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

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

Ronald A Merrill et al. FEBS J. 2013 Jan.

Abstract

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 protein kinase A-A kinase anchoring protein 1 complex. We further show that translocation of PP2A/Bβ2 to mitochondria is regulated by phosphorylation of Bβ2 at three N-terminal serines. Phosphomimetic substitution of Ser20, Ser21, and Ser22 renders Bβ2 cytosolic, blocks Drp1 dephosphorylation and mitochondrial fragmentation, and abolishes the ability of Bβ2 overexpression to induce apoptosis in cultured hippocampal neurons. Alanine substitution of Ser20-Ser22 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-Ser22 are available for phosphorylation, suggesting that PP2A/Bβ2 autodephosphorylation is necessary for OMM association, probably 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.

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Figures

Fig. 1
Fig. 1

Bβ2 can be phosphorylated on N-terminal residues in vitro and in intact cells. (A) N-terminal fragments of Bβ2 fused to GFP were in vitro phosphorylated with purified CaMKII and [γ-33P] ATP. Major 33P incorporation occurs between residues 20 and 26 (drop from 92% phosphorylation of Bβ21-26 to 18% of Bβ21-19). Percent phosphorylation (% phos) was determined by densitometry as the ratio of 33P to protein signals (Ponceau S total protein stain) relative to Bβ21-35 (100%). (B-D) Full-length Bβ1, Bβ2, and Bβ2 SSS20AAA (FLAG-tagged in (B), V5-tagged in (C)) or Bβ21-35-FLAG-GFP ((D), wild-type and SSS20AAA) was metabolically labeled with ortho-32P phosphate in transfected COS1 cells and immunoprecipitated. Cells expressing full-length regulatory subunits (B-C) were treated with the phosphatase inhibitor calyculin A (25 nM, 30 min) prior to lysis. % phos is the ratio of 32P to immunoblot signals relative to wild-type Bβ2. Bβ2 is more heavily phosphorylated than Bβ1, and Ser20-22 substitution reduces 32P incorporation into Bβ2.

Fig. 2
Fig. 2

N-terminal serines influence subcellular localization of Bβ2. (A,B) The indicated Bβ-GFP proteins (green) were expressed in HeLa cells, and colocalization with mitochondria (cytochrome oxidase II antibody, red) was determined by epifluorescence microcopy. Representative images (A) show that wild-type (WT) Bβ2 has a mixed cytosolic/mitochondrial distribution, whereas dephospho (SSS20AAA) and phospho (SS21DD) Bβ2 are largely mitochondrial and cytosolic, respectively. Colocalization with mitochondria is quantified in (B) as the Pearson’s coefficient (mean ± SEM of ca. 400 cells from typically three independent experiments). (C) HEK293 cells expressing the indicated GFP-tagged B subunits were fractionated into membrane and cytosolic proteins and immunoblotted for the indicated antigens. Percent mitochondrial localization (% mito.) of B subunits was calculated as the ratio of mitochondrial to total (mitochondrial plus cytosolic) signals normalized to input signals. Statistics: unpaired Student’s t-test compared to Bβ2 wild-type; **p < 0.01, ***p < 0.001.

Fig. 3
Fig. 3

Mitochondrial localization of PP2A/Bβ2 requires intrinsic phosphatase activity. (A) Space filling model of PP2A/Bα (PDB 3DW8) highlighting residues that align with Bβ2 residues examined in this study. (B,C) Colocalization of Bβ2-GFP with mitochondria in HeLa cells was assessed as in Fig. 2. The SSS20AAA mutation rescues mitochondrial targeting of catalytically impaired (K87A) Bβ2. (D) Immunoprecipitation shows that Bβ2 mutations do not affect association with the PP2A catalytic subunit. Statistics: unpaired Student’s t-test compared to wild-type Bβ2; *p < 0.05, ***p < 0.001.

Fig. 4
Fig. 4

N-terminal phosphorylation of PP2A/Bβ2 influences mitochondrial morphology and Drp1 S656 dephosphorylation. (A,B) Hela cells were transfected with Bβ2-GFP (WT, SS21AA, and SS21DD) and OMM-PKA, fixed, and immunofluorescently labeled for cytochrome oxidase II (red). Mitochondrial morphology was determined from epifluorescence micrographs (representatives in (A)) and is expressed as form factor (circular mitochondria = 1) ((B), mean ± SEM of 57, 24, and 29 cells). (C-F) Drp1 S656 phosphorylation levels were assessed by phospho-specific antibody in COS1 cell lysates expressing GFP-Drp1 and the indicated Bβ subunits. Phospho S656 Drp1 signals were boosted by PKA activation via forskolin (2-7.5μM) and rolipram (2 μM) treatment for 60 min prior to cell lysis. Cells in (E,F) additionally received vehicle or FK506 (2 μM, 60 min) to inhibit calcineurin. (C,E) show representative immunoblots and (D,F) show quantification as the ratio of phospho- to total Drp1 (mean ± SEM of 6 (D) and 5 (F) experiments). Statistics: (B,D) unpaired Student’s t-test compared to wild-type Bβ2; (F) one-way analysis of variance (ANOVA) followed by pairwise tests with Bonferroni adjustments; *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5
Fig. 5

PP2A/Bβ2 dephosphorylates Drp1 in vitro. (A,B) PP2A holoenzymes containing the indicated FLAG-GFP tagged regulatory subunits were immunoisolated from transfected COS1 and assayed for dephosphorylation of a model phosphopeptide (A) or GST-Drp1582-736 that had been in vitro 32P-phosphorylated on S656 by PKA (B, 15 and 45 min assay time). Raw phosphatase activities were adjusted for relative PP2A catalytic subunit levels obtained by immunoblotting (inset in (A)). Shown are means ± SEM of 5 experiments ((A), normalized to Bβ2) and means ± SEM of quadruplicate reactions from a representative experiment (B). Statistics: unpaired Student’s t-test compared to Bβ2; **p < 0.01.

Fig. 6
Fig. 6

N-terminal phosphorylation modulates PP2A/Bβ2 subcellular localization and survival in neurons. (A) Primary hippocampal neurons were transfected as indicated and GFP-positive neurons were imaged after labeling mitochondria with an antibody to TOM20 (mito, red). (B,C) Hippocampal neurons were scored for viability 72 h after transfection by examining nuclear morphology, neurite integrity, and propidium iodide (PI) exclusion. (B), representative images; (C) quantification of neuronal death normalized to wild-type Bβ2-induced death (~40%) as means ± SEM from 3-4 experiments. (D) Model (see discussion). Statistics: (B) one-way analysis of variance (ANOVA) followed by pairwise test with Bonferroni adjustments; *p < 0.05, **p < 0.01, ***p < 0.001.

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