Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin

Abstract

Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Commonly, phosphorylation of S579 is considered activating, while S600 phosphorylation is considered inhibiting. However, direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. Here, we compare effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and in vitro phosphorylation. Both phospho-mimetic mutants are shifted toward smaller oligomers. Both phospho-mimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wildtype Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant (K38A) lacking GTPase activity stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.

Systemic phospho-defective and phospho-mimetic Drp1 mice exhibit normal growth and development with altered anxiety-like behavior

“…Previous studies have extensively explored the mechanism and function of Drp1 S579 phosphorylation using cell culture systems and purified proteins. 33 , 38 , 39 , 40 , 41 , 43 , 44 , 54 In this current study, we have for the first time systematically analyzed Drp1 S579 phosphorylation in mouse tissues. We also examined the whole-body, physiological impact of this phosphorylation by generating Drp1 S579 phospho-mimetic (S579D) and phospho-defective (S579R) mice using the CRISPR-Cas genome-editing system.…”

“… 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 Interestingly, a recent biochemical study using purified Drp1 revealed that S579 phosphorylation does not enhance Drp1’s activity but rather suppresses its activation by actin, receptors, and cardiolipin. 33 It has been proposed that the regulatory mechanism of mitochondrial division via S579 phosphorylation may involve isoform-specific effects, as Drp1 generates various isoforms in different cell types and tissues, 14 , 15 , 34 and other factors such as nucleoside diphosphate kinases, which could supply GTP to dynamin and dynamin-related GTPase family proteins. 33 , 35 , 36 , 37 Moreover, this phosphorylation might coordinate and orchestrate these multiple regulatory processes with other post-translational signaling mechanisms of Drp1, such as ubiquitination, SUMOylation, O-GlcNAcylation, and S-nitrosylation.…”

“… 33 It has been proposed that the regulatory mechanism of mitochondrial division via S579 phosphorylation may involve isoform-specific effects, as Drp1 generates various isoforms in different cell types and tissues, 14 , 15 , 34 and other factors such as nucleoside diphosphate kinases, which could supply GTP to dynamin and dynamin-related GTPase family proteins. 33 , 35 , 36 , 37 Moreover, this phosphorylation might coordinate and orchestrate these multiple regulatory processes with other post-translational signaling mechanisms of Drp1, such as ubiquitination, SUMOylation, O-GlcNAcylation, and S-nitrosylation. 16

Figure 1 Drp1 S579 is strongly phosphorylated in neurons (A) A table shows the UniProt ID, amino acid length, alternative exons, and phosphorylation sites of mouse Drp1 (isoform 3, the most widely expressed) 14 , 15 and human Drp1 (canonical isoform 1).

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Serine 39 in the GTP‐binding domain of Drp1 is involved in shaping mitochondrial morphology

“…Ser39 does not make significant interactions with the β‐phosphate binding [22]. Single amino acid mutagenesis of Ser39 within the GTPase domain completely abolished the GTPase activity of Drp1, similar to the well‐studied K38A mutation [22–25].…”