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PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3 Seo Jung Mo a, 1, Yongsang Cho b, 1, Byung-il Choi a, Dongmin Lee a, **, Hyun Kim a, * a b
Department of Anatomy, College of Medicine, Korea University, Seoul 02841, Republic of Korea Gachon Liberal Arts College, Gachon University, Seongnam-si, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2018 Accepted 7 November 2018 Available online xxx
Microtubule-associated end-binding protein 3 (EB3) accumulates asymmetrically at the tip-end of growing microtubules, providing a central platform for linking various cellular components. EB3 orchestrates microtubule dynamics and targeting, enabling diverse processes within neurons. Inositol 1, 4, 5-trisphosphate 3-kinase A (IP3K-A; also known as ITPKA) is a neuron-enriched protein that binds to microtubules by PKA-dependent manners. In this study, we found that IP3K-A binds to EB3 and their binding affinity is precisely regulated by protein kinase A (PKA)-dependent phosphorylation of IP3K-A at Ser119 (pSer119). We also revealed that the complex of IP3K-A and EB3 dissociates and reassociates rapidly during chemically induced LTP (cLTP) condition. This dynamic rearrangement of IP3K-A and EB3 complex will contribute remodeling of microtubule cytoskeleton allowing effective structural plasticity in response to synaptic stimulations. © 2018 Elsevier Inc. All rights reserved.
Keywords: IP3K-A EB3 Cytoskeleton PKA Phosphorylation Neuron
1. Introduction The versatile functions of microtubules are achieved via a myriad of microtubule-associated proteins (MAPs), including microtubule plus-end-tracking proteins (þTIPs) [1,2]. þTIPs target the growing microtubule tip and this asymmetrical þ TIP distribution contributes to microtubule polarity. End-binding protein 3 (EB3), one of the most enriched þ TIPs in mature neurons, coordinates microtubule dynamics and functions through association with various subcellular components and cytoskeleton-related proteins. Inositol 1, 4, 5-trisphosphate 3-kinase A (IP3K-A, also known as ITPKA), which converts inositol 1, 4, 5-trisphosphate (IP3) into inositol 1, 3, 4, 5-tetrakisphosphate (IP4) with its catalytic domain [3], is a neuron-enriched cytoskeleton binding protein for both microtubules and F-actin [4,5]. The levels of IP3K-A mRNA and protein expression are regulated by neuronal activity and were shown to be altered by electroconvulsive shocks [6], spatial learning [7], and kainic acid [8]. Transient overexpression of IP3K-A induces morphological changes of dendritic spines via Rac1-
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (D. Lee), [email protected] (H. Kim). 1 These authors contributed equally to this work.
dependent pathway [9] and calcium signaling [10]. Recently, transgenic mice conditionally overexpressing IP3K-A shows impaired hippocampal-dependent learning and memory tasks [11]. Although several IP3K-A signaling pathways have been suggested [3], the activity-dependent roles of IP3K-A are still unclear. Here, we found that IP3K-A binds to EB3 and the binding affinity of IP3K-A and EB3 is negatively regulated by PKA-dependent pSer119. During cLTP, pSer119 was increased rapidly in early phase of cLTP and decreased in late phase. Through in situ PLA during cLTP condition, we also revealed that IP3K-A and EB3 complex are rapidly dissociated and reassociated in consistent to the dynamic profile of pSer119 IP3K-A. These findings indicate that the interaction of IP3K-A and EB3 is a potent regulator of microtubule cytoskeleton, contributing the structural remodeling of microtubule cytoskeleton during neuronal activation. 2. Materials and methods 2.1. Subcloning of GST-EB3 and His-EB3 To generate GST-EB3 constructs, EB3 sequences were amplified from pEGFP-N1-EB3, a kind gift from Dr. Casper C. Hoogenraad (Utrecht University, Netherlands). PGEX-5X-1 (GE Healthcare Life Sciences, USA) was used as a backbone vector. His-EB3 was amplified from the pEGFP-N1-EB3 vector. All constructs were
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Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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confirmed by DNA sequence analysis (Macrogen, Republic of Korea).
15 63 15 mm, n ¼ 1.522, Korea Electro-Optics Co., Ltd., Korea) was used to align the total internal reflection (TIR) of the light source.
2.2. Overexpression and purification of GST-IP3K-A 2.8. Immunoblotting analysis Overexpression and purification of GST-IP3K-A was performed as described previously [12]. Briefly, the pCold1-GST-IP3K-A-His vector was introduced into E. coli BL21(DE3). His affinity purification of pCold1-GST-IP3K-A-His was conducted on the basis of interactions between the polyhistidine tag at the C-terminus of the enzyme and metal ions. Affinity columns (Bio-Rad, Cat# 731e1550) were packed with Ni2þ-charged agarose resin (2 ml) (Elpis Biotech, Cat# EBE-1031). Recombinant pCold1-GST-IP3K-A-His was then eluted with elution buffer (5 ml; 0.5 M NaCl, 250 mM imidazole, and 20 mM Tris-HCl, pH 7.9), and the eluted protein solution (5 ml) was collected as five separate 1 ml fractions. 2.3. Overexpression and purification of His-EB3 The His-pProEX-HTa-NcoI-EB3 construct was transformed into E. coli BL21 (CodonPlus; Stratagene). After induction for 3 h with IPTG, collected E. coli BL-21 cells were suspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.9). The cells were lysed by sonication and centrifuged at 10,000g for 20 min. The separated supernatant was applied to a 0.2 ml Ni-NTA column (Qiagen) and the bound His-EB3 protein was then collected by adding elution buffer (20 mM Tris-HCl, 500 mM NaCl, 1 M imidazole, pH 7.9). 2.4. GST-EB3 pull-down assay Bacterially expressed GST-EB3 was purified with glutathioneSepharose 4B beads (GE Healthcare Life Sciences, USA). The mixture of E. coli lysate and beads was incubated overnight in a rotator at 4 C. After three washes, GST-EB3 immobilized on Sepharose beads was mixed with rat whole-brain lysates. The mixture was incubated for 4 h in a rotator at 4 C. After additional three washes, the remaining mixture was removed with a 31G insulin syringe (BD, USA) and 2 SDS-sample buffer was added for further analysis.
After gel electrophoresis, proteins were transferred onto a nitrocellulose membrane (Whatman Protran, Germany) using an electrophoretic transfer cell (Bio-Rad). The membrane was blocked with 5% skim milk in TBST buffer. Then, the membrane was incubated with primary antibodies, which were diluted in blocking buffer for 1 h at RT or overnight at 4 C. Membranes were then washed three times with TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at RT. 2.9. HEK293T cell culture and primary hippocampal neuronal culture HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). Primary hippocampal neuronal culture was performed as previously described [13]. Briefly, rat hippocampi were rapidly dissected and digested with 0.25% trypsin-EDTA (Invitrogen) for 10 min at 37 C. After the remaining trypsin-EDTA was removed, cells were carefully triturated by passing through a narrow opening of 1 ml pipet tip. Digested cells were counted and plated on 50 mg/ml poly-D-lysine (PDL) (Sigma-Aldrich) pre-coated coverslips. Media consisted of neurobasal medium (Invitrogen) and the following reagents: 0.5 mM L-glutamine (Invitrogen), 1:50 (v/v) B27 supplement (Gibco), and 1% penicillin-streptomycin. All procedures were carried out in accordance with the ethical guidelines of Korea University and with the approval of the Animal Care and Use Committee of Korea University. 2.10. Forskolin-induced cLTP in dissociated hippocampal culture neurons
Cell lysates were prepared with NETT buffer (50 mM Tri-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM DTT, protease inhibitor cocktail). A mixture of the lysate and protein G beads was incubated at 4 C for 30 min to reduce non-specific binding. The cleared lysate was incubated with 10 mg of FLAG antibody (M2, Sigma). The mixture of lysate and antibody was incubated overnight at 4 C. In turn, this mixture and washed protein G beads were incubated in the rotator at 4 C for an additional 4 h.
The procedures for forskolin-induced cLTP consisted of three steps: pre-incubation, cLTP induction, and post-incubation. The original medium was replaced with pre-warmed pre-incubation medium [Hank's Balanced Salt Solution (HBSS, Gibco Cat. #14175) supplemented with 1 mM MgCl2, 2 mM CaCl2, and 33 mM Dglucose]. After 20 min pre-incubation at 37 C and 5% CO2, the preincubation medium was replaced by the induction medium, which contained 50 mM forskolin (Sigma Cat. #F6886), 0.1 mM rolipram (Sigma Cat. #R6520), 2 mM CaCl2, and 33 mM D-glucose, and incubated for 10 min. Lastly, the induction medium was replaced by the post-incubation medium, which was identical to the preincubation medium.
2.6. Immunocytochemistry, and confocal and N-SIM microscopy
2.11. PLA for detection of proteineprotein interaction
Super-resolution SIM (Nikon N-SIM) with a CFI Apo TIRF 100 oil objective lens (NA1.49) and iXon DU-897 EMCCD camera was used to image the neurons. Image processing was performed in Zen software (Carl Zeiss, Germany). Sample preparation for N-SIM is the same as for conventional microscopy.
Duolink in situ fluorescence (Olink Bioscience, Uppsala, Sweden) was used for detection of EB3 and IP3K-A interactions under cLTP. All procedures were performed as described in the manufacturer's manuals. Briefly, neurons were fixed with 4% PFA for 20 min. For cell permeabilization, cells were incubated in TBST containing 0.5% triton X-100. The following antibodies were used in the PLA: mouse anti-IP3K-A (1:500; homemade II4E) and rabbit anti-EB3 (1:500; Millipore, Cat. #AB6033). Neurons were incubated in blocking buffer containing primary antibodies for 1 h at RT. After extensive washing, the prepared PLA probes were added to the neurons, and the neurons were incubated in a humidity chamber for 1 h at 37 C.
2.5. Immunoprecipitation
2.7. Total internal reflection microscopy The TIRFM optics were incorporated into an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan). A transmitting all-side polished dove-type prism (BK7,
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 1. IP3K-A binds to EB3. (A) Ponceau stained membrane (top) and Western blot (bottom) images of a GST pull-down assay with rat brain lysates. Full expression of GST and GSTEB3 is indicated by white arrows (GST) and yellow arrows (GST-EB3) at the expected size. (B) Schematic figure showing the immunoprecipitation assay for HEK293T cells expressing EGFP-EB3 and/or FLAG-IP3K-A. Western blot analysis against EGFP shows the interaction of EB3 and IP3K-A. (C) Representative total internal reflection fluorescence (TIRF) microscopy images of rat hippocampal neurons expressing EB3-EGFP and IP3K-A-mCherry. Yellow arrowheads indicate dendritic spines. (D) Representative structured illumination microscopy (SIM; Nikon N-SIM) images of rat hippocampal neurons. White arrows indicate co-localization of IP3K-A and EB3 in dendritic spine regions. Scale bars: 20 mm (C), 5 mm (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
The prepared ligationeligase solution was added, and neurons were incubated in a humidity chamber for 30 min at 37 C. Lastly, amplification-polymerase solution was added to the washed neurons. 2.12. Statistics Statistical significance was calculated by Student's t-test or oneway ANOVA with post hoc Tukey HSD or Dunnett's test using SPSS 12.0 (IBM) software. 3. Results 3.1. IP3K-A binds to EB3 EB3, well-characterized þ TIP protein in mature neurons, interacts with a wide spectrum of proteins. Particularly, most of EB3binding partners are cytoskeleton-related proteins. Considering that our protein of interest, IP3K-A, binds to both F-actin and microtubules [4,5], we deemed it highly plausible that IP3K-A
interacts with EB3. To test this possibility, we first conducted a glutathione S-transferase (GST) pull-down assay and found that the specific IP3K-A band was blotted only in the GST-EB3 lane and not in the GST-alone lane (Fig. 1A), suggesting that IP3K-A interacts with GST-EB3. Similarly, we carried out an immunoprecipitation assay with HEK293T cells expressing EGFP-EB3 and FLAG-IP3K-A. The Western blot results showed that GFP-EB3 was also precipitated by the FLAG antibody (Fig. 1B). To acquire visual evidence of the interaction of IP3K-A and EB3, we used two advanced imaging techniques that enabled fine spatial localization of IP3K-A and EB3. The first technique was total internal reflection fluorescence (TIRF) microscopy, which provides outstanding image quality with a reduced background. Many studies of dynamic microtubules have used TIRF microscopy to track EB3 [14,15]. When we doubly transfected primary hippocampal cultured neurons with IP3K-AmCherry and EB3-EGFP, TIRF imaging showed prominent colocalization of IP3K-A and EB3 in dendritic spine regions (Fig. 1C). The second technique was structured illumination microscopy (SIM; N-SIM, Nikon, Japan), which acquires super-resolution im patterns produced under different ages by analyzing the moire
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 2. Structural mapping of binding regions for EB3 and IP3K-A. (A) Schematic figure showing the functional domains of the EB3 and GST-EB3 constructs used in the pull-down assay. (B) GST-EB3 pull-down assay for mapping the IP3K-A-binding region. Full expression of various GST-EB3 constructs was confirmed by the Ponceau stained membrane (upper panel). White arrows indicate GST-EB3 constructs at the expected size. Western blot of GST-EB3 pull-down assay probed with a specific antibody against IP3K-A (lower panel). (C) Schematic figure displaying the functional domains of the IP3K-A and GST-IP3K-A constructs used in the pull-down assay. (D) Western blot analysis of the GST-IP3K-A pull-down assay with purified His-EB3.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 3. The binding affinity between IP3K-A and EB3 is regulated by PKA-dependent phosphorylation of IP3K-A at Ser119. (A) GST-EB3 pull-down assay of HEK293T cell lysates expressing EGFP or EGFP-IP3K-A constructs [wild-type (WT), S119E, and S119A]. Purified GST-EB3 was incubated with overexpressed HEK293T lysates. A Ponceau stained membrane (top) shows equal loading of IP3K-A in each lane. A Western blot (bottom) against the EGFP antibody shows the different binding affinities between EB3 and the IP3K-A constructs. Notably, a phosphorylation-dependent band shift of IP3K-A is observed in only the WT input lane (second lane; yellow arrow). (B) In vitro binding assay for purified IP3K-A and EB3 with the catalytic PKA subunit. The red arrow indicates the decreased EB3 band intensity (second and third panels). PKA-dependent phosphorylation of IP3K-A at Ser119 was confirmed by an anti-pSer119 antibody (fourth panel), while IP3K-A bands were the same (fifth panel). (C) Schematic figure shows changes in IP3K-A phosphorylation by PKA (top). The PKA inhibitor H89 decreases the phosphorylation level of EGFP-IP3K-A, resulting in a decrease in EB3-binding affinity. The PKA agonist forskolin (FSK) works in the opposite direction. Western blot analysis of GST-EB3 pull-down assay. (D) Summary graph of the GST-EB3 pull-down assay (WT-H89: 0.79 ± 0.06, WT-FSK: 0.41 ± 0.05, S119A-H89: 1.44 ± 0.25, S119A-FSK: 1.33 ± 0.07, mean ± SEM; one-way ANOVA [F (3, 8) ¼ 37.739, ***p < 0.001], Tukey HSD post-hoc test [WT-H89/WT-FSK: *p ¼ 0.035; WT-H89/S119A-H89: **p ¼ 0.002; WT-H89/S119A-H89: **p ¼ 0.006; WT-FSK/S119A-H89: ***p < 0.001; WT-FSK/S119A-FSK: ***p < 0.001; S119A-H89/S119A-FSK: p ¼ 0.766]). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 4. Rapid dissociation and reassociation of IP3K-A and EB3 complex during neuronal activation. (A) Temporal profile of pSer119 IP3K-A over the time course of cLTP induction. The first and second panels show the blot against the pSer119 IP3K-A antibody and its intensity profile. Anti-pSer119 peaked at 0 min (at the end of 10 min of cLTP induction) and reverted to basal levels within 30 min. (B) Quantification of phosphorylation levels of IP3K-A Ser119. Statistic values were calculated from three independent experiments (**p < 0.01, *p < 0.05, one-way ANOVA followed by post-hoc Dunnett's test). (C, D) Representative confocal images of hippocampal cultured neurons after a proximity ligation assay (PLA) in control (C) and cLTP (D) conditions. Red duolink spots indicate direct interaction of IP3K-A and EB3. Phalloidin 488 (green channel) staining was used to visualize the general structure of the neurons. Scale bar: 20 mm. (E) Quantification of the total number of duolink spots during cLTP induction (control: 47.5 ± 14.6, cLTP 0‘: 104.1 ± 27.0, cLTP 15‘: 136.2 ± 33.6, and cLTP 30‘: 150.1 ± 8.7; *p < 0.05, **p < 0.01; mean ± SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
phase-structured illumination [16]. We also found significant colocalization of IP3K-A and EB3 with this method (Fig. 1D), consistent with the results from TIRF imaging. 3.2. IP3K-A N-terminus is necessary for the binding of IP3K-A and EB3 Next, we conducted a domain-based mapping study using
several deletion mutants of EB3 and IP3K-A (Fig. 2A and C). Structurally, EB3 contains well-characterized functional domains, including a microtubule-binding domain, a coiled coil domain, and a þTIP-binding domain [2]. Six GST-EB3 constructs were designed with different combinations of these three domains to determine which domain is involved in the interaction with IP3K-A (Fig. 2A). The results of the GST-EB3 pull-down assay shows that IP3K-A bound to all deletion mutants of GST-EB3 except for the 130e251
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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deletion mutant that lacked both the microtubule-binding domain and the þTIP-binding domain (Fig. 2B). Inversely, we investigated the EB3-binding region of IP3K-A using purified His-EB3 and GSTIP3K-A constructs (GST-IP3K-A WT, 81e459, and 191e459). We found that EB3 binds to only the full-length wild-type (WT) construct (Fig. 2D), indicating that the IP3K-A N-terminus is necessary for the interaction of IP3K-A and EB3. 3.3. Binding affinity of IP3K-A and EB3 is regulated by PKAdependent phosphorylation In several studies, the binding affinity of EB proteins is regulated by post-translational modifications such as detyrosination and phosphorylation [17]. Phosphorylation of serine residues near the binding regions blocks interactions with EB proteins [18e20]. IP3KA contains well-characterized phosphorylation sites for PKA, protein kinase C (PKC), and Ca2þ/calmodulin-dependent protein kinase II (CaMKII) [3,21e23]. Among these, we focused on PKA-dependent Ser119 phosphorylation because this site is close to the N-terminal EB3-binding site and because Ser119 also controls the binding affinity between microtubules and IP3K-A [4]. To investigate the relevance of Ser119 phosphorylation to the binding affinity of IP3KA and EB3, we generated a phosphodeficient form of Ser119 (S119A; Ser / Ala) and a phosphomimic form of Ser119 (S119E; Ser / Glu). HEK293T cell lysates expressing EGFP, EGFP-IP3K-A WT, EGFP-IP3KA S119A, or EGFP-IP3K-A S119E were incubated with immobilized GST-EB3. As expected, EGFP-IP3K-A S119A showed stronger affinity for EB3 than S119E, suggesting that phosphorylation at IP3K-A Ser119 interferes with the binding between EB3 and IP3K-A (Fig. 3A). Interestingly, we observed a phosphorylation-related band shift in the GFP-IP3K-A WT construct (Fig. 3A; yellow arrow). This phospho-regulatory interaction is also possible during binding of microtubules and IP3K-A [4]. Therefore, we needed to check for a microtubule-independent interaction between EB3 and IP3K-A. To address this, we designed an in vitro direct-binding assay using purified His-EB3 and GST-IP3K-A recombinant protein together with the catalytic subunit of PKA (Fig. 3B). For GST-IP3K-A WT, the amount of bound EB3 was decreased when PKA was added with ATP (Fig. 3B, red arrow indicates the decreased intensity in the EB3 blot) but, for GST-IP3K-A S119A, there was no change. Phosphorylation of IP3K-A Ser119 (pSer119 IP3K-A) by PKA was detected with a specific phosphor antibody [4]. We further tested for a direct correlation between phosphorylation levels of IP3K-A at Ser119 and binding affinity with EB3. To bidirectionally adjust the phosphorylation level of IP3K-A at Ser119, we treated cells with forskolin (an adenyl-cyclase activator) or H-89 (a selective PKA inhibitor) (Fig. 3C). In the Western blot analysis of the GST-EB3 pull-down assay, reduced binding affinity was observed in the forskolintreated EGFP-IP3K-A WT group compared with the H-89-treated EGFP-IP3K-A WT group (Fig. 3C and D). No significant changes were observed in the EGFP-IP3K-A S119A groups regardless of treatment with forskolin or H89 (Fig. 3C and D), suggesting that pSer119 IP3K-A is essential for the regulation of EB3 binding.
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phosphorylation restored back to a base line level at late phase (Fig. 4A and B). We predicted that these dynamic changes of phosphorylation would affect to rapid rearrangement of IP3K-A and EB3 through cLTP induction. To confirm this hypothesis, we performed in situ PLA to investigate the total number of IP3K-A and EB3 complex at specific time points along cLTP induction. The result of PLA assay presents a prominent increase in the number of PLA spot which corresponds to the interaction of IP3K-A and EB3 (Fig. 4D) compared with the control (Fig. 4C). As predicted, the number of PLA spot gradually increased along the time course of cLTP induction (Fig. 4E), suggesting that more new interactions between IP3K-A and EB3 are formed during neuronal activation.
4. Discussion Here, we demonstrated that IP3K-A interacts directly with EB3 in a microtubule-independent manner (Figs. 1 and 2). EB3 binds to various proteins including þ TIPs and MAPs through a its C-terminus-binding motif. In general, many þ TIPs and MAPs bind to microtubules and/or EB family proteins using their basic and serine-rich regions [17]. These regions interact with the negatively charged sequences in microtubules and EB family proteins. Phosphorylation of þTIPs or MAPs reduces their binding affinity to microtubules and EB family proteins through changes in the electrostatic interactions [2,18]. Indeed, many þ TIPs or MAPs are the substrates of various kinases. For instance, glycogen synthase kinase 3 (GSK3)-dependent phosphorylation of residues surrounding the SxIP motif of CLASP2 interferes with binding to EB and microtubule tips [19]. In addition, PKA phosphorylates p150Glued and mediates its microtubule interactions [25]. As shown in this study, the binding affinity of IP3K-A to EB3 is regulated by PKA-dependent phosphoregulation of IP3K-A at Ser119 (Fig. 3). This similarity in regulatory mechanisms might result from the negatively charged EEY/F motif that is observed in both microtubules and EB3. During neuronal activation in response to synaptic stimulation, various signaling pathways including phosphorylation and dephosphorylation affect wide spectrum of cellular events such as cytoskeletal remodeling [26] and synaptic targeting of specific biomolecules [27] causing synaptic plasticity and morphological changes [24]. Precise spatiotemporal controls of signaling direction and intensity are crucial for delicate neuronal functions to transmit information effectively. Considering EB3 functions as the central hub of microtubule [28], transient phosphorylation by PKA triggers rapid dissociations and reassociations of IP3K-A and EB3, providing an activity-dependent rearrangement and recruitment of cytoskeletons to lead morphological remodeling of neuron during synaptic plasticity. Taken together, our results present that the activity-dependent binding of IP3K-A and EB3 and this interaction functions as a potent regulator of rearrangement or recruitment of microtubule cytoskeleton contributing synaptic plasticity during neuronal activation.
3.4. Activity-dependent interaction of IP3K-A and EB3
Author contributions
PKA is one of the most powerful regulators during neuronal activation and phosphorylation level of its substrates are tightly regulated to tune functional and morphological changes of neurons depending on the synaptic input [24]. To acquire the temporal profile of phosphorylation level of IP3K-A Ser119 during neuronal activation, we introduced cLTP protocol with Forskolin and prepared lysate samples at different time points of cLTP. The Western blot image shows the intensity of Ser119 phosphorylation reaches the peak level at early phase of cLTP, but the level of
D.L. and H.K. designed research. D.L., S.M., Y.C., and B.C. performed the research and analyzed the data. D.L., S.M., Y.C., and H.K. wrote the paper.
Conflicts of interest The authors declare no competing financial interests or potential conflicts of interest.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Acknowledgements We thank all members of Hyun Kim's lab for critical comments on the manuscript. This work was supported by National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (NRF-2017M3C7A1079696 to H. Kim). Bioedit edited the manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.042. References [1] L.C. Kapitein, C.C. Hoogenraad, Building the neuronal microtubule cytoskeleton, Neuron 87 (2015) 492e506. [2] A. Akhmanova, M.O. Steinmetz, Tracking the ends: a dynamic protein network controls the fate of microtubule tips, Nat. Rev. Mol. Cell Biol. 9 (2008) 309e322. [3] M.J. Schell, Inositol trisphosphate 3-kinases: focus on immune and neuronal signaling, Cell. Mol. Life Sci. 67 (2010) 1755e1778. [4] D. Lee, H.W. Lee, S. Hong, B.I. Choi, H.W. Kim, S.B. Han, I.H. Kim, J.Y. Bae, Y.C. Bae, I.J. Rhyu, W. Sun, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity, J. Biol. Chem. 287 (2012) 15981e15995. [5] M.J. Schell, C. Erneux, R.F. Irvine, Inositol 1,4,5-trisphosphate 3-kinase A associates with F-actin and dendritic spines via its N terminus, J. Biol. Chem. 276 (2001) 37537e37546. [6] H. Kim, J.P. Ko, U.G. Kang, J.B. Park, H.L. Kim, Y.H. Lee, Y.S. Kim, Electroconvulsive shock reduces inositol 1,4,5-trisphosphate 3-kinase mRNA expression in rat dentate gyrus, J. Neurochem. 63 (1994) 1991e1994. [7] I.H. Kim, S.K. Park, W. Sun, Y. Kang, H.T. Kim, H. Kim, Spatial learning enhances the expression of inositol 1,4,5-trisphosphate 3-kinase A in the hippocampal formation of rat, Brain Res. Mol. Brain Res. 124 (2004) 12e19. [8] W. Sun, Y. Kang, I.H. Kim, E.H. Kim, I.J. Rhyu, H.J. Kim, H. Kim, Inhibition of rat brain inositol 1,4,5-trisphosphate 3-kinase A expression by kainic acid, Neurosci. Lett. 392 (2006) 181e186. [9] I.H. Kim, S.K. Park, S.T. Hong, Y.S. Jo, E.J. Kim, E.H. Park, S.B. Han, H.S. Shin, W. Sun, H.T. Kim, S.H. Soderling, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase a functions as a scaffold for synaptic Rac signaling, J. Neurosci. 29 (2009) 14039e14049. [10] S. Windhorst, D. Minge, R. Bahring, S. Huser, C. Schob, C. Blechner, H.Y. Lin, G.W. Mayr, S. Kindler, Inositol-1,4,5-trisphosphate 3-kinase A regulates dendritic morphology and shapes synaptic Ca2þ transients, Cell. Signal. 24 (2012) 750e757. [11] B. Choi, H.W. Lee, S. Mo, J.Y. Kim, H.W. Kim, I.J. Rhyu, E. Hong, Y.K. Lee, J.S. Choi, C.H. Kim, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase A overexpressed in mouse forebrain modulates synaptic transmission and mGluR-LTD
of CA1 pyramidal neurons, PloS One 13 (2018), e0193859. [12] D. Lee, S. Han, S. Woo, H.W. Lee, W. Sun, H. Kim, Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coli, Protein Expr. Purif. 97 (2014) 72e80. [13] D. Lee, J.H. Hyun, K. Jung, P. Hannan, H.B. Kwon, A calcium- and light-gated switch to induce gene expression in activated neurons, Nat. Biotechnol. 35 (2017) 858e863. [14] X. Hu, C. Viesselmann, S. Nam, E. Merriam, E.W. Dent, Activity-dependent dynamic microtubule invasion of dendritic spines, J. Neurosci. 28 (2008) 13094e13105. [15] J. Jaworski, L.C. Kapitein, S.M. Gouveia, B.R. Dortland, P.S. Wulf, I. Grigoriev, P. Camera, S.A. Spangler, P. Di Stefano, J. Demmers, H. Krugers, P. Defilippi, A. Akhmanova, C.C. Hoogenraad, Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity, Neuron 61 (2009) 85e100. [16] M.G. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 13081e13086. [17] A. Akhmanova, M.O. Steinmetz, Microtubule þTIPs at a glance, J. Cell Sci. 123 (2010) 3415e3419. [18] S. Honnappa, S.M. Gouveia, A. Weisbrich, F.F. Damberger, N.S. Bhavesh, H. Jawhari, I. Grigoriev, F.J. van Rijssel, R.M. Buey, A. Lawera, I. Jelesarov, F.K. Winkler, K. Wuthrich, A. Akhmanova, M.O. Steinmetz, An EB1-binding motif acts as a microtubule tip localization signal, Cell 138 (2009) 366e376. [19] P. Kumar, K.S. Lyle, S. Gierke, A. Matov, G. Danuser, T. Wittmann, GSK3beta phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment, J. Cell Biol. 184 (2009) 895e908. [20] T. Watanabe, J. Noritake, M. Kakeno, T. Matsui, T. Harada, S. Wang, N. Itoh, K. Sato, K. Matsuzawa, A. Iwamatsu, N. Galjart, K. Kaibuchi, Phosphorylation of CLASP2 by GSK-3beta regulates its interaction with IQGAP1, EB1 and microtubules, J. Cell Sci. 122 (2009) 2969e2979. [21] D. Communi, V. Vanweyenberg, C. Erneux, D-myo-inositol 1,4,5-trisphosphate 3-kinase A is activated by receptor activation through a calcium:calmodulindependent protein kinase II phosphorylation mechanism, EMBO J. 16 (1997) 1943e1952. [22] X. Hu, L. Ballo, L. Pietila, C. Viesselmann, J. Ballweg, D. Lumbard, M. Stevenson, E. Merriam, E.W. Dent, BDNF-induced increase of PSD-95 in dendritic spines requires dynamic microtubule invasions, J. Neurosci. 31 (2011) 15597e15603. [23] S.S. Sim, J.W. Kim, S.G. Rhee, Regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent protein kinase and protein kinase C, J. Biol. Chem. 265 (1990) 10367e10372. [24] K.P. Giese, K. Mizuno, The roles of protein kinases in learning and memory, Learn. Mem. 20 (2013) 540e552. [25] P.S. Vaughan, P. Miura, M. Henderson, B. Byrne, K.T. Vaughan, A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport, J. Cell Biol. 158 (2002) 305e319. [26] P.R. Gordon-Weeks, A.E. Fournier, Neuronal cytoskeleton in synaptic plasticity and regeneration, J. Neurochem. 129 (2014) 206e212. [27] F.T. Crump, K.S. Dillman, A.M. Craig, cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors, J. Neurosci. 21 (2001) 5079e5088. [28] G. Lansbergen, A. Akhmanova, Microtubule plus end: a hub of cellular activities, Traffic 7 (2006) 499e507.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3 Seo Jung Mo a, 1, Yongsang Cho b, 1, Byung-il Choi a, Dongmin Lee a, **, Hyun Kim a, * a b
Department of Anatomy, College of Medicine, Korea University, Seoul 02841, Republic of Korea Gachon Liberal Arts College, Gachon University, Seongnam-si, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 October 2018 Accepted 7 November 2018 Available online xxx
Microtubule-associated end-binding protein 3 (EB3) accumulates asymmetrically at the tip-end of growing microtubules, providing a central platform for linking various cellular components. EB3 orchestrates microtubule dynamics and targeting, enabling diverse processes within neurons. Inositol 1, 4, 5-trisphosphate 3-kinase A (IP3K-A; also known as ITPKA) is a neuron-enriched protein that binds to microtubules by PKA-dependent manners. In this study, we found that IP3K-A binds to EB3 and their binding affinity is precisely regulated by protein kinase A (PKA)-dependent phosphorylation of IP3K-A at Ser119 (pSer119). We also revealed that the complex of IP3K-A and EB3 dissociates and reassociates rapidly during chemically induced LTP (cLTP) condition. This dynamic rearrangement of IP3K-A and EB3 complex will contribute remodeling of microtubule cytoskeleton allowing effective structural plasticity in response to synaptic stimulations. © 2018 Elsevier Inc. All rights reserved.
Keywords: IP3K-A EB3 Cytoskeleton PKA Phosphorylation Neuron
1. Introduction The versatile functions of microtubules are achieved via a myriad of microtubule-associated proteins (MAPs), including microtubule plus-end-tracking proteins (þTIPs) [1,2]. þTIPs target the growing microtubule tip and this asymmetrical þ TIP distribution contributes to microtubule polarity. End-binding protein 3 (EB3), one of the most enriched þ TIPs in mature neurons, coordinates microtubule dynamics and functions through association with various subcellular components and cytoskeleton-related proteins. Inositol 1, 4, 5-trisphosphate 3-kinase A (IP3K-A, also known as ITPKA), which converts inositol 1, 4, 5-trisphosphate (IP3) into inositol 1, 3, 4, 5-tetrakisphosphate (IP4) with its catalytic domain [3], is a neuron-enriched cytoskeleton binding protein for both microtubules and F-actin [4,5]. The levels of IP3K-A mRNA and protein expression are regulated by neuronal activity and were shown to be altered by electroconvulsive shocks [6], spatial learning [7], and kainic acid [8]. Transient overexpression of IP3K-A induces morphological changes of dendritic spines via Rac1-
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (D. Lee), [email protected] (H. Kim). 1 These authors contributed equally to this work.
dependent pathway [9] and calcium signaling [10]. Recently, transgenic mice conditionally overexpressing IP3K-A shows impaired hippocampal-dependent learning and memory tasks [11]. Although several IP3K-A signaling pathways have been suggested [3], the activity-dependent roles of IP3K-A are still unclear. Here, we found that IP3K-A binds to EB3 and the binding affinity of IP3K-A and EB3 is negatively regulated by PKA-dependent pSer119. During cLTP, pSer119 was increased rapidly in early phase of cLTP and decreased in late phase. Through in situ PLA during cLTP condition, we also revealed that IP3K-A and EB3 complex are rapidly dissociated and reassociated in consistent to the dynamic profile of pSer119 IP3K-A. These findings indicate that the interaction of IP3K-A and EB3 is a potent regulator of microtubule cytoskeleton, contributing the structural remodeling of microtubule cytoskeleton during neuronal activation. 2. Materials and methods 2.1. Subcloning of GST-EB3 and His-EB3 To generate GST-EB3 constructs, EB3 sequences were amplified from pEGFP-N1-EB3, a kind gift from Dr. Casper C. Hoogenraad (Utrecht University, Netherlands). PGEX-5X-1 (GE Healthcare Life Sciences, USA) was used as a backbone vector. His-EB3 was amplified from the pEGFP-N1-EB3 vector. All constructs were
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Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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confirmed by DNA sequence analysis (Macrogen, Republic of Korea).
15 63 15 mm, n ¼ 1.522, Korea Electro-Optics Co., Ltd., Korea) was used to align the total internal reflection (TIR) of the light source.
2.2. Overexpression and purification of GST-IP3K-A 2.8. Immunoblotting analysis Overexpression and purification of GST-IP3K-A was performed as described previously [12]. Briefly, the pCold1-GST-IP3K-A-His vector was introduced into E. coli BL21(DE3). His affinity purification of pCold1-GST-IP3K-A-His was conducted on the basis of interactions between the polyhistidine tag at the C-terminus of the enzyme and metal ions. Affinity columns (Bio-Rad, Cat# 731e1550) were packed with Ni2þ-charged agarose resin (2 ml) (Elpis Biotech, Cat# EBE-1031). Recombinant pCold1-GST-IP3K-A-His was then eluted with elution buffer (5 ml; 0.5 M NaCl, 250 mM imidazole, and 20 mM Tris-HCl, pH 7.9), and the eluted protein solution (5 ml) was collected as five separate 1 ml fractions. 2.3. Overexpression and purification of His-EB3 The His-pProEX-HTa-NcoI-EB3 construct was transformed into E. coli BL21 (CodonPlus; Stratagene). After induction for 3 h with IPTG, collected E. coli BL-21 cells were suspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, pH 7.9). The cells were lysed by sonication and centrifuged at 10,000g for 20 min. The separated supernatant was applied to a 0.2 ml Ni-NTA column (Qiagen) and the bound His-EB3 protein was then collected by adding elution buffer (20 mM Tris-HCl, 500 mM NaCl, 1 M imidazole, pH 7.9). 2.4. GST-EB3 pull-down assay Bacterially expressed GST-EB3 was purified with glutathioneSepharose 4B beads (GE Healthcare Life Sciences, USA). The mixture of E. coli lysate and beads was incubated overnight in a rotator at 4 C. After three washes, GST-EB3 immobilized on Sepharose beads was mixed with rat whole-brain lysates. The mixture was incubated for 4 h in a rotator at 4 C. After additional three washes, the remaining mixture was removed with a 31G insulin syringe (BD, USA) and 2 SDS-sample buffer was added for further analysis.
After gel electrophoresis, proteins were transferred onto a nitrocellulose membrane (Whatman Protran, Germany) using an electrophoretic transfer cell (Bio-Rad). The membrane was blocked with 5% skim milk in TBST buffer. Then, the membrane was incubated with primary antibodies, which were diluted in blocking buffer for 1 h at RT or overnight at 4 C. Membranes were then washed three times with TBST buffer and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at RT. 2.9. HEK293T cell culture and primary hippocampal neuronal culture HEK293T cells were grown in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum and 1% penicillin-streptomycin (Invitrogen). Primary hippocampal neuronal culture was performed as previously described [13]. Briefly, rat hippocampi were rapidly dissected and digested with 0.25% trypsin-EDTA (Invitrogen) for 10 min at 37 C. After the remaining trypsin-EDTA was removed, cells were carefully triturated by passing through a narrow opening of 1 ml pipet tip. Digested cells were counted and plated on 50 mg/ml poly-D-lysine (PDL) (Sigma-Aldrich) pre-coated coverslips. Media consisted of neurobasal medium (Invitrogen) and the following reagents: 0.5 mM L-glutamine (Invitrogen), 1:50 (v/v) B27 supplement (Gibco), and 1% penicillin-streptomycin. All procedures were carried out in accordance with the ethical guidelines of Korea University and with the approval of the Animal Care and Use Committee of Korea University. 2.10. Forskolin-induced cLTP in dissociated hippocampal culture neurons
Cell lysates were prepared with NETT buffer (50 mM Tri-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 1 mM DTT, protease inhibitor cocktail). A mixture of the lysate and protein G beads was incubated at 4 C for 30 min to reduce non-specific binding. The cleared lysate was incubated with 10 mg of FLAG antibody (M2, Sigma). The mixture of lysate and antibody was incubated overnight at 4 C. In turn, this mixture and washed protein G beads were incubated in the rotator at 4 C for an additional 4 h.
The procedures for forskolin-induced cLTP consisted of three steps: pre-incubation, cLTP induction, and post-incubation. The original medium was replaced with pre-warmed pre-incubation medium [Hank's Balanced Salt Solution (HBSS, Gibco Cat. #14175) supplemented with 1 mM MgCl2, 2 mM CaCl2, and 33 mM Dglucose]. After 20 min pre-incubation at 37 C and 5% CO2, the preincubation medium was replaced by the induction medium, which contained 50 mM forskolin (Sigma Cat. #F6886), 0.1 mM rolipram (Sigma Cat. #R6520), 2 mM CaCl2, and 33 mM D-glucose, and incubated for 10 min. Lastly, the induction medium was replaced by the post-incubation medium, which was identical to the preincubation medium.
2.6. Immunocytochemistry, and confocal and N-SIM microscopy
2.11. PLA for detection of proteineprotein interaction
Super-resolution SIM (Nikon N-SIM) with a CFI Apo TIRF 100 oil objective lens (NA1.49) and iXon DU-897 EMCCD camera was used to image the neurons. Image processing was performed in Zen software (Carl Zeiss, Germany). Sample preparation for N-SIM is the same as for conventional microscopy.
Duolink in situ fluorescence (Olink Bioscience, Uppsala, Sweden) was used for detection of EB3 and IP3K-A interactions under cLTP. All procedures were performed as described in the manufacturer's manuals. Briefly, neurons were fixed with 4% PFA for 20 min. For cell permeabilization, cells were incubated in TBST containing 0.5% triton X-100. The following antibodies were used in the PLA: mouse anti-IP3K-A (1:500; homemade II4E) and rabbit anti-EB3 (1:500; Millipore, Cat. #AB6033). Neurons were incubated in blocking buffer containing primary antibodies for 1 h at RT. After extensive washing, the prepared PLA probes were added to the neurons, and the neurons were incubated in a humidity chamber for 1 h at 37 C.
2.5. Immunoprecipitation
2.7. Total internal reflection microscopy The TIRFM optics were incorporated into an upright Olympus BX51 microscope (Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan). A transmitting all-side polished dove-type prism (BK7,
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 1. IP3K-A binds to EB3. (A) Ponceau stained membrane (top) and Western blot (bottom) images of a GST pull-down assay with rat brain lysates. Full expression of GST and GSTEB3 is indicated by white arrows (GST) and yellow arrows (GST-EB3) at the expected size. (B) Schematic figure showing the immunoprecipitation assay for HEK293T cells expressing EGFP-EB3 and/or FLAG-IP3K-A. Western blot analysis against EGFP shows the interaction of EB3 and IP3K-A. (C) Representative total internal reflection fluorescence (TIRF) microscopy images of rat hippocampal neurons expressing EB3-EGFP and IP3K-A-mCherry. Yellow arrowheads indicate dendritic spines. (D) Representative structured illumination microscopy (SIM; Nikon N-SIM) images of rat hippocampal neurons. White arrows indicate co-localization of IP3K-A and EB3 in dendritic spine regions. Scale bars: 20 mm (C), 5 mm (D). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
The prepared ligationeligase solution was added, and neurons were incubated in a humidity chamber for 30 min at 37 C. Lastly, amplification-polymerase solution was added to the washed neurons. 2.12. Statistics Statistical significance was calculated by Student's t-test or oneway ANOVA with post hoc Tukey HSD or Dunnett's test using SPSS 12.0 (IBM) software. 3. Results 3.1. IP3K-A binds to EB3 EB3, well-characterized þ TIP protein in mature neurons, interacts with a wide spectrum of proteins. Particularly, most of EB3binding partners are cytoskeleton-related proteins. Considering that our protein of interest, IP3K-A, binds to both F-actin and microtubules [4,5], we deemed it highly plausible that IP3K-A
interacts with EB3. To test this possibility, we first conducted a glutathione S-transferase (GST) pull-down assay and found that the specific IP3K-A band was blotted only in the GST-EB3 lane and not in the GST-alone lane (Fig. 1A), suggesting that IP3K-A interacts with GST-EB3. Similarly, we carried out an immunoprecipitation assay with HEK293T cells expressing EGFP-EB3 and FLAG-IP3K-A. The Western blot results showed that GFP-EB3 was also precipitated by the FLAG antibody (Fig. 1B). To acquire visual evidence of the interaction of IP3K-A and EB3, we used two advanced imaging techniques that enabled fine spatial localization of IP3K-A and EB3. The first technique was total internal reflection fluorescence (TIRF) microscopy, which provides outstanding image quality with a reduced background. Many studies of dynamic microtubules have used TIRF microscopy to track EB3 [14,15]. When we doubly transfected primary hippocampal cultured neurons with IP3K-AmCherry and EB3-EGFP, TIRF imaging showed prominent colocalization of IP3K-A and EB3 in dendritic spine regions (Fig. 1C). The second technique was structured illumination microscopy (SIM; N-SIM, Nikon, Japan), which acquires super-resolution im patterns produced under different ages by analyzing the moire
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 2. Structural mapping of binding regions for EB3 and IP3K-A. (A) Schematic figure showing the functional domains of the EB3 and GST-EB3 constructs used in the pull-down assay. (B) GST-EB3 pull-down assay for mapping the IP3K-A-binding region. Full expression of various GST-EB3 constructs was confirmed by the Ponceau stained membrane (upper panel). White arrows indicate GST-EB3 constructs at the expected size. Western blot of GST-EB3 pull-down assay probed with a specific antibody against IP3K-A (lower panel). (C) Schematic figure displaying the functional domains of the IP3K-A and GST-IP3K-A constructs used in the pull-down assay. (D) Western blot analysis of the GST-IP3K-A pull-down assay with purified His-EB3.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 3. The binding affinity between IP3K-A and EB3 is regulated by PKA-dependent phosphorylation of IP3K-A at Ser119. (A) GST-EB3 pull-down assay of HEK293T cell lysates expressing EGFP or EGFP-IP3K-A constructs [wild-type (WT), S119E, and S119A]. Purified GST-EB3 was incubated with overexpressed HEK293T lysates. A Ponceau stained membrane (top) shows equal loading of IP3K-A in each lane. A Western blot (bottom) against the EGFP antibody shows the different binding affinities between EB3 and the IP3K-A constructs. Notably, a phosphorylation-dependent band shift of IP3K-A is observed in only the WT input lane (second lane; yellow arrow). (B) In vitro binding assay for purified IP3K-A and EB3 with the catalytic PKA subunit. The red arrow indicates the decreased EB3 band intensity (second and third panels). PKA-dependent phosphorylation of IP3K-A at Ser119 was confirmed by an anti-pSer119 antibody (fourth panel), while IP3K-A bands were the same (fifth panel). (C) Schematic figure shows changes in IP3K-A phosphorylation by PKA (top). The PKA inhibitor H89 decreases the phosphorylation level of EGFP-IP3K-A, resulting in a decrease in EB3-binding affinity. The PKA agonist forskolin (FSK) works in the opposite direction. Western blot analysis of GST-EB3 pull-down assay. (D) Summary graph of the GST-EB3 pull-down assay (WT-H89: 0.79 ± 0.06, WT-FSK: 0.41 ± 0.05, S119A-H89: 1.44 ± 0.25, S119A-FSK: 1.33 ± 0.07, mean ± SEM; one-way ANOVA [F (3, 8) ¼ 37.739, ***p < 0.001], Tukey HSD post-hoc test [WT-H89/WT-FSK: *p ¼ 0.035; WT-H89/S119A-H89: **p ¼ 0.002; WT-H89/S119A-H89: **p ¼ 0.006; WT-FSK/S119A-H89: ***p < 0.001; WT-FSK/S119A-FSK: ***p < 0.001; S119A-H89/S119A-FSK: p ¼ 0.766]). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Fig. 4. Rapid dissociation and reassociation of IP3K-A and EB3 complex during neuronal activation. (A) Temporal profile of pSer119 IP3K-A over the time course of cLTP induction. The first and second panels show the blot against the pSer119 IP3K-A antibody and its intensity profile. Anti-pSer119 peaked at 0 min (at the end of 10 min of cLTP induction) and reverted to basal levels within 30 min. (B) Quantification of phosphorylation levels of IP3K-A Ser119. Statistic values were calculated from three independent experiments (**p < 0.01, *p < 0.05, one-way ANOVA followed by post-hoc Dunnett's test). (C, D) Representative confocal images of hippocampal cultured neurons after a proximity ligation assay (PLA) in control (C) and cLTP (D) conditions. Red duolink spots indicate direct interaction of IP3K-A and EB3. Phalloidin 488 (green channel) staining was used to visualize the general structure of the neurons. Scale bar: 20 mm. (E) Quantification of the total number of duolink spots during cLTP induction (control: 47.5 ± 14.6, cLTP 0‘: 104.1 ± 27.0, cLTP 15‘: 136.2 ± 33.6, and cLTP 30‘: 150.1 ± 8.7; *p < 0.05, **p < 0.01; mean ± SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
phase-structured illumination [16]. We also found significant colocalization of IP3K-A and EB3 with this method (Fig. 1D), consistent with the results from TIRF imaging. 3.2. IP3K-A N-terminus is necessary for the binding of IP3K-A and EB3 Next, we conducted a domain-based mapping study using
several deletion mutants of EB3 and IP3K-A (Fig. 2A and C). Structurally, EB3 contains well-characterized functional domains, including a microtubule-binding domain, a coiled coil domain, and a þTIP-binding domain [2]. Six GST-EB3 constructs were designed with different combinations of these three domains to determine which domain is involved in the interaction with IP3K-A (Fig. 2A). The results of the GST-EB3 pull-down assay shows that IP3K-A bound to all deletion mutants of GST-EB3 except for the 130e251
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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deletion mutant that lacked both the microtubule-binding domain and the þTIP-binding domain (Fig. 2B). Inversely, we investigated the EB3-binding region of IP3K-A using purified His-EB3 and GSTIP3K-A constructs (GST-IP3K-A WT, 81e459, and 191e459). We found that EB3 binds to only the full-length wild-type (WT) construct (Fig. 2D), indicating that the IP3K-A N-terminus is necessary for the interaction of IP3K-A and EB3. 3.3. Binding affinity of IP3K-A and EB3 is regulated by PKAdependent phosphorylation In several studies, the binding affinity of EB proteins is regulated by post-translational modifications such as detyrosination and phosphorylation [17]. Phosphorylation of serine residues near the binding regions blocks interactions with EB proteins [18e20]. IP3KA contains well-characterized phosphorylation sites for PKA, protein kinase C (PKC), and Ca2þ/calmodulin-dependent protein kinase II (CaMKII) [3,21e23]. Among these, we focused on PKA-dependent Ser119 phosphorylation because this site is close to the N-terminal EB3-binding site and because Ser119 also controls the binding affinity between microtubules and IP3K-A [4]. To investigate the relevance of Ser119 phosphorylation to the binding affinity of IP3KA and EB3, we generated a phosphodeficient form of Ser119 (S119A; Ser / Ala) and a phosphomimic form of Ser119 (S119E; Ser / Glu). HEK293T cell lysates expressing EGFP, EGFP-IP3K-A WT, EGFP-IP3KA S119A, or EGFP-IP3K-A S119E were incubated with immobilized GST-EB3. As expected, EGFP-IP3K-A S119A showed stronger affinity for EB3 than S119E, suggesting that phosphorylation at IP3K-A Ser119 interferes with the binding between EB3 and IP3K-A (Fig. 3A). Interestingly, we observed a phosphorylation-related band shift in the GFP-IP3K-A WT construct (Fig. 3A; yellow arrow). This phospho-regulatory interaction is also possible during binding of microtubules and IP3K-A [4]. Therefore, we needed to check for a microtubule-independent interaction between EB3 and IP3K-A. To address this, we designed an in vitro direct-binding assay using purified His-EB3 and GST-IP3K-A recombinant protein together with the catalytic subunit of PKA (Fig. 3B). For GST-IP3K-A WT, the amount of bound EB3 was decreased when PKA was added with ATP (Fig. 3B, red arrow indicates the decreased intensity in the EB3 blot) but, for GST-IP3K-A S119A, there was no change. Phosphorylation of IP3K-A Ser119 (pSer119 IP3K-A) by PKA was detected with a specific phosphor antibody [4]. We further tested for a direct correlation between phosphorylation levels of IP3K-A at Ser119 and binding affinity with EB3. To bidirectionally adjust the phosphorylation level of IP3K-A at Ser119, we treated cells with forskolin (an adenyl-cyclase activator) or H-89 (a selective PKA inhibitor) (Fig. 3C). In the Western blot analysis of the GST-EB3 pull-down assay, reduced binding affinity was observed in the forskolintreated EGFP-IP3K-A WT group compared with the H-89-treated EGFP-IP3K-A WT group (Fig. 3C and D). No significant changes were observed in the EGFP-IP3K-A S119A groups regardless of treatment with forskolin or H89 (Fig. 3C and D), suggesting that pSer119 IP3K-A is essential for the regulation of EB3 binding.
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phosphorylation restored back to a base line level at late phase (Fig. 4A and B). We predicted that these dynamic changes of phosphorylation would affect to rapid rearrangement of IP3K-A and EB3 through cLTP induction. To confirm this hypothesis, we performed in situ PLA to investigate the total number of IP3K-A and EB3 complex at specific time points along cLTP induction. The result of PLA assay presents a prominent increase in the number of PLA spot which corresponds to the interaction of IP3K-A and EB3 (Fig. 4D) compared with the control (Fig. 4C). As predicted, the number of PLA spot gradually increased along the time course of cLTP induction (Fig. 4E), suggesting that more new interactions between IP3K-A and EB3 are formed during neuronal activation.
4. Discussion Here, we demonstrated that IP3K-A interacts directly with EB3 in a microtubule-independent manner (Figs. 1 and 2). EB3 binds to various proteins including þ TIPs and MAPs through a its C-terminus-binding motif. In general, many þ TIPs and MAPs bind to microtubules and/or EB family proteins using their basic and serine-rich regions [17]. These regions interact with the negatively charged sequences in microtubules and EB family proteins. Phosphorylation of þTIPs or MAPs reduces their binding affinity to microtubules and EB family proteins through changes in the electrostatic interactions [2,18]. Indeed, many þ TIPs or MAPs are the substrates of various kinases. For instance, glycogen synthase kinase 3 (GSK3)-dependent phosphorylation of residues surrounding the SxIP motif of CLASP2 interferes with binding to EB and microtubule tips [19]. In addition, PKA phosphorylates p150Glued and mediates its microtubule interactions [25]. As shown in this study, the binding affinity of IP3K-A to EB3 is regulated by PKA-dependent phosphoregulation of IP3K-A at Ser119 (Fig. 3). This similarity in regulatory mechanisms might result from the negatively charged EEY/F motif that is observed in both microtubules and EB3. During neuronal activation in response to synaptic stimulation, various signaling pathways including phosphorylation and dephosphorylation affect wide spectrum of cellular events such as cytoskeletal remodeling [26] and synaptic targeting of specific biomolecules [27] causing synaptic plasticity and morphological changes [24]. Precise spatiotemporal controls of signaling direction and intensity are crucial for delicate neuronal functions to transmit information effectively. Considering EB3 functions as the central hub of microtubule [28], transient phosphorylation by PKA triggers rapid dissociations and reassociations of IP3K-A and EB3, providing an activity-dependent rearrangement and recruitment of cytoskeletons to lead morphological remodeling of neuron during synaptic plasticity. Taken together, our results present that the activity-dependent binding of IP3K-A and EB3 and this interaction functions as a potent regulator of rearrangement or recruitment of microtubule cytoskeleton contributing synaptic plasticity during neuronal activation.
3.4. Activity-dependent interaction of IP3K-A and EB3
Author contributions
PKA is one of the most powerful regulators during neuronal activation and phosphorylation level of its substrates are tightly regulated to tune functional and morphological changes of neurons depending on the synaptic input [24]. To acquire the temporal profile of phosphorylation level of IP3K-A Ser119 during neuronal activation, we introduced cLTP protocol with Forskolin and prepared lysate samples at different time points of cLTP. The Western blot image shows the intensity of Ser119 phosphorylation reaches the peak level at early phase of cLTP, but the level of
D.L. and H.K. designed research. D.L., S.M., Y.C., and B.C. performed the research and analyzed the data. D.L., S.M., Y.C., and H.K. wrote the paper.
Conflicts of interest The authors declare no competing financial interests or potential conflicts of interest.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042
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Acknowledgements We thank all members of Hyun Kim's lab for critical comments on the manuscript. This work was supported by National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (NRF-2017M3C7A1079696 to H. Kim). Bioedit edited the manuscript. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.11.042. References [1] L.C. Kapitein, C.C. Hoogenraad, Building the neuronal microtubule cytoskeleton, Neuron 87 (2015) 492e506. [2] A. Akhmanova, M.O. Steinmetz, Tracking the ends: a dynamic protein network controls the fate of microtubule tips, Nat. Rev. Mol. Cell Biol. 9 (2008) 309e322. [3] M.J. Schell, Inositol trisphosphate 3-kinases: focus on immune and neuronal signaling, Cell. Mol. Life Sci. 67 (2010) 1755e1778. [4] D. Lee, H.W. Lee, S. Hong, B.I. Choi, H.W. Kim, S.B. Han, I.H. Kim, J.Y. Bae, Y.C. Bae, I.J. Rhyu, W. Sun, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase A is a novel microtubule-associated protein: PKA-dependent phosphoregulation of microtubule binding affinity, J. Biol. Chem. 287 (2012) 15981e15995. [5] M.J. Schell, C. Erneux, R.F. Irvine, Inositol 1,4,5-trisphosphate 3-kinase A associates with F-actin and dendritic spines via its N terminus, J. Biol. Chem. 276 (2001) 37537e37546. [6] H. Kim, J.P. Ko, U.G. Kang, J.B. Park, H.L. Kim, Y.H. Lee, Y.S. Kim, Electroconvulsive shock reduces inositol 1,4,5-trisphosphate 3-kinase mRNA expression in rat dentate gyrus, J. Neurochem. 63 (1994) 1991e1994. [7] I.H. Kim, S.K. Park, W. Sun, Y. Kang, H.T. Kim, H. Kim, Spatial learning enhances the expression of inositol 1,4,5-trisphosphate 3-kinase A in the hippocampal formation of rat, Brain Res. Mol. Brain Res. 124 (2004) 12e19. [8] W. Sun, Y. Kang, I.H. Kim, E.H. Kim, I.J. Rhyu, H.J. Kim, H. Kim, Inhibition of rat brain inositol 1,4,5-trisphosphate 3-kinase A expression by kainic acid, Neurosci. Lett. 392 (2006) 181e186. [9] I.H. Kim, S.K. Park, S.T. Hong, Y.S. Jo, E.J. Kim, E.H. Park, S.B. Han, H.S. Shin, W. Sun, H.T. Kim, S.H. Soderling, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase a functions as a scaffold for synaptic Rac signaling, J. Neurosci. 29 (2009) 14039e14049. [10] S. Windhorst, D. Minge, R. Bahring, S. Huser, C. Schob, C. Blechner, H.Y. Lin, G.W. Mayr, S. Kindler, Inositol-1,4,5-trisphosphate 3-kinase A regulates dendritic morphology and shapes synaptic Ca2þ transients, Cell. Signal. 24 (2012) 750e757. [11] B. Choi, H.W. Lee, S. Mo, J.Y. Kim, H.W. Kim, I.J. Rhyu, E. Hong, Y.K. Lee, J.S. Choi, C.H. Kim, H. Kim, Inositol 1,4,5-trisphosphate 3-kinase A overexpressed in mouse forebrain modulates synaptic transmission and mGluR-LTD
of CA1 pyramidal neurons, PloS One 13 (2018), e0193859. [12] D. Lee, S. Han, S. Woo, H.W. Lee, W. Sun, H. Kim, Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coli, Protein Expr. Purif. 97 (2014) 72e80. [13] D. Lee, J.H. Hyun, K. Jung, P. Hannan, H.B. Kwon, A calcium- and light-gated switch to induce gene expression in activated neurons, Nat. Biotechnol. 35 (2017) 858e863. [14] X. Hu, C. Viesselmann, S. Nam, E. Merriam, E.W. Dent, Activity-dependent dynamic microtubule invasion of dendritic spines, J. Neurosci. 28 (2008) 13094e13105. [15] J. Jaworski, L.C. Kapitein, S.M. Gouveia, B.R. Dortland, P.S. Wulf, I. Grigoriev, P. Camera, S.A. Spangler, P. Di Stefano, J. Demmers, H. Krugers, P. Defilippi, A. Akhmanova, C.C. Hoogenraad, Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity, Neuron 61 (2009) 85e100. [16] M.G. Gustafsson, Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 13081e13086. [17] A. Akhmanova, M.O. Steinmetz, Microtubule þTIPs at a glance, J. Cell Sci. 123 (2010) 3415e3419. [18] S. Honnappa, S.M. Gouveia, A. Weisbrich, F.F. Damberger, N.S. Bhavesh, H. Jawhari, I. Grigoriev, F.J. van Rijssel, R.M. Buey, A. Lawera, I. Jelesarov, F.K. Winkler, K. Wuthrich, A. Akhmanova, M.O. Steinmetz, An EB1-binding motif acts as a microtubule tip localization signal, Cell 138 (2009) 366e376. [19] P. Kumar, K.S. Lyle, S. Gierke, A. Matov, G. Danuser, T. Wittmann, GSK3beta phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment, J. Cell Biol. 184 (2009) 895e908. [20] T. Watanabe, J. Noritake, M. Kakeno, T. Matsui, T. Harada, S. Wang, N. Itoh, K. Sato, K. Matsuzawa, A. Iwamatsu, N. Galjart, K. Kaibuchi, Phosphorylation of CLASP2 by GSK-3beta regulates its interaction with IQGAP1, EB1 and microtubules, J. Cell Sci. 122 (2009) 2969e2979. [21] D. Communi, V. Vanweyenberg, C. Erneux, D-myo-inositol 1,4,5-trisphosphate 3-kinase A is activated by receptor activation through a calcium:calmodulindependent protein kinase II phosphorylation mechanism, EMBO J. 16 (1997) 1943e1952. [22] X. Hu, L. Ballo, L. Pietila, C. Viesselmann, J. Ballweg, D. Lumbard, M. Stevenson, E. Merriam, E.W. Dent, BDNF-induced increase of PSD-95 in dendritic spines requires dynamic microtubule invasions, J. Neurosci. 31 (2011) 15597e15603. [23] S.S. Sim, J.W. Kim, S.G. Rhee, Regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent protein kinase and protein kinase C, J. Biol. Chem. 265 (1990) 10367e10372. [24] K.P. Giese, K. Mizuno, The roles of protein kinases in learning and memory, Learn. Mem. 20 (2013) 540e552. [25] P.S. Vaughan, P. Miura, M. Henderson, B. Byrne, K.T. Vaughan, A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport, J. Cell Biol. 158 (2002) 305e319. [26] P.R. Gordon-Weeks, A.E. Fournier, Neuronal cytoskeleton in synaptic plasticity and regeneration, J. Neurochem. 129 (2014) 206e212. [27] F.T. Crump, K.S. Dillman, A.M. Craig, cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors, J. Neurosci. 21 (2001) 5079e5088. [28] G. Lansbergen, A. Akhmanova, Microtubule plus end: a hub of cellular activities, Traffic 7 (2006) 499e507.
Please cite this article as: S.J. Mo et al., PKA-dependent phosphorylation of IP3K-A at Ser119 regulates a binding affinity with EB3, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.042