N‐terminal phosphorylation of protein phosphatase 2A/Bβ2 regulates translocation to mitochondria, dynamin‐related protein 1 dephosphorylation, and neuronal survival

Abstract

Summary The neuron-specific Bβ2 regulatory subunit of protein phosphatase 2A (PP2A), a product of the spinocerebellar ataxia type 12 disease gene PPP2R2B, recruits heterotrimeric PP2A to the outer mitochondrial membrane (OMM) through its N-terminal mitochondrial targeting sequence. OMM-localized PP2A/Bβ2 induces mitochondrial fragmentation, thereby increasing susceptibility to neuronal insults. Here, we report that PP2A/Bβ2 activates the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) by dephosphorylating Ser656, a highly conserved inhibitory phosphorylation site targeted by the neuroprotective PKA/AKAP1 kinase complex. We further show that translocation of PP2A/Bβ2 to mitochondria is regulated by phosphorylation of Bβ2 at three N-terminal Ser residues. Phosphomimetic substitution of Ser20-22 renders Bβ2 cytosolic, blocks Drp1 dephosphorylation and mitochondrial fragmentation, and abolishes the ability of Bβ2 overexpression to induce apoptosis in cultured hippocampal neurons. Ala substitution of Ser20-22 to prevent phosphorylation has the opposite effect, promoting association of Bβ2 with mitochondria, Drp1 dephosphorylation, mitochondrial fission, and neuronal death. OMM translocation of Bβ2 can be attenuated by mutation of residues in close proximity to the catalytic site, but only if Ser20-22 are available for phosphorylation, suggesting that PP2A/Bβ2 autodephosphorylation is necessary for OMM association, likely by uncovering the net positive charge of the mitochondrial targeting sequence. These results reveal another layer of complexity in the regulation of the mitochondrial fission/fusion equilibrium and its physiological and pathophysiological consequences in the nervous system.

A Calcineurin Docking Motif (LXVP) in Dynamin-related Protein 1 Contributes to Mitochondrial Fragmentation and Ischemic Neuronal Injury

“…Recombinant GST-Drp1-(585-662) constructs immobilized on glutathione-agarose were prepared as described above and phosphorylated on Ser-656 with [␥-32 P]ATP and recombinant PKA (gift of Susan Taylor, University of California, San Diego) and used for phosphatase reactions as previously described (16).…”

“…24 h later cells were treated with the adenylate cyclase agonist forskolin (10 M) and the phosphodiesterase IV inhibitor rolipram (2 M) to stimulate PKA-dependent phosphorylation of Drp1 at Ser-656. Drp1 Ser-656 phosphorylation was then assessed as previously described (16).…”

“…Phosphorylation of Ser-656 is catalyzed by the cAMP-dependent protein kinase (PKA) (11,12) and has been shown to influence many interconnected facets of Drp1 activity including intermolecular assembly of larger Drp1 oligomers (13,14), GTPase activity (12), and translocation to the outer mitochondrial membrane (15). Two phosphatases have been identified to be active at Drp1 Ser-656, protein phosphatase 2A in complex with its regulatory subunit B␤2 (PP2A/B␤2) (16), and calcineurin (CaN) (11,15). In neurons, PP2A/B␤2-mediated dephosphorylation of Drp1 regulates neuronal maturation (4) and survival following injury (16,17).…”

“…Two phosphatases have been identified to be active at Drp1 Ser-656, protein phosphatase 2A in complex with its regulatory subunit B␤2 (PP2A/B␤2) (16), and calcineurin (CaN) (11,15). In neurons, PP2A/B␤2-mediated dephosphorylation of Drp1 regulates neuronal maturation (4) and survival following injury (16,17). In cardiac tissue, expression of CaN negatively correlates with phosphorylation of Drp1 Ser-656 and survival of cardiomyocytes following ischemic injury (18).…”

Mitochondrial dynamics in neuronal injury, development and plasticity

“…Furthermore, the importance of mitochondrial dynamics has been documented in neuronal development (Li et al, 2004;Dickey and Strack, 2011;Chan, 2006;Ishihara et al, 2009) and survival Merrill et al, 2013;Dagda et al, 2008;Merrill et al, 2011;Cho et al, 2010;Nakamura et al, 2010). However, also there, the specific mechanisms often await elucidation.…”