sites that controlled the switch from cytosolic to mitochondrial form. Labeling with ³²P clearly confirmed this hypothesis and we processed Drp1 sequence for consensus PKG phosphorylation sites through bioinformatic analysis and we identified two possible amino acidic as target of PKG: Thr⁷⁹and Ser⁵⁹⁶. We generated mutant forms in which phosphomimetic substitutions resulted in loss of PKG-mediated phosphorylation or in constitutive phosphorylation. Preliminary results of Drp1 localization seemed indicate both sites as responsible for PKG inhibitory effects on Drp1 functions.
So we can conclude that NO, through PKG, is a new regulator of Drp1 and this action is likely due to double phosphorylation of the protein.