Research ArticleSynaptic Plasticity

Pin1 and PKMζ Sequentially Control Dendritic Protein Synthesis

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Science Signaling  09 Mar 2010:
Vol. 3, Issue 112, pp. ra18
DOI: 10.1126/scisignal.2000451

Abstract

Some forms of learning and memory and their electrophysiologic correlate, long-term potentiation (LTP), require dendritic translation. We demonstrate that Pin1 (protein interacting with NIMA 1), a peptidyl-prolyl isomerase, is present in dendritic spines and shafts and inhibits protein synthesis induced by glutamatergic signaling. Pin1 suppression increased dendritic translation, possibly through eukaryotic translation initiation factor 4E (eIF4E) and eIF4E binding proteins 1 and 2 (4E-BP1/2). Consistent with increased protein synthesis, hippocampal slices from Pin−/− mice had normal early LTP (E-LTP) but significantly enhanced late LTP (L-LTP) compared to wild-type controls. Protein kinase C ζ (PKCζ) and protein kinase M ζ (PKMζ) were increased in Pin1−/− mouse brain, and their activity was required to maintain dendritic translation. PKMζ interacted with and inhibited Pin1 by phosphorylating serine 16. Therefore, glutamate-induced, dendritic protein synthesis is sequentially regulated by Pin1 and PKMζ signaling.

Introduction

Long-term memory and long-lasting forms of synaptic plasticity, such as late-phase long-term potentiation (L-LTP), require the synthesis of new protein on dendritic polyribosomes (13) at or near activated synapses (1). By contrast, short-term forms of synaptic plasticity, such as early-phase LTP (E-LTP), are independent of protein synthesis. Multiple dendritic kinases, including calcium/calmodulin-dependent protein kinase II (CaMKII), mitogen-activated protein kinase (MAPK), mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK1), protein kinase C ζ (PKCζ), and protein kinase M ζ (PKMζ), have been implicated in the induction or maintenance of hippocampal L-LTP (47). Blockade of PKC, MEK1, or MAPK prevents the induction of L-LTP and the initial phases of learning and memory consolidation (8, 9). In contrast, PKMζ is essential for the maintenance of L-LTP and the persistence of spatial memory storage in the hippocampus (7, 8, 10). PKMζ is an atypical PKC isoform that is unique in lacking an autoinhibitory, pseudosubstrate regulatory domain (11) and is activated by phosphoinositide-dependent protein kinase-1 (PDK1) (12, 13). How PKMζ maintains L-LTP and spatial memory is largely unknown but may involve AMPA-type glutamate receptor (AMPAR) phosphorylation and trafficking, with subsequent changes in excitatory postsynaptic potential (EPSP) amplitude, and likely requires ongoing dendritic translation (14, 15).

The regulation of dendritic translation is predominantly at the level of initiation through eukaryotic translation initiation factor 4E (eIF4E), the component of the multisubunit eIF4F complex that binds to the 5′ methylguanosine cap of messenger RNAs (mRNAs) (16). eIF4F assembly is inhibited through direct interaction of eIF4E with partially phosphorylated eIF4E binding proteins (4E-BPs), preventing cap-dependent translation (16, 17). Additional phosphorylation of 4E-BPs on Thr37 or Thr46 by mammalian target of rapamycin [mTOR, also known as FKBP-12-rapamycin–associated protein (FRAP)], S6 kinase p70 (S6K), MAPKs, or PKC causes release of 4E-BP1 from eIF4E, leading to the subsequent assembly of the translation initiation complex (719). Both Thr37 and Thr46 are immediately N-terminal to a proline and are thus potential recognition sites for Pin1 (protein interacting with NIMA 1) (20), a peptidyl-prolyl isomerase (PPIase). Pin1, which is highly abundant in the cytoplasm and nucleus of neurons (2022), is composed of an N-terminal type IV WW domain connected by a flexible linker to a C-terminal PPIase domain. The WW domain mediates specific interactions with target substrates containing dipeptide Ser-Pro or Thr-Pro motifs, distinguishing its actions from those of the related but less selective PPIases cyclophilinA and FK506-binding protein (FKBP) (20). Pin1 preferentially isomerizes the Ser/Thr-Pro peptide bond after phosphorylation of Ser or Thr, leading to alterations in target protein conformation and biological activity (20, 22). Pin1 has been linked to cellular transformation and tumorigenesis, apoptosis, neurodegeneration, and the transcriptional regulation of cytokines (2224).

FKBPs and cyclophilins have been implicated in protein synthesis in dendrites and in L-LTP (2528). FKBP12 binds to mTOR, the inhibition of which with rapamycin inhibits dendritic protein synthesis and LTP (19, 25, 28). Deletion of FKBP in rodents leads to enhanced L-LTP that is resistant to rapamycin but sensitive to inhibitors of protein synthesis (25). Knockout of cyclophilinD improves learning, memory, and synaptic function in a mouse model of Alzheimer’s disease (26, 27). Thus, PPIases likely play a role in plasticity and memory by regulating the initiation of dendritic protein synthesis.

We now demonstrate that Pin1 is present and constitutively active in dendritic shafts and spines, where it interacts with 4E-BP and eIF4E and, under basal conditions, suppresses protein synthesis. Pin1 activity was rapidly decreased by glutamatergic signaling. Genetic ablation or pharmacologic inhibition of Pin1 increased dendritic translation. Both PKMζ and the related PKCζ increased in abundance in dendrites after Pin1 inhibition or knockout and functioned in feedforward loops to maintain translation and feedback loops to inhibit Pin1 activity. Hippocampal slices from Pin1−/− mice displayed normal E-LTP but enhanced protein synthesis–dependent L-LTP. These results implicate Pin1 and PKMζ in the regulation of glutamate-induced dendritic protein synthesis and clarify how PKMζ functions to modulate synaptic plasticity.

Results

Pin1 is present and catalytically active in dendrites

Pin1 has been identified in the nucleus and cytoplasm of neurons (21, 22, 29) but not in dendritic shafts or spines. Immunofluorescence analysis of hippocampal sections revealed strong Pin1 staining in CA1, CA3, and the dentate gyrus (Fig. 1A). Synaptoneurosomes (SNs, resealed vesicles containing pre- and postsynaptic structures) prepared from total mouse cortex were positive for Pin1 by Western blot (Fig. 1B). SNs include ribosomes, as well as functional glutamate receptors, making them a convenient and powerful system to study dendritic protein synthesis (30, 31). Confocal analysis of cultured cortical neurons prepared from the brains of wild-type embryonic day 17 to 18 (E17 to E18) mouse embryos showed punctate Pin1 staining along dendritic shafts, which colocalized with postsynaptic density protein 95 (PSD95) (Fig. 1C). Immunoblot (Fig. 1B) and immunofluorescence (fig. S1A) analyses of Pin1−/− brain tissue were negative, indicating that the antisera were specific for Pin1. These results suggest that Pin1 is present in dendritic spines and shafts of rodent neurons and that SNs are markedly enriched for pre- and postsynaptic structures.

Fig. 1

Pin1 is present and active in postsynaptic terminals. (A) Representative 100× confocal images of 30-μm hippocampal slices from P21 to P25 mice immunostained with antibodies against PSD95-rhodamine (anti–Pin1-rhodamine) and Pin1-FITC (anti–Pin1-FITC), and To-Pro3. The merge panel shows uncolocalized (Pin1-red, PSD95-green, and To-Pro3-blue) and colocalized (yellow and magenta) points, whereas the colocalization panel shows only points of Pin1 and PSD95 colocalization (yellow). (B) Pin1 and SNAP25 were detected by Western blot of 10 μg of SN protein from wild-type (WT) and Pin1−/− mice. (C) Representative confocal image (600x) of E17 cortical neuron dendrites, 18 DIV. Cells were labeled with anti–Pin1-rhodamine and anti–PSD95-Cy5 to identify colocalization (magenta). (D) Electron microscopy of SNs stained with anti-Pin1 coupled to gold beads (63). Left and center: vesicles (scale bars, 1 μm left; 200 nm, center) containing pre- and postsynaptic terminals. Right: representative region (scale bar, 500 nm) stained with anti–Pin1-gold. Open arrows denote postsynaptic densities. Long, narrow arrows denote Pin1 staining. Small, narrow arrows denote synaptic vesicles. (E) SNs were untreated (□) or pretreated with 1 μM juglone (⋄) or 1 μM CsA (○) for 10 min before lysis and isomerase activity assay. No SN control (x) contained complete reaction except SN, n = 3, ± SEM; *P < 0.001. Initial slopes were calculated to determine K (see Table 1).

To precisely identify the subcellular location of Pin1, we performed immunoelectron microscopy. SNs from Pin1+/+ mice [C57BL6 background, age postnatal day 12 to 22 (P12 to P22)] were isolated by Percoll gradient centrifugation, fixed, and stained with antibody directed against Pin1 (anti-Pin1) coupled to gold beads. As typical for SN preparations, various organelles, including mitochondria, were present along with the resealed vesicles, which contained pre- and postsynaptic terminals (Fig. 1D). Immunogold reactivity in vesicles was most pronounced postsynaptically, with much weaker presynaptic staining (Fig. 1D, right panel). In contrast, mitochondria failed to stain with anti-Pin1. We thus conclude that Pin1 is present in dendritic shafts and spines in rodent cortex and hippocampus and is preferentially located in the postsynaptic region.

Pin1 activity can be measured in SNs or cell lysates by means of a protease-coupled, cis-to-trans isomerization assay (24, 32, 33). Conversion of the peptide substrate (Suc-Ala-Glu-Pro-Phe-pNA) from cis to trans permits cleavage of the C-terminal nitroaniline by chymotrypsin and detection at 390 nm. Untreated SNs contained substantial basal PPIase activity (Fig. 1E and Table 1). Although Suc-Ala-Glu-Pro-Phe-pNA is isomerized by Pin1 (21, 3335), other PPIases, including cyclophilinA, may also act on it. Therefore, we performed isomerase assays on SNs in the presence of the selective PPIase inhibitors cyclosporin A (CsA), FK506, and juglone. We also tested the effects of a membrane-permeable, catalytically inactive form of Pin1 created by fusion of a transactivator of transcription (TAT) penetrating tag to the N terminus and substitution of lysine 63 with alanine (TAT-Pin1-K63A). CsA inhibits cyclophilinA, FK506 blocks FKBP, and juglone and TAT-Pin1-K63A specifically inhibit endogenous Pin1 by covalent modification and prevention of substrate access, respectively (32, 3436). Neither CsA (Fig. 1E and Table 1, set A) nor FK506 (Table 1, set A) reduced the rate of substrate isomerization. However, TAT-Pin1-K63A or the Pin1 inhibitor juglone (32) dose-dependently inhibited PPIase activity (Fig. 1E and Table 1, set B). Recombinant TAT-Pin1-K63A also dose-dependently prevented recombinant WT-Pin1 from isomerizing substrate, supporting the conclusion that the former can function as a dominant-negative form of Pin1 (fig. S2E). Therefore, we conclude that constitutively active Pin1 is present in dendrites and that the observed isomerization of target substrate is independent of cyclophilinA or FKBP. Thus, unlike the immune system where Pin1 is inactive in unstimulated cells (24), dendritic Pin1 shows increased activity under basal conditions.

Table 1

PPIase activity in SN is mediated by Pin1 and regulated by glutamate and PKCζ/PKMζ. SNs were untreated (untreated control) or pretreated with the indicated drugs before lysis and assay of isomerase activity. The initial slopes were calculated to determine K [K = kobsk0/k0 (36)]. No SN control (k0) contained the complete reaction mixture including protease but excluding SNs, n = 3, ± SEM. Individual groups are compared to no SN control (P valuec) in all cases; P compared to CsA, β-Gal, or myr-peptide controls is shown as appropriate. Set A: SNs were untreated (control) or pretreated with 1 μM juglone or 1 μM CsA for 10 min. Set B: SNs were untreated (control) or incubated with 1 μM CsA for 5 min alone or before the addition of TAT-Pin1-K63A or TAT-β-Gal for an additional 5 min. Set C: SNs were untreated or treated with Glu for 30 min. Set D: SNs were untreated or treated with 10 μM myr-PKC/PKMζ or myr-control peptide for 5 min.

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Pin1 inhibits protein synthesis and is itself inhibited by glutamate stimulation

Dendritic translation is required for the induction of L-LTP and for memory consolidation, which presumably reflects a requirement for the creation of new proteins that underlie synaptic remodeling (37). We modeled in vivo events by evaluating SN translation by measuring [35S]methionine ([35S]Met) incorporation into protein after addition of glutamate. SNs were translationally active, producing a broad spectrum of proteins, the abundance of which increased significantly in response to treatment with 50 μM glutamate and 10 μM glycine (Glu) (Fig. 2, A and B) and decreased in response to cycloheximide (fig. S2A), and which were not apparent after treatment with anisomycin (Aniso) (Fig. 2, A and B). Translation was progressively reduced after treatment with increasing concentrations of Triton X-100, which is consistent with the notion that protein synthesis occurred within a membrane-encased organelle (fig. S2D). The rate of protein synthesis in unstimulated SNs transduced with dominant-negative TAT-Pin1-K63A was nearly identical with that in untransduced SNs treated with Glu (Fig. 2, A and B). Similar effects were seen after treatment of SNs with the Pin1 inhibitor juglone (fig. S2B). We also evaluated protein synthesis in SNs from Pin1−/− mouse brains at P16 to P22. SNs isolated from Pin1−/− mice showed twice the basal rate of [35S]Met incorporation than did those from Pin1+/+ littermate controls (Fig. 2, C and D). Moreover, glutamate had no effect on translation in Pin1−/− SNs. Thus, these results suggest that Pin1 normally suppresses dendritic translation, an inhibition that can be overcome through glutamate-mediated signaling or Pin1 ablation.

Fig. 2

Pin1 suppresses dendritic translation. (A) SNs were preincubated for 15 min without (lanes 1 to 4) or with 40 μM anisomycin (Aniso, lanes 5 to 8) before addition of [35S]Met and no additional treatment, transduction with 50 nM TAT-β-Gal (β-Gal) or 50 nM TAT-Pin1-K63A (K63A), or treatment with Glu for 30 min before lysis and SDS-PAGE. (B) Total [35S]Met incorporation into protein was quantitated by phosphorimaging. Relative translation compared to control SN (□) was 1.25 ± 0.03 for β-Gal, 1.59 ± 0.09 for K63A and 1.62 ± 0.01 for Glu, 0.58 ± 0.06 for Aniso (▪), 0.58 ± 0.07 for Aniso plus β-Gal, 0.55 ± 0.07 for Aniso plus K63A, 0.54 ± 0.12 for Aniso plus Glu, n = 3, ± SEM, *P < 0.019 between K63A and β-Gal; P < 0.003 between K63A and control. (C) Wild-type (WT) or Pin1 KO SN were untreated (Control) or treated with Glu for 30 min. Total [35S]Met incorporation was quantitated (D). n = 3, ± SEM, *P < 0.04.

Glutamatergic signaling could activate dendritic translation by inducing Pin1 catabolism or by suppressing Pin1 PPIase activity. Western analysis revealed no significant changes in the abundance of Pin1 after glutamate stimulation of SNs (fig. S2C), although Pin1 abundance significantly increased in dendritic spines of cultured cortical neurons treated with glutamate (Fig. 3B and fig. S3A), demonstrating Pin1 can be locally synthesized in response to glutamatergic signaling. However, PPIase activity was significantly reduced in Glu-treated SNs compared to that in untreated controls (Table 1, set C). The degree of Glu-induced suppression of Pin1 isomerase activity and stimulation of Pin1 translation was comparable to that seen after TAT-Pin1-K63A transduction (Table 1, set B, and Fig. 2A). Therefore, Pin1 appears to repress translation under basal conditions through mechanisms that require its isomerase activity. Glutamatergic signaling rapidly suppresses Pin1 activity as a prerequisite for translational up-regulation.

Fig. 3

Pin1 associates with signaling proteins, and Pin1−/− hippocampal slices show increased L-LTP. (A) Wild-type brain homogenates (600 μg) were lysed and immunoprecipitated with pre-immune IgG, anti-Pin1, or anti–4E-BP1 followed by immunoblot, n = 3. Homogenate (50 to 200 μg, input) was used as a positive control. (B) E17 cortical neurons, DIV18, were untreated (Control), stimulated with Glu, or pretreated with 40 μM Aniso before Glu, and then fixed, stained, and the dendrites visualized. The fluorescence of ~300 random puncta was quantitated and SEM was determined. (C) L-LTP was induced in Pin1−/− (O; n = 11 slices, n = 3 mice) or Pin1+/+ (•; n = 9 slices, n = 3 mice, P = 0.0295) hippocampal slices with four trains of high-frequency stimulation. (D) L-LTP was induced in anisomycin-treated hippocampal slices from Pin1+/+ [•; n = 12 slices, n = 3 mice, P = 0.0053 (WT with and without Aniso)] and Pin1−/− [O; n = 11 slices, n = 3 mice, P = 0.0003 (KO with and without Aniso)].

Pin1 interacts with 4EBP1/2 and eIF4E, but not with mTOR or p70-S6K

4E-BP1 and 2 (4E-BP1/2), mTOR, ribosomal p70-S6K, and eIF4E all contain Ser/Thr-Pro sites that can potentially be recognized and isomerized by Pin1. Immunoprecipitation of cortical lysates with antibodies directed against Pin1 or 4E-BP1 followed by immunoblot revealed that Pin1 interacts with 4E-BP1/2 and eIF4E (Fig. 3A) but not p70-S6K, mTOR, or MEK1 (fig. S3B). The interaction of Pin1 with 4E-BP1/2 was unaffected by brief (10 min) treatment of SN with Glu, rapamycin, or a combination of the two (fig. S3C). To visualize the location of the interactions between Pin1 and translational regulators, we analyzed cultured cortical neurons prepared from E17 embryos by confocal microscopy. Pin1 was present in a punctate pattern along dendrites, where its abundance was modestly increased by Glu and decreased by Aniso (Fig. 3B and fig. S3A). eIF4E, 4E-BP1/2, and p70-S6K displayed similar staining patterns along the dendritic shafts and spines as Pin1 (fig. S3A). Overlay of the images with ImageJ (38) demonstrated considerable colocalization of Pin1 with eIF4E, 4E-BP1/2, and p70-S6K, which was decreased after Aniso treatment (fig. S3A). These results demonstrate that Pin1 is present in dendritic shafts and spines, where it is associated with 4E-BP1/2 and eIF4E, known regulators of global protein synthesis, suggesting that Pin1 modulates these proteins to influence translation initiation.

Pin1−/− mice show enhanced L-LTP

Protein synthesis, especially in dendrites, is essential for the formation of long-term memory and the maintenance of long-term forms of synaptic plasticity, such as L-LTP (2, 9). Thus far, our data indicate that Pin1 is a mediator of glutamate-induced dendritic protein synthesis. Therefore, we investigated the possibility that Pin1 was involved in LTP. Although basal synaptic transmission, as defined by the field EPSP (fEPSP) slope versus amplitude of the presynaptic volley, were equivalent in hippocampal slices from wild-type and Pin1−/− mice (fig. S4A), paired-pulse facilitation, a form of short-term synaptic plasticity, was enhanced at 10- and 20-ms interstimulus intervals in Pin1−/− slices (10-ms interstimulus interval, ***P < 0.001; 20-ms interstimulus interval, **P < 0.01) (fig. S4B), indicating that Pin1 may affect neurotransmitter release. Next we evaluated the role of Pin1 during a protocol designed to induce L-LTP (four high-frequency trains of stimuli). Slices isolated from Pin1−/− animals showed a similar degree of LTP induction compared to those from wild-type mice, and showed a similar fEPSP slope for the first 60 min (Fig. 3C). After 1 hour, Pin1−/− slices showed a significant increase in fEPSP slope compared to that in wild-type slices [P = 0.0214; repeated-measures analysis of variance (ANOVA), 60 to 180 min after stimulation], which was prevented by protein synthesis inhibitors (Fig. 3D). In contrast, when L-LTP was induced with a theta burst stimulation (TBS) protocol, we found no significant differences between wild-type and knockout (KO) slices in LTP induction or the magnitude of the fEPSP slope (fig. S4C). The differences found in Pin1 KO slices between the two stimulation protocols may reflect the different biochemical pathways induced by high-frequency stimulation and TBS (3942). In addition, our results are distinct from those observed in mice lacking 4E-BP2, the eIF2a kinase GCN2, or MEK1. In these mice, a single stimulus that normally leads to E-LTP induces plasticity that mimics L-LTP (9, 43, 44).

PKMζ and PKCζ are increased in abundance in Pin1−/− mice

Multiple kinases (PKC, MEK1, MAPK, PKCζ, and PKMζ) and translational regulators (4E-BP2 and GCN2) have been implicated in the induction or maintenance (or both) of L-LTP (810, 12, 43, 44) or persistent memory storage (45). Given the increased maintenance of L-LTP we observed in the Pin1−/− hippocampal slices, we evaluated PKCζ and PKMζ abundance in brain lysates. Western blot revealed that the abundance of both PKC isoforms was increased by up to 200% in the cortex and hippocampus (Fig. 4A) of Pin1−/− mice compared to that in the cortex and hippocampus of wild-type mice. In contrast, mGluR1, GABAβR1, and actin abundances were unchanged (Fig. 4A). PKMζ, which is predominantly expressed in the hippocampus (46), rapidly increases by ~50% after the induction of L-LTP in wild-type hippocampal slices (15, 45).

Fig. 4

PKCζ/PKMζ show increased abundance in Pin−/− brain and control Pin1 activity through Ser16 phosphorylation. (A) Cortex (C) and hippocampus (H) were dissected from 4 week-old wild-type (WT) and Pin1−/− mouse brains and lysates were analyzed by Western blot. (B) WT and Pin1 KO SNs were pretreated with 10 μM myr-control or myr-PKCζ/PKMζ inhibitor peptides and left untreated or treated with Glu for 30 min. Total [35S]Met incorporation was quantitated. *P < 0.018. (C) Upper panel: isomerase activity of recombinant TAT-Pin1 (1 μg) plus 1 μM juglone or plus 400 ng of TAT-PKMζ and ATP, after a 2-min preincubation; n = 3, ± SEM, *P < 0.01. Lower panel: isomerase activity of recombinant TAT-Pin1, TAT-Pin1-S16A, and TAT-Pin1-S16E (2 μg each), n = 3, ± SEM, *P < 0.01. (D) Kinase activity assays were performed with 2 μg TAT-PKMζ, [γ-32P]ATP, and increasing amounts of TAT-Pin1 (0 to 4 μg), n = 3. (E) Full-length TAT-Pin1, TAT-Pin1-WW, or TAT-Pin1-WW-S16A was incubated with TAT-PKMζ and [γ-32P]ATP for 15 min before SDS-PAGE, Western blotting with antibody against His tag (Ab), and autoradiography, n = 3. (F) Recombinant GST, albumin, or no additions were incubated with TAT-PKMζ and [γ-32P]ATP for 15 min before SDS-PAGE.

PKCζ and PKMζ inhibit Pin1 and promote protein synthesis

The induction of L-LTP can be prevented by inhibitors of protein synthesis, whereas L-LTP maintenance is sensitive to inhibition of PKCζ and PKMζ (9, 10). Whether PKCζ and PKMζ also participate in the regulation of dendritic translation is unknown. We treated SNs from Pin1−/− and Pin1+/+ mouse brains with a membrane-permeable myristoylated PKCζ peptide inhibitor (myr-PKCζ/PKMζ) or an irrelevant myristoylated control peptide (myr-control) and assessed translation. Myr-PKCζ/PKMζ mimics the pseudosubstrate domain that binds the PKCζ catalytic domain and suppresses its catalytic activity (10, 45). Because PKCζ and PKMζ share a common catalytic domain (11), the same myr-peptide inhibits both isoforms. Basal or glutamate-induced translation by Pin1+/+ SN was unaffected by myr-control but reduced by >75% by myr-PKCζ/PKMζ (Fig. 4B). Translation by Pin1−/− SN was also sensitive to myr-PKCζ/PKMζ (Fig. 4B), suggesting that PKCζ and PKMζ are downstream of Pin1 and, once produced, are required to maintain translation. The requirement for PKCζ and PKMζ activity in the maintenance of L-LTP may reflect their involvement in the regulation of dendritic protein synthesis.

Because Pin1 activity is inhibited by reversible phosphorylation (24, 47), we asked if Pin1 was a target of PKCζ or PKMζ (Fig. 3A). Both PKCζ and PKMζ immunoprecipitated with Pin1 (Fig. 3A); moreover, myr-PKCζ/PKMζ markedly increased Pin1 activity (Table 1, set D). Therefore, we evaluated the ability of recombinant PKMζ to inactivate recombinant Pin1. In the presence of adenosine triphosphate (ATP), recombinant PKMζ was capable of phosphorylating itself, glutathione S-transferase (GST), and albumin (Fig. 4F). When incubated with ATP and PKMζ, recombinant Pin1 was phosphorylated and its activity was significantly decreased to a similar degree as with juglone treatment (Fig. 4, C to E). Phosphorylation of the WW domain of Pin1 at Ser16 prevents its interaction with Ser/Thr-Pro substrates, leading to a functional loss of Pin1 PPIase activity (47). Therefore, we performed PKMζ kinase activity assays with different TAT-linked, Pin1 recombinant proteins including full-length wild type (TAT-Pin1), the N-terminal WW domain (TAT-Pin1WW), or a mutant WW domain with Ala substituted for Ser16 (TAT-Pin1WWS16A). Both TAT-Pin1 and TAT-Pin1WW were phosphorylated, whereas TAT-Pin1WWS16A was not (Fig. 4E). To determine whether Ser16 phosphorylation affected Pin1 activity, we compared the in vitro PPIase activity of recombinant TAT-Pin1 constructs with Ala16 (TAT-Pin1-S16A) or Glu16 (TAT-Pin1-S16E) to TAT-Pin1. The Glu substitution mimics phosphorylated Ser, whereas Ala cannot be phosphorylated. TAT-Pin1-S16A and TAT-Pin1 showed equivalent PPIase activity, whereas TAT-Pin1-S16E showed none (Fig. 4C). Therefore, we conclude that PKMζ phosphorylates Pin1 on Ser16. This modification is sufficient to prevent the WW domain of Pin1 from binding substrate and leads to a functional loss of PPIase activity.

Discussion

Pin1 is a molecular switch that binds and isomerizes phosphorylated Ser/Thr-Pro containing proteins, thereby regulating their biological activity (20). The function of Pin1, in neurons in general and at synapses in particular, is largely unknown. Here, we show that Pin1, by regulating translation and inhibiting PKMζ production, is involved in LTP and synaptic plasticity.

Pin1 abundance is increased during cell division and in continuously dividing tumor cell lines and is an independent risk factor for increased tumor grade and metastatic potential (48). Pin1 has been implicated in the pathogenesis of Alzheimer’s disease, possibly by mislocalization to hyperphosphorylated tau (22, 29). Accelerated apoptotic neurodegeneration occurs in Pin−/− mice, but not in wild-type animals, likely through misregulation of Bim-EL, a BH3-containing, proapoptotic member of the Bcl-2 family (20). These effects of Pin1 depend on its cytoplasmic or nuclear location; here, we show that Pin1 is highly abundant and constitutively active in postsynaptic terminals of rodent brain where, under basal conditions, it suppresses protein synthesis. These results suggest that Pin1 may also antagonize neurodegeneration by decreasing the synthesis of potentially toxic proteins such as the transcription factor E-26–like protein 1 (Elk-1), whose dendritic translation induces neuronal apoptosis (49).

Under basal conditions, 4E-BP is partially phosphorylated (50, 51), which is associated with maximal affinity for eIF4E and suppression of translation (1719, 50, 51). Glutamate activates p70-S6K, PKC, and mTOR (52, 53), all of which phosphorylate 4E-BP at multiple sites (1719). Akt does not directly act on 4E-BP, although it is required for 4E-BP inhibition after growth factor–mediated signaling (18). Therefore, we propose that under basal conditions in dendrites, active Pin1 binds to and isomerizes minimally (hypo)phosphorylated 4E-BP1, preventing additional phosphorylation and facilitating its inhibitory interactions with eIF4E. Pin1 isomerization inhibits hyperphosphorylation of tau by restricting its access to protein phosphatase 2A (PP2A), and neurons from Pin1−/− mice accumulate hyperphosphorylated proteins (35). Glutamatergic signaling rapidly increases dendritic translation by inducing phosphorylation of Pin1 at Ser16 and 4E-BP at multiple sites including Thr37, Thr46, Ser65, Ser101, and Ser112 (1719, 50), which inactivate Pin1 and 4E-BP, respectively. Despite glutamatergic signaling, we did not observe a change in the degree of interaction between Pin1 and 4E-BP. Similar events were observed between Pin1 and heterogenous nuclear ribonucleoprotein (hnRNP D) in activated immune cells (24). Because PKMζ-mediated Ser16 phosphorylation of Pin1 prevents Pin1 interactions with its targets, these results suggest Pin1 PPIase activity may be inactivated through additional mechanisms or that distinct pools of differentially modified Pin1 exist in dendritic compartments.

Classical PKCs indirectly cause the dephosphorylation of 4E-BP1/2 (54, 55) and consequently suppress translation. After eIF4E dissociates from 4E-BP, it is activated by PKC-mediated phosphorylation (56). It is also possible that Pin1 isomerizes hypophosphorylated eIF4E, enhancing its binding to hypophosphorylated 4E-BP1/2 and suppressing translation. Either model is consistent with the increase in basal translation and insensitivity to glutamate activation of translation found in SNs derived from Pin1−/− mice. Because Pin1 has not been implicated in translational control in other cell types, these functional attributes may be unique to neurons.

PKMζ, an atypical PKC, is transcribed independently under the control of an internal promoter within the PKCζ gene (11), and its mRNA is subsequently transported to dendrites and dendritic spines (57). We show that PKCζ/PKMζ, known regulators of synaptic plasticity, influence Pin1 function. Consistent with a direct regulatory role, Pin1 activity was increased in SNs treated with myristoylated PKMζ inhibitor peptides and Pin1 activity was directly inhibited by PKMζ in vitro. Ser16, which has previously been shown to inactivate Pin1 substrate-binding activity, is one likely site of Pin1 modification by PKMζ (47).

In addition to regulating Pin1, PKCζ/PKMζ promote protein synthesis in SN. Specific blockade of PKCζ/PKMζ reduced both basal and Glu-induced translation in wild-type and Pin1 KO preparations. These results suggest that PKCζ/PKMζ function independently and downstream from Pin1 to maintain dendritic translation after glutamatergic signaling. These results may also explain how PKCζ/PKMζ blockade can markedly and quickly reverse the maintenance phase of L-LTP, as well as memory persistence (7, 12, 45). The classical view that protein synthesis is not required for the maintenance of L-LTP (58, 59) is at odds with this interpretation. However, memory can last a lifetime, whereas the turnover of dendritic proteins is usually on the order of several hours, indicating that ongoing protein synthesis must be involved (60). Thus, the physiologic significance of this previously unrecognized function of PKMζ is yet to be fully resolved.

PKCζ/PKMζ has been implicated in several cellular signaling cascades, but little is known about their specific substrates. Our data identify Pin1 as a PKCζ/PKMζ substrate and possibly 4E-BP or eIF4E. We propose that Pin1 regulates LTP through bidirectional interactions with PKMζ, whereby GluR-mediated signaling decreases Pin1 activity, leading to an increase in PKMζ abundance. PKMζ maintains translation and suppresses Pin1 through phosphorylation on Ser16. Thus, PKMζ likely contributes to the maintenance of L-LTP through the induction of dendritic translation. Pin1, as an upstream modulator of PKMζ abundance, plays an integral role in the maintenance of L-LTP.

Materials and Methods

Materials

Reagents were obtained from the following companies. Antibodies, see table S1 for manufacturers and antibody dilutions used for immunoblots and immunofluorescence; anisomycin, antibody against β-actin, cycloheximide, glutamate, glycine, mouse and rabbit immunoglobulin G (IgG), okadaic acid, and protease inhibitor cocktail were from Sigma; juglone was from Calbiochem; antibody against rabbit and HRP-conjugated secondary antibodies against mouse, ECL+ detection reagents, Percoll, Redivue Pro-Mix L-[35S] were from Amersham; rhodamine-conjugated goat antibody against mouse and Prolong Gold Antifade were from Invitrogen; MagnaBind Protein A and G beads, PAGEprep Advance Kit, micro BCA (bicinchoninic acid) protein assay reagent kit, and Seize Primary Mammalian Immunoprecipitation kit were from Pierce Biotechnology; myristoylated-PKCζ peptide (P-219, N-myr-SIYRRGARRWRKL) and myristoylated-PKC peptide (P-205, N-myr-FARKGALRQ) were from Biomol; myristoylated control peptide (#1776, N-myr-QPPASNPRVR) was from Tocris.

Recombinant TAT proteins

The complementary DNA (cDNA) encoding Pin1 (provided by K. P. Lu, Harvard University), WW-Pin1, and PKMζ were cloned in-frame into pHisTAT (provided by S. Dowdy, University of California, San Diego). The cDNA was mutated with a QuikChange XL Site-Directed Mutagenesis kit (Stratagene), as described in the manufacturer’s manual, to produce TAT-Pin1-K63A, TAT-Pin1-S16A, TAT-Pin1-S16E, TAT-WW-S16A, and TAT-WW-S16E. Proteins were expressed in Escherichia coli and purified on a Ni2+ chelate column (Qiagen), as described by the manufacturer, with and without urea. TAT-linked proteins were more than 95% pure on the basis of Ponceau S and Coomassie staining, and Western blots.

Mouse husbandry

C57BL/6 mice were used for all experiments described herein except where it was specified that Pin1−/− and Pin1+/+ were used. Wild-type and Pin1−/− mice in the C57BL/6J background were a gift from A. Means of Duke University Medical Center. All husbandry and euthanasia procedures were performed in accordance with National Institutes of Health guidelines and an approved University of Wisconsin Madison animal care protocol through the Research Animal Resources Center, as described previously (31). Pin1 genotypes were determined by polymerase chain reaction analysis of DNA extracted from tail biopsies.

SN preparation and treatment

SNs were prepared from wild-type and Pin1−/− mouse cortical tissue as described (31, 61, 62) (pups age, 16 to 21 days). Briefly, P16 to P21 mouse pups were killed by carbon dioxide asphyxiation followed by removal of the brain cortices. The cortices were washed in ice-cold gradient medium [GM buffer: 0.25 M sucrose, 5 mM tris (pH 7.5), and 0.1 mM EDTA), transferred to a glass dounce homogenizer containing ice-cold GM buffer, and gently homogenized with seven strokes of the loose pestle followed by five strokes of the tight pestle. The homogenate was spun at 1000g for 10 min at 4°C in round-bottomed tubes to pellet cellular debris and nuclei. The supernatant (2-ml aliquots) was applied to Percoll gradients (layers = 2 ml each of 23, 15, 10, and 3% isosmotic Percoll) and spun at 32,500g for 5 min at 4°C. The third band from the top of the gradient (the 23%/15% interface) containing intact SNs was removed and pooled for the experiments. The two higher-molecular-weight bands at the 15%/10% and 10%/3% interfaces contain broken membranes. The salt concentration of the SNs was adjusted by adding one-tenth volume of 10× stimulation buffer [100 mM tris (pH 7.5), 5 mM Na2HPO4, 4 mM KH2PO4, 40 mM NaHCO3, 800 mM NaCl]. In addition, CaCl2 was added to a final concentration of 12 nM. To suppress nonspecific excitation, 1 μM tetrodotoxin was added. The protein concentration of the SNs was determined by BCA assay and ranged from 200 to 500 ng/μl.

SNs were pretreated with 40 μM Aniso, 100 nM Rap, 10 μM myr-proteins, TAT-proteins or appropriate solvent control and equilibrated at 37°C for 10 to 15 min on a nutator before treatment with 50 μM glutamate and 10 μM glycine (Glu) or no additive (and [35S]Met for translation experiments). Samples were mixed at 37°C in 1.5-ml Eppendorf tubes for 5, 10, 15, or 30 min and then snap-frozen. To characterize the SN preparation, we performed immunoblots of cortical homogenate, postnuclear supernatants, and post-Percoll gradient SNs for nuclear, cytoplasmic, mitochondrial, astrocytic, and synaptic markers. The protein concentration was determined by the BCA assay and ranged from 200 to 500 ng/μl. Synaptic and neuronal markers were maintained in purified SN fractions (fig. S1B) but the abundance of all other markers was significantly reduced.

Immunogold electron microscopy

SNs prepared from C57BL/6 mice (age P15 to P22) were fixed with paraformaldehyde, inactivated with sodium borohydride to neutralize the aldehyde groups, permeabilized with Triton X-100, blocked with normal goat serum, incubated with anti-Pin1, and stained with secondary antibody conjugate, which was ultrasmall gold-conjugated F(ab′)2 fragments as previously described (63).

[35S]Met incorporation

For radiolabeling, 500 μl of SNs were pretreated for 15 min with drug before addition of [35S]Met, 15 μl Redivue Pro-Mix L-[35S], plus or minus Glu. To analyze new protein synthesis, SN lysates were cleared of free isotope, Percoll, and sucrose by purification with the PAGEprep Advance kit per the manufacturer’s instructions. PAGEprep samples were denatured and run on 15% SDS gels. The gels were dried, exposed to a phosphorimager screen, and scanned on a Storm 860 phosphorimager (Molecular Dynamics). The bands from three independent experiments were quantitated with ImageQuant software.

Pin1 activity assays

Pin1 activity was measured as previously described (24), with a few modifications. Briefly, for analysis of Pin1 inhibitors, SNs were pretreated for 15 min at 37°C with drugs before lysates were prepared by addition of Triton X-100 to a final concentration of 1%, followed by three freeze-thaw cycles and 20 min of incubation on ice. Lysed SNs were diluted 5- to 10-fold with 50 mM Hepes and 100 mM NaCl (pH 7.0) and allowed to further incubate on ice. Inhibitors were added back to retain the original concentration before assay of protein lysates (0.05 μg). For analysis of PKCζ and PKMζ inhibitors, 10 μM okadaic acid was added before Triton X-100 addition, freeze-thaw cycles were excluded, and incubations on ice were reduced to 5 min each. The substrate, Suc-AEPF-pNA was prepared in a LiCl/triflouroethane (TFE) solution, as described previously (24), to preserve the cis conformation. The final reaction concentration was 12.8 μM. Readings were taken for the first 1 to 2 min of the reaction and plotted as A390 (absorbance at 390 nm) versus time (minutes). Spontaneous cis-trans isomerization of the substrate (k0) was measured with the complete reaction mixture minus SN protein. The initial reaction slopes were used to determine K values as described by Küllertz et al. with the equation K = (kobsk0)/k0 (36).

Kinase activity assays

PKMζ activity was measured as previously described (64), with a few modifications. Briefly, for analysis of phosphorylation activity, TAT-PKMζ was added to 0 to 4 μg of various TAT-Pin1 substrates. The final reaction buffer was 50 mM tris (pH 7.5), 5 mM MgCl2, 1 mM NaVaO4, 1 mM NaF, 500 μM β-glycerol phosphate, 1× protease inhibitor cocktail, and bovine serum albumin (0.1 mg/ml). [γ-32P]ATP (10 μCi) was added to initiate reactions, which were incubated at 37°C for 60 min. Phosphorylation was stopped by addition of reducing Laemmli buffer and proteins were separated by 15 or 20% SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and analyzed by phosphorimaging and Western blotting.

Immunoprecipitation

After drug treatment, SNs were snap-frozen at −80°C and protein concentration was determined by BCA assay (Pierce). For immunoprecipitation, 10 μg of antibody was added to each SN lysate (600 μg), followed by rocking for 2 hours at 4°C. Protein A or G beads were added and rotation continued overnight. Beads were washed three times with lysis buffer. SDS loading buffer was added directly to the beads and the sample was analyzed by Western blot. For immunoprecipitation of phosphoproteins, okadaic acid (10 μM) was added to the SNs.

Neuronal cell culture, confocal microscopy, and image analysis

Primary neuronal embryonic cultures were prepared as described (31). After 18 to19 days in vitro (DIV), the primary neurons were pretreated with or without 40 μM Aniso at 37°C for 15 min, and then treated with 300 μM Glu, 50 nM TAT-β-Gal, or 50 nM TAT-Pin1-K63A. Cells were stained and imaged as described (31) with rhodamine-labeled anti-Pin1 (anti-Pin1-rhodamine), FITC-labeled antibody against eIF4E (anti-eIF4E-FITC), FITC-labeled antibody against 4E-BP1 (anti-4E-BP1-FITC), FITC-labeled antibody against 4E-BP2 (anti-4E-BP2-FITC), Cy5-labeled (anti-PSD95-Cy5) and/or To-Pro 3-iodide (To-Pro3). See table S1 for antibody concentrations. Colocalization images were generated by ImageJ RG2B colocalization plug-in.

Hippocampal slices and immunostaining

Hippocampal slices from 21- to 25-day-old mice were prepared and stained as described previously (65). In brief, P21 to P25 mice were killed by CO2 asphyxiation and the brains were removed and immersion-fixed in 10% formalin overnight. The fixed brains were then cryoprotected in 30% sucrose/0.1 M phosphate-buffered saline (PBS) for at least 2 days. Frozen 30-μm-thick coronal sections of the hippocampus were cut in a microtome cryostat and placed in PBS for storage. Slices were permeabilized in blocking solution for 2 hours with 2 to 10% fetal bovine serum (FBS), 0.4% Triton X-100, and 0.02% sodium azide in 0.1 M PBS (65). The subsequent staining and imaging were similar to that described above for primary neurons except the wash buffer used was PBS containing 2% FBS and 0.4% Triton X-100.

Hippocampal slice electrophysiology

Hippocampal slices were isolated from wild-type and Pin1−/− mice, and electrophysiology was performed as described (66). Slices were subjected to high-frequency stimulation consisting of a 100-Hz, 1-s-long tetanic stimulus, repeated four times, with an interstimulus interval of 2 min to generate L-LTP in the presence or absence of 25 μM Aniso. Theta burst stimulation was induced by three trains of theta burst stimulation (10 bursts at 5 Hz per train, four pulses (1 s) at 100 Hz per burst) separated by 20 s.

Statistical analysis

P values were calculated with the Student’s t test with a two-tailed distribution on samples, except for LTP studies, which used two-way ANOVA with repeated measures (mixed model) and Bonferroni posttests.

Acknowledgments

Acknowledgments: We thank the members of the laboratory for their comments and suggestions, B. K. August from the University of Wisconsin-Madison Electron Microscope Facility for the immunoelectron microscopy, A. Means of Duke University Medical Center for providing the C57BL/6J Pin1+/− mice, E. Whitesel for constructing Pin1 mutant cDNAs, and K. A. Hanson for helping construct TAT-PKMζ cDNA.

Funding: This work was supported by NIH grants R01-DA026067 and P30-HD03352 (to J.S.M.).

Author contributions: P.R.W., J.L., C.B., and J.S.M. generated the hypotheses; P.R.W., C.J.W., S.W., J.L., K.J.O., and C.B. performed experiments; P.R.W., K.J.O., C.B., and J.S.M. analyzed data, and P.R.W., J.L., C.B., and J.S.M. wrote the manuscript.

Competing interests: The authors declare that they have no competing financial interests.

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/3/112/ra18/DC1

Fig. S1. SNs are enriched for synaptic markers.

Fig. S2. Pin1 regulates dendritic translation.

Fig. S3. Pin1 associates with translational regulators.

Fig. S4. Pin−/− hippocampal slices show normal, basal synaptic transmission, but enhanced paired-pulse facilitation.

Table S1. Antibody manufacturers and dilutions.

References and Notes

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