S. Strack, Department of Pharmacology, 2-432 BSB, University of Iowa, Iowa City, IA 52242, USA Fax: +1 319 335 8930 Tel: +1 319 384 4439 E-mail: firstname.lastname@example.org
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.
Mitochondria play a vital role in nearly every facet of eukaryotic life, and many mitochondrial functions including, ATP synthesis and calcium buffering, can be influenced by their size and shape. Mitochondrial shape is a product of the opposing processes of fission and fusion, which are carried out by a group of large GTPases of the dynamin superfamily . Dynamin-related protein 1 (Drp1) is an evolutionarily ancient motor protein that catalyzes the mitochondrial fission reaction. In analogy to dynamin, Drp1 is thought to form spirals around mitochondria that, upon GTP hydrolysis, constrict to sever the organelle . Drp1-mediated mitochondrial fission is essential in both mice and humans, and Drp1 activity is tightly regulated by several post-translational mechanisms [3–6]. A conserved and well-established regulatory site is Ser656, which, upon phosphorylation by protein kinase A (PKA), decreases Drp1 activity to elongate mitochondria by unopposed fusion [7,8]. We have recently reported that targeting of PKA to the outer mitochondrial membrane (OMM) by A kinase anchoring protein 1 (AKAP1) confers neuroprotection that depends on Drp1 Ser656 phosphorylation and elongation of mitochondria .
In opposition to PKA, the ubiquitously expressed calcium-dependent phosphatase calcineurin (CaN or protein phosphatase 2B) has been shown to activate Drp1 via dephosphorylation of Ser656 [8,10]. However, our previous work also implicated protein phosphatase 2A (PP2A) as an important regulator of mitochondrial shape and its physiological and pathophysiological sequelae [11,12]. Heterotrimeric PP2A consists of a dimer containing a scaffolding A subunit bound to a catalytic C subunit plus a variable regulatory B subunit. Encoded by 15 genes in mammals, regulatory B subunits direct the localization and substrate specificity of the heterotrimeric complex [13,14]. The Bβ gene (PPP2R2B) encodes two neuron-specific PP2A regulatory subunits, Bβ1 and Bβ2, and is associated with a debilitating neurodegenerative disease, spinocerebellar ataxia type 12. This disease is caused by a CAG repeat expansion within the promoter that drives expression of the Bβ1 variant . Bβ1 and Bβ2 differ only in their first exons, encoding 21 and 24 amino acids, respectively. The Bβ1 splice variant directs the heterotrimeric PP2A complex to the cytosol, whereas Bβ2 localizes PP2A to both cytosol and mitochondria . Bβ2 is recruited to mitochondria via its alternatively spliced N-terminus, with conserved basic and hydrophobic residues playing critical roles. Bβ21–24 acts as a cryptic mitochondrial import signal, as these residues promote matrix import and cleavage by signal peptidases when fused to green fluorescent protein (GFP). However, full-length Bβ2 is retained at the OMM via low-affinity interactions with receptor components of the translocase of outer membrane (TOM) complex, because Bβ2’s C-terminal β-propeller domain resists the partial unfolding step required for transfer through the TOM40 channel . Knockdown of Bβ2 in hippocampal neurons leads to elongation of mitochondria and confers neuroprotection in models of excitotoxic/ischemic injury. Conversely, overexpression of wild-type, but not OMM targeting-defective, PP2A/Bβ2 results in Drp1-dependent mitochondrial fission and increases basal rates of neuronal apoptosis [11,16]. PP2A/Bβ2 also plays a role in dendrite and synapse development, as knockdown of Bβ2 increases dendritic branch complexity but decreases synapse number in cultured hippocampal neurons. Bβ2 overexpression promotes synaptogenesis, but this effect could be blocked with pseudophosphorylated Drp1 (S656D mutant) . However, the question of whether PP2A/Bβ2 can dephosphorylate Drp1 was left unanswered.
We have previously shown that various cell stressors (rotenone, glutamate, and growth factor withdrawal) cause a dramatic redistribution of Bβ2 from the cytosol to the OMM in PC12 cells and hippocampal neurons . Here, we report on the mechanism of this translocation. We show that Bβ2 localization is determined by the phosphorylation state of its N-terminus, with phosphorylation of Ser20–Ser22 causing sequestration in the cytosol and autodephosphorylation promoting translocation of PP2A/Bβ2 to the OMM. We also provide evidence that Bβ2 N-terminal phosphorylation is neuroprotective, as it inhibits Drp1 dephosphorylation/activation, mitochondrial fragmentation, and neuronal death.
Bβ2 is phosphorylated on N-terminal serines
Mitochondrial import sequences generally carry a net positive charge that is important for translocation into the negatively charged mitochondrial matrix by an electrophoretic mechanism . The cryptic import sequence of Bβ2 (residues 1–24) includes one threonine, two tyrosines, and four serines, phosphorylation of which could neutralize the +3 charge of the N-terminus. In initial experiments, we fused Bβ2 N-terminal sequences of increasing length to the N-terminus of GFP, and isolated fusion proteins from transfected COS cells by immunoprecipitation. Bβ2–GFP fusion proteins were used as substrates in in vitro phosphorylation reactions with calcium/calmodulin-dependent kinase II and [γ-33P]ATP under conditions that favor promiscuous phosphorylation of nonconsensus sites. These experiments revealed that residues between positions 20 and 26 can be phosphorylated (Fig. 1A). To investigate whether Bβ2 is phosphorylated in intact cells, we immunoprecipitated the FLAG-tagged regulatory subunit from transiently transfected COS1 cells after metabolic labeling with 32PO43–. Bβ2 incorporated approximately twice as much 32P as the cytosolic N-terminal splice variant Bβ1, indicating that Bβ2 is phosphorylated at residues in the differentially spliced N-terminal tail and the common C-terminal β-propeller domain (Fig. 1B). Additional metabolic labeling experiments with mutant Bβ2 carrying alanines in place of Ser20–Ser22 confirmed phosphorylation of N-terminal residues (Fig. 1C). To obtain further evidence, we examined phosphorylation of the isolated Bβ2 N-terminus (Bβ21–35–GFP) in intact cells. The wild-type N-terminus was appreciably phosphorylated, but mutation of Ser20–Ser22 eliminated almost all 32P incorporation (Fig. 1D). 32P labeling of Bβ21–35–GFP could be detected without inhibition of protein phosphatases. In contrast, 32P incorporation into full-length Bβ2 (or Bβ1), which incorporates into the PP2A heterotrimer, required treatment with the cell-permeant PP1/PP2A inhibitor calyculin A (25 nm, 30 min) prior to cell lysis and immunoprecipitation. These results indicate that Bβ2 is phosphorylated on one or more of three N-terminal serines, but that these phosphates turn over rapidly, presumably because of autodephosphorylation by the PP2A holoenzyme.
N-terminal serines regulate the subcellular localization of Bβ2
To examine the functional consequence of Bβ2 phosphorylation, we mutated Ser20–Ser22 and Thr25 to alanines to mimic the unphosphorylated state of these amino acids. We then expressed Bβ2–GFP fusion proteins in HeLa cells, fixed cells for immunofluorescence labeling of mitochondria, and examined the colocalization of Bβ2–GFP with mitochondria by measuring Pearson’s coefficients (PCs) (PC = 1 is perfect colocalization). Wild-type Bβ2 colocalized well with mitochondria (PC = 0.46), whereas neutralization of a positive charge (R6A)  reduced PCs to levels similar to those of cytosolic Bβ1 (PC = 0.14; Fig. 2A,B). Analysis of single Thr→Ala and Ser→Ala substitutions in the Bβ2 N-terminus revealed that only S21A affected the localization of Bβ2, resulting in a small but significant increase in targeting to mitochondria (PC = 0.5; Fig. 2B). In contrast, alanine substitution of all three vicinal serines (SSS20 AAA) resulted in a robust increase of Bβ2 recruitment to mitochondria (PC = 0.64; Fig. 2A,B). Alanine substitution of Ser21 and Ser22 (SS21 AA) was nearly as effective (Fig. 4A, and data not shown). To provide complementary evidence for phosphorylation regulating Bβ2’s subcellular localization, Ser20–Ser22 were replaced with aspartic acid to mimic phosphoserine. Phosphomimetic substitution of two (SS21 DD) or three (SSS20 DDD) serines rendered Bβ2 completely cytosolic (PC approximately equal to that of Bβ1; Fig. 2A,B). As Bβ2 SSS20 DDD sometimes formed nonmitochondrial aggregates in cells, we instead analyzed Bβ2 SS21 DD in subsequent experiments.
To support the microscopy studies by biochemistry, we fractionated transfected HEK293 cells into cytosol and heavy membranes (including mitochondria), assessing fraction purity by immunoblotting for β--tubulin and TOM40. Twice as much Bβ2 as Bβ1–GFP or B′β–GFP was found in the heavy membrane fraction. Twenty per cent mitochondrial association is probably a low estimate for Bβ2, as the protein interacts weakly and transiently with receptor components of the TOM complex, and dissociates from mitochondria during the fractionation process [16,17]. Dephospho-Bβ2 (SSS20 AAA) displayed enhanced association with the membrane fraction, whereas pseudophospho-Bβ2 (SS21 DD) had a fractionation profile similar to cytosolic Bβ1 and B′β (Fig. 2C). Together, these observations suggest that serine phosphorylation counteracts positive charges in the N-terminal mitochondrial import sequence to maintain cytosolic localization of Bβ2.
Autodephosphorylation of the PP2A/Bβ2 N-terminus is necessary for OMM translocation
We reasoned that intrinsic PP2A/Bβ2 phosphatase activity may mediate dephosphorylation of the Bβ2 N-terminus. Guided by the crystal structure of PP2A/Bα, Bα residues Glu27 and Asp197 were identified as being critical for tau dephosphorylation by PP2A . Because these residues are conserved in Bβ2, we hypothesized that they might regulate activity-dependent subcellular distribution of the PP2A/Bβ2 holoenzyme. To address this hypothesis, we mutated these residues (Bβ2 E26R and D196K), as well as a third residue that forms the tip of a loop extending from Bβ2 to contact the catalytic site on the C subunit (K87A; Fig. 3A). If PP2A/Bβ2 mediated its own N-terminal dephosphorylation, then mutation of residues involved with the phosphatase reaction should result in reduced mitochondrial localization. Indeed, substitution of Glu26 and Lys87 resulted in a dramatic loss of mitochondrial localization of Bβ2; however, the D196K substitution had no effect (Fig. 3B,C).
If substrate-binding residues mediated mitochondrial localization of Bβ2 by allowing for dephosphorylation of the N-terminus, then dephospho-Bβ2 (SSS20 AAA) should be refractory to mutation of these residues. As expected, SSS20 AAA/K87A double-mutant Bβ2 displayed constitutive mitochondrial association indistinguishable from that of Bβ2 SSS20 AAA (Fig. 3B,C). Coimmunoprecipitation with the PP2A catalytic subunit confirmed that the Bβ2 mutations did not disrupt PP2A heterotrimer formation (Fig. 3D). These results suggest that autodephosphorylation of Ser20–Ser22 is necessary for OMM translocation of Bβ2. Autodephosphorylation could occur either within the same PP2A/Bβ2 heterotrimer (intracomplex) or between holoenzymes (intercomplex).
N-terminal phosphorylation of Bβ2 impacts on Drp1 dephosphorylation and mitochondrial shape
Mitochondrial fragmentation by Bβ2 overexpression requires recruitment of PP2A holoenzymes to mitochondria [11,12]. To determine the effects of the nonphosphorylatable and pseudophosphorylated Bβ2 mutants on mitochondrial morphology, we performed digital morphometric analyses of transfected and fixed HeLa cells. As compared with untransfected cells or cytosolic Bβ1, expression of wild-type Bβ2 resulted in a reduction in the form factor of mitochondria, indicative of enhanced mitochondrial fragmentation (Fig. 4A,B). Blocking Bβ2 phosphorylation with the SS21 AA substitution amplified this effect, whereas pseudophosphorylation of Bβ2 (SS21 DD) increased form factors to control levels, consistent with impaired mitochondrial fragmentation (Fig. 4A,B).
We recently reported that reversible phosphorylation of Drp1 at Ser656 regulates dendritic outgrowth and synapse formation in cultured hippocampal neurons . Epistasis experiments with Ser656-mutant Drp1 indicated that both mitochondrial fusion by the OMM-targeted PKA–AKAP1 complex and mitochondrial fission by PP2A/Bβ2 require Drp1 Ser656 [9,12]. To investigate whether PP2A/Bβ2 can dephosphorylate Ser656 of Drp1 in cells, we coexpressed in COS1 cells PP2A/B subunits along with a construct that replaces the endogenous Drp1 protein with a GFP-tagged version , allowing us to assess Drp1 phosphorylation in the transfected cell population only. To increase signals and thus allow for more robust quantification with the phospho-Ser656 antibody, Drp1 phosphorylation was boosted by treating cells with the adenylate cyclase agonist forskolin and the phosphodiesterase inhibitor rolipram 60 min prior to lysis. As compared with empty vector, expression of Bβ1 had no effect on Drp1 phosphorylation; however, wild-type Bβ2 reduced phospho-Ser656 Drp1 immunoreactivity by 34%. Rendering Bβ2 nonphosphorylatable (SSS20 AAA) and pseudophosphorylated (SS21 DD) augmented and abrogated, respectively, Drp1 dephosphorylation by Bβ2. Mutation of the lysine that contacts the catalytic site (K87A) also attenuated Bβ2 activity towards Drp1 Ser656 (Fig. 4C,D).
CaN was previously shown to dephosphorylate Drp1 at Ser656 [8,10]. Furthermore, mitochondrial dysfunction and fragmentation in cells deficient in PTEN-induced kinase 1, a mitochondrial protein kinase mutated in familial Parkinson’s disease, was proposed to enhance CaN-mediated Drp1 dephosphorylation via increased [Ca2+]i . To investigate whether Drp1 Ser656 dephosphorylation involves a PP2A/Bβ2 → CaN ‘phosphatase cascade’, we inhibited CaN pharmacologically in transfected COS1 cells. Treatment with the CaN inhibitor FK506/tacrolimus resulted in an overall increase in Drp1 Ser656 phosphorylation, but ectopic expression of Bβ2 still dephosphorylated Drp1 as compared with Bβ1 (Fig. 4E,F). These data indicate that CaN and mitochondria-targeted PP2A/Bβ2 dephosphorylate Drp1 independently.
We performed in vitro phosphatase assays to confirm that PP2A/Bβ2 can dephosphorylate Drp1 directly. To this end, PP2A complexes were immunoisolated from COS1 cells transfected with regulatory B subunits carrying C-terminal FLAG–GFP epitopes . With a model phosphopeptide substrate [RRA(pT)VA], rates of phosphate release were equivalent for Bβ1-containing and Bβ2-containing PP2A heterotrimers, but two times greater for PP2A/B′β after normalization for catalytic subunit levels (Fig. 5A). To assay Drp1 dephosphorylation, GST–Drp1582–736 was phosphorylated with PKA and [γ-33P]ATP at Ser656 . PP2A/Bβ1 and Bβ2 dephosphorylated phospho-Ser656 Drp1 equally well, whereas PP2A/B′β activity was fourfold lower (Fig. 5B). These results demonstrate that Drp1 Ser656 is a preferred substrate for PP2A heterotrimers containing Bβ (and perhaps other B-family) regulatory subunits. Because PP2A/Bβ2 is a better Drp1 phosphatase than PP2A/Bβ1 in intact cells, but not in vitro, we further conclude that the divergent N-terminus of Bβ2 mediates specific dephosphorylation of Drp1 via OMM localization of PP2A/Bβ2, rather than via direct substrate recognition.
Bβ2-induced neuronal death is modulated by N-terminal phosphorylation
We next assessed the ability of N-terminal phosphorylation to modulate mitochondrial localization of Bβ2–GFP in dendrites of primary hippocampal neurons 24 h after transfection. As compared with the mixed cytosolic/mitochondrial distribution of wild-type Bβ2, Bβ2 SS21 DD was largely excluded from areas of high mitochondrial content (Fig. 6A). In contrast, Bβ2 SSS20 AAA colocalized with dendritic mitochondria; most strikingly so in dying neurons with fragmented mitochondria (Fig. 6A, bottom right).
We previously showed that high-level overexpression of Bβ2 in cultured hippocampal neurons can induce apoptosis outright . We therefore investigated whether phosphorylation of N-terminal serines can modulate neuronal survival by scoring Bβ2–GFP-positive neurons for integrity of the nucleus and neurites. At 72 h post-transfection, expression of wild-type Bβ2 increased basal cell death by twofold (to ∼ 40%), whereas pseudophosphorylated Bβ2 (SS21 DD) was no more lethal than Bβ1. Conversely, dephospho-Bβ2 (SSS20 AAA) killed neurons more effectively than wild-type Bβ2 (Fig. 6B,C).
The goals of this study were to understand how translocation of PP2A/Bβ2 from the cytosol to mitochondria is regulated and whether Bβ2 translocation modulates Drp1 activity and neuronal survival. Metabolic labeling identified Ser20–Ser22 in the N-terminal mitochondrial targeting sequence of Bβ2 as phosphorylation sites in intact cells. Phosphomimetic substitutions of these residues: (a) maintain Bβ2 in the cytosol; (b) inhibit Drp1 dephosphorylation at Ser656; (c) attenuate mitochondrial fragmentation; and (d) abolish the proapoptotic activity of PP2A/Bβ2 in hippocampal neurons. As expression of constitutively dephosphorylated Bβ2 (SSS20 AAA) resulted in opposite, gain-of-function phenotypes, we conclude that reversible phosphorylation of Bβ2 at Ser20–Ser22 is an important regulatory mechanism in Drp1-mediated mitochondrial fission in neurons.
According to the model shown in Fig. 6D, PP2A/Bβ2 holoenzymes in healthy, unstressed neurons are sequestered in the cytosol via phosphorylation by as yet unidentified, prosurvival kinases. Upon cell stress, such as excitotoxic glutamate treatment or bioenergetic impairment with rotenone , Ser20–Ser22 phosphorylation levels drop to reveal the net positive charge of Bβ2’s mitochondrial signal sequence, allowing for accumulation of the PP2A holoenzyme near the TOM import complex, in turn driving Drp1 activation by dephosphorylation, mitochondrial fission, and ultimately cell death. Several examples of phosphorylation enhancing mitochondrial protein import have been reported [21–24]. To the best of our knowledge, the present study is the first to demonstrate inhibition of mitochondrial targeting by phosphorylation of a leader sequence. Whether stress-mediated dephosphorylation of the Bβ2 N-terminus is a consequence of kinase inhibition or enhanced intermolecular or intramolecular autodephosphorylation of the PP2A/Bβ2 holoenzyme is an important question that deserves further study.
We also provide evidence that a pivotal inhibitory phosphorylation site in Drp1, Ser656 [7,8], is targeted not only by CaN, but also by PP2A/Bβ2. PP2A/Bβ2-mediated Drp1 dephosphorylation is independent of CaN in intact cells, and both Bβ splice variants, but not a structurally unrelated PP2A regulatory subunit, efficiently dephosphorylate Drp1 Ser656 in vitro. Intriguingly, PP2A inhibition by okadaic acid or calyculin A does not increase Drp1 Ser656 phosphorylation levels in PC12 cells, which express most PP2A regulatory subunits, but not Bβ2 . As point mutations that block mitochondrial targeting of Bβ2 also prevent Drp1 dephosphorylation, we conclude that PP2A-mediated dephosphorylation and activation of Drp1 strictly depends on mitochondrial localization of PP2A by Bβ2. Because cytosolic (Bβ1) and mitochondrial (Bβ2) splice forms mediate equally efficient Drp1 dephosphorylation in vitro, local concentration at the OMM most likely accounts for the specific dephosphorylation of Drp1 by the PP2A/Bβ2 holoenzyme in vivo.
Whereas PP2A/Bβ2 increases susceptibility to neuronal insults, as shown by knockdown and overexpression approaches , the phosphatase also plays important physiological roles, stimulating mitochondrial division to foster synaptogenesis and curb dendritic hyperplasia in cultured hippocampal neurons . Regulation of OMM translocation of PP2A/Bβ2 by N-terminal phosphorylation therefore provides neurons with temporal and spatial control of mitochondrial morphogenesis in development, plasticity, and survival.
Reagents and cDNA constructs
The following commercial antibodies were used: mouse anti-GFP IgG2a clone 86/8 (NeuroMab, Davis, CA, USA), rabbit anti-GFP IgG (ab290; Abcam, Cambridge, MA, USA), mouse anti-MTCO2 Ig (cytochrome oxidase subunit II; Neomarkers, Fremont, CA, USA), mouse anti-Drp1 Ig (BD Transduction Laboratories, San Jose, CA, USA), mouse anti-β-tubulin Ig (E7; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), and rabbit anti-TOM40 Ig (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse anti-phospho-Ser656 Drp1 Ig was raised at the Iowa State Hybridoma Facility . For immunofluorescence staining, we purchased Alexa fluorophore-coupled secondary antibodies (Invitrogen, Grand Island, NY, USA). For quantitative immunoblot analysis, infrared fluorophore-coupled secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA). The GFP–Drp1 , Bβ1–FLAG–GFP, Bβ2–FLAG–GFP, B′β–FLAG–GFP [16,17,25] and OMM-PKA  vectors have been described previously. Bβ2 point mutants were generated by PCR-based methods, taking advantage of a nearby, unique EcoRI site.
In vitro phosphorylation and metabolic labeling
For in vitro assays, COS1 cells were transiently transfected with Bβ2 N-terminus–FLAG–GFP fusion proteins, using Lipofectamine 2000 . After 48 h, proteins were immunoisolated with FLAG-directed antibodies  and in vitro phosphorylated (30 min, 30 °C) with 0.5 μm CaMKIIα and 2 μm calmodulin (kind gift of R. Colbran, Vanderbilt University) in buffer containing 25 mm Hepes (pH 7.4), 2 mm CaCl2, 10 mm MgCl2, 0.2 μCi·μL−1 [γ-33P]ATP, and 200 μm unlabeled ATP. Kinase reactions were stopped with 25 mm EDTA and analyzed by SDS/PAGE, total protein staining, and phosphorimaging.
For metabolic 32P-labeling, COS1 cells were transfected with Bβ splice variants carrying C-terminal FLAG or V5 epitopes or Bβ21–35–FLAG–GFP. Twenty-four hours later, the cells were washed once with phosphate-free RPMI (MP Biomedical) and incubated in medium containing phosphate-free RPMI, 1% dialyzed fetal bovine serum and 0.5 mCi·mL−1 32P-labeled orthophosphate (PerkinElmer, Waltham, MA, USA) for 3.5 h at 37 °C. When full-length Bβ was expressed, 25 nm calyculin A was added to the medium for the last 30 min. Cells were washed with phosphate-free RPMI, and Bβ21–35–GFP was immunoprecipitated with antibodies against FLAG or V5 in the presence of phosphatase inhibitors, as described previously . Immunoprecipitates were analyzed by phosphorimaging and by immunoblotting for GFP, FLAG or V5 tags.
Subcellular fractionation and Drp1 Ser656 phosphorylation analysis
For subcellular fractionation, HEK293 cells expressing GFP-tagged B subunit were permeabilized in 0.5 mg·mL−1 digitonin, 20 mm Hepes (pH 7.4), 1 mm EDTA, 1 mm EGTA, 1 mm benzamidine, 5 mg·mL−1 leupeptin, 1 mm dithiothreitol, and 1 mm phenylmethanesulfonyl fluoride. Lysates were centrifuged (10 min, 20 000 g, 4 °C) to separate the cytosolic and heavy membrane fractions, essentially as described previously .
To quantify Drp1 phosphorylation in cells, COS cells were cotransfected with GFP-tagged B subunits or empty vector and GFP–Drp1 at a 1 : 1 plasmid mass ratio, using Lipofectamine 2000. The GFP–Drp1 plasmid replaces endogenous Drp1 with the GFP-tagged protein by coexpression of RNA interference-resistant cDNA and H1 promoter-driven small hairpin RNA . After 24 h, cells were treated with forskolin/rolipram (10 : 1 μm, 1 h) to upregulate PKA activity, lysed in SDS sample buffer containing 2 mm EDTA and 1 mm microcystin, and sonicated with a probe tip to shear DNA.
Subcellular fractions or total cell lysates were resolved on 10% polyacrylamide gels and transferred to nitrocellulose membrane. Blots were probed with primary and infrared fluorophore-labeled secondary antibodies, and bands were visualized with a LI-COR Odyssey infrared fluorescence scanner. Band intensities were quantified with the imagej gel analysis macro set (National Institutes of Health). Phosphorylation of GFP–Drp1 was quantified as the ratio of phospho-Ser656 Drp1 antibody immunoreactivity  to GFP antibody immunoreactivity of the same band.
Mitochondrial colocalization and morphology analysis
HeLa cells were cultured on No. 1 cover glasses (20-mm2 chamber; Nalgen Nunc, Thermo Scientific, Rochester, NY, USA) and transfected with LipofectAmine 2000. Cells were fixed with 4% paraformaldehyde and subjected to immunofluorescence staining with antibodies against cytochrome oxidase II and GFP, as reported previously . Images were captured at × 630 magnification with an epifluorescence microscope (Leica Microsystems, Wetzlar, Germany), and processed for local contrast enhancement (contrast-limited adaptive histogram equalization) with imagej. Images subjected to red/green colocalization analysis underwent a second processing step involving iterative 2D deconvolution (macro code and algorithm parameters are available upon request). Colocalization of GFP-tagged Bβ subunits with mitochondria (PC) was quantified with the JaCoP plug-in for imagej. To quantify mitochondrial morphology, mitochondria channel images were analyzed with a custom imagej macro described previously [9,26].
GST–Drp1 purification, in vitro phosphorylation, and phosphatase assays
A plasmid expressing GST–Drp1582–736 was created by ligation of a human Drp1 (NCBI accession no. NP_036192) PCR fragment into pGEX-6P1 digested with BamHI and XhoI. GST–Drp1 was expressed in Escherichia coli BL21/DE3 and purified on glutathione–agarose according to standard protocols. GST–Drp1582–736 (13.5 μm) was specifically phosphorylated on Ser656 (30 min, 30 °C) with 0.27 μm PKA catalytic subunit (kind gift of S. Taylor, University of California at San Diego) in buffer containing 20 mm Tris (pH 7.5), 10 mm MgCl2, 0.1 μCi·μL−1 [γ-33P]ATP, 100 μm unlabeled ATP, and 1 mm benzamidine. Reactions were arrested by the addition of 25 mm EDTA, and free ATP was removed by two passages through desalting columns (Zeba; Thermo Scientific, Rochester, NY, USA).
PP2A holoenzymes containing transfected and epitope-tagged regulatory subunits complexed with endogenous A and C subunits were immunoisolated from COS1 cells as described previously . Phosphatase complexes on agarose beads were resuspended in reaction buffer containing 50 mm Tris (pH 7.5), 0.1% Triton X-100, 2 mm EDTA, 2 mm EGTA, 1 mm benzamidine, 0.05%β-mercaptoethanol, and 2 mg·mL−1 BSA. Phosphatase reactions were started by the addition of 32P-labeled GST–Drp1582–736, and incubated at 30 °C with intermittent agitation. Reactions were stopped at 15 and 45 min by addition to a 40% trichloroacetic acid solution for a final concentration of 20%. After centrifugation at 22 000 g, acid-soluble 32P was quantified by liquid scintillation counting. For peptide dephosphorylation experiments, phosphatase activities towards a phosphopeptide derived from myosin light chain [RRA(pT)VA, 100 μm] were determined with a molybdate/malachite green-based colorimetric assay (Promega, Madison, WI, USA).
Mitochondrial localization and survival assays in hippocampal neurons
Hippocampal neurons from embryonic day 18 rat embryos were cultured in Neurobasal medium with B27 supplement (Invitrogen, Grand Island, NY, USA), transfected with GFP-tagged regulatory B subunits with LipofectAmine 2000 (0.1%, 2 μg·mL−1 DNA) at 10–14 days in vitro, and fixed with 3.7% paraformaldehyde 24 or 72 h later. For the localization experiments, cultures were immunofluorescently labeled for GFP to enhance the intrinsic fluorescence of the Bβ–GFP proteins, and for the mitochondrial protein TOM20. Neurons were imaged at × 1000 magnification with a Leica epifluorescence microscope. For survival assays, hippocampal cultures were fixed 72 h after transfection, and labeled with GFP-directed antibodies and Alexa 488-coupled secondary antibodies. Nuclei were stained with 1 μg·mL−1 Hoechst 33342. In some experiments, cultures were also incubated with propidium iodide (1 μg·mL−1) 5 min prior to fixation, in order to stain the nuclei of necrotic cells that had lost membrane integrity. Death was quantified as the percentage of transfected neurons with condensed, irregular or fragmented nuclei or dystrophic neurites by experimenters blinded to transfection conditions.
The unpaired Student’s t-tests were performed with Excel. The one-way ANOVA and the pairwise tests with Bonferroni adjustments were carried out with the statistical software package r . Unless indicated otherwise, data are representative of three or more independent experiments.
This work was supported by National Institute of Health grants NS043254, NS056244 and NS057714 (to S. Strack) and National Research Service Award Predoctoral Fellowship NS077563 (to A. M. Slupe).