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bioRxiv · 10.1101/2023.08.20.554022

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

Liu, A.·Hatch, A. L.·Higgs, H.
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Abstract

Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane in a GTP hydrolysis-dependent manner. 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. Phosphorylation of S579 is widely regarded as activating, while S600 phosphorylation is commonly considered inhibiting. However, the direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. In this study, we compare the effects of S579 and S600 phosphorylation on purified Drp1, using phospho-mimetic mutants and in vitro phosphorylation. The oligomerization state of both phospho-mimetic mutants is 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 wild-type Drp1, both S579D and S600D phospho-mimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant that lacks GTPase activity, the K38A mutant, stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions, similar to previous results for dynamin-1. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly, and likely requires additional factors for stimulation of mitochondrial fission in cells. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.

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Liu, A., Hatch, A. L., Higgs, H.. 2023-08-20. Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin. https://doi.org/10.1101/2023.08.20.554022

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