Response by Arezu JahaniAsl & Ruth S. Slack

emphasize the important roles of protein phosphorylation by cAMP-dependent protein kinase (PKA) in the regulation of the dynamin-related protein 1 (Drp1) GTPase and mitochondrial fission. However, the Literature Report ( Jahani-Asl & Slack, 2007) that accompanied the Cribbs & Strack article led to incorrect interpretations, which we would like to clarify here. Foremost among these, the Literature Report discusses cAMP-dependent phosphorylation at Ser 637 in human Drp1 splice variant 1 (Chang & Blackstone, 2007) and Ser 656 in rat Drp1 splice variant 1 (Cribbs & Strack, 2007) as if they are distinct sites. However, both our study and that of Cribbs & Strack present sequence alignments that clearly indicate that these sites are the same; thus there is only one PKA phosphorylation site in Drp1 (Fig 1). In this regard, it is important to emphasize that there are several splice variants in the Drp1 protein and that protein size varies among species, making protein sequence alignments important when comparing results of studies investigating different species or variants. In addition, the Literature Report states that the Cribbs & Strack study showed that phosphorylation at this site attenuates GTPase activity. In fact, these authors reported no effect using a phosphomimetic substitution (Supplementary Figure 1C in Cribbs & Strack, 2007), although our study did report attenuation of GTPase activity in response to both direct phosphorylation by cAMP-dependent protein kinase, as well as with the same phosphomimetic mutant (Figure 3 in Chang & Blackstone, 2007). The reason for this discrepancy, despite using similar in vitro approaches and the same phosphomimetic mutation, is unclear; however, the attenuation of Drp1 GTPase activity that we observed would provide a mechanistic explanation for the findings in both papers that mitochondrial fission is impaired in cells, as well as for the resistance to pro-apoptotic stimuli reported by Cribbs & Strack. Our study also showed that the intramolecular association of Drp1 is altered by the phosphomimetic substitution (Figure 2 in Chang & Blackstone, 2007); other mutations in the GTPase effector domain that alter intramolecular interactions also attenuate GTPase activity (Zhu et al, 2004). Even so, we cannot eliminate the possibility that conformational changes owing to phosphorylation at this site also affect interactions with other proteins involved in mitochondrial fission. Importantly, the two-dimensional phosphopeptide mapping experiments in our study showed that there is no basal phosphorylation at the PKA phosphorylation site in HeLa cells (Figure 1 in Chang & Blackstone, 2007), indicating that any regulation of Drp1 function by dephosphorylation at this site in these widely studied cells would need to occur in conjunction with activation of cAMP-PKA signalling. Conversely, we visualized other basal sites of Drp1 phosphorylation, one of which might correspond to a site of mitotic phosphorylation previously identified by Taguchi et al (2007). The functional regulation of Drp1 by post-translational modifications such as protein phosphorylation, ubiquitination and sumoylation clearly provide cells with an impressive array of regulatory mechanisms to modulate mitochondrial morphology within cells. Further studies promise to clarify the role of these mechanisms in the dynamic regulation of mitochondrial morphology and distribution within cells. REFERENCES Chang CR, Blackstone C (2007) Cyclic AMPdependent protein kinase phosphorylation of Drp1 regulates its GTPase activity and mitochondrial morphology. J Biol Chem 282: 21583–21587 Cribbs JT, Strack S (2007) Reversible phosphorylation of Drp1 by cyclic AMPdependent protein kinase and calcineurin regulates mitochondrial fission and cell death. EMBO Rep 8: 939–944 Jahani-Asl A, Slack RS (2007) The phosphorylation state of Drp1 determines its cell fate. EMBO Rep 8: 912–913 Taguchi N, Ishihara N, Jokufu A, Oka T, Mihara K (2007) Mitotic phosphorylation of dynaminrelated GTPase participates in mitochondrial fission. J Biol Chem 282: 11521–11529 Zhu P-P, Patterson A, Stadler J, Seeburg DP, Sheng M, Blackstone C (2004) Intraand intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1. J Biol Chem 279: 35967–35974