Opposing mitochondrial fission and fusion reactions determine the shape and interconnectivity of mitochondria. Dynamin‐related protein 1 (Drp1) is an ancient mechanoenzyme that uses GTP hydrolysis to power the constriction and division of mitochondria. Although Drp1‐mediated mitochondrial fragmentation is recognized as an early event in the apoptotic programme, acute regulation of Drp1 activity is poorly understood. Here, we identify a crucial phosphorylation site that is conserved in all metazoan Drp1 orthologues. Ser 656 is phosphorylated by cyclic AMP‐dependent protein kinase and dephosphorylated by calcineurin, and its phosphorylation state is controlled by sympathetic tone, calcium levels and cell viability. Pseudophosphorylation of Drp1 by mutation of Ser 656 to aspartic acid leads to the elongation of mitochondria and confers resistance to various pro‐apoptotic insults. Conversely, the constitutively dephosphorylated Ser656Ala mutant Drp1 promotes mitochondrial fragmentation and increases cell vulnerability. Thus, Drp1 phosphorylation at Ser 656 provides a mechanism for the integration of cAMP and calcium signals in the control of mitochondrial shape, apoptosis and other aspects of mitochondrial function.

The mitochondrial signaling complex PKA/AKAP1 protects neurons against mitochondrial fragmentation and cell death by phosphorylating and inactivating the mitochondrial fission enzyme Drp1.

The predominant forms of protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, are dimers of catalytic (C) and scaffolding (A) subunits and trimers with an additional variable regulatory subunit. In mammals, catalytic and scaffolding subunits are encoded by two genes each (␣/␤), whereas three gene families (B, B , and B؆) with a total of 12 genes contribute PP2A regulatory subunits. We generated stable PC12 cell lines in which the major scaffolding A␣ subunit can be knocked down by inducible RNA interference (RNAi) to study its role in cell viability. A␣ RNAi decreased total PP2A activity as well as protein levels of C, B, and B but not B؆ subunits. Inhibitor experiments indicate that monomeric C and B subunits are degraded by the proteosome. Knock-down of A␣ triggered cell death by redundant apoptotic and non-apoptotic mechanisms because the inhibition of RNAi-associated caspase activation failed to stall cell death. PP2A holoenzymes positively regulate survival kinase signaling, because RNAi reduced basal and epidermal growth factor-stimulated Akt phosphorylation. RNAi-resistant A␣ cDNAs rescued RNAi-induced loss of the C subunit, and A␣ point mutants prevented regulatory subunit degradation as predicted from each mutant's binding specificity. In transient, stable, and stable-inducible rescue experiments, both wild-type A␤ and A␣ mutants capable of binding to at least one family of regulatory subunits were able to delay A␣ RNAi-induced death of PC12 cells. However, only the expression of wild-type A␣ restored viability completely. Thus, heterotrimeric PP2A holoenzymes containing the A␣ subunit and members of all three regulatory subunit families are necessary for mammalian cell viability.

The splice isoform Drp1-x01 promotes mitochondrial fission and is regulated by Cdk phosphorylation-dependent changes in microtubule association.

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