ATP Enhances Neuronal Differentiation of PC12 Cells by Activating PKCr Interactions with Cytoskeletal Proteins Consuelo Marı´n-Vicente,†,‡ Marta Guerrero-Valero,†,§ Michael L. Nielsen,| Mikhail M. Savitski,| Juan C. Go ´ mez-Ferna´ndez,§ Roman A. Zubarev,*,‡ and Senena Corbala´n-Garcı´a*,§ Departamento de Bioquı´mica y Biologı´a Molecular (A), Facultad de Veterinaria, Universidad de Murcia, Apdo. 4021, E-30100 Murcia, Spain, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden, and Division of Molecular Biometry, Institute for Cell and Molecular Biology, Uppsala University, SE-75 124 Uppsala, Sweden Received July 19, 2010
PKCR is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCR, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and β-actin as potential interacting proteins. The colocalization of PKCR and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of β-actin were dramatically affected when PKCR was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCR signaling. Taken together, these data demonstrate that PKCR is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin. Keywords: cytoskeleton • proteomics • PKC;plectin • peripherin • ATP receptors
Introduction Protein Kinase C (PKC) is a family of Ser/Thr kinases that play an essential role in various cellular processes such as cell survival, proliferation and differentiation.1 In the nervous system, PKC has been linked to the regulation of axonal and dendritic growth mediated by neuronal growth factor (NGF),2 neural cell adhesion mediated by NCAM (neuronal cell adhesion mediated protein),3 neurotransmission4 and axonal regeneration,5 among others. Neurite formation is a primary morphological event in neuronal differentiation, which ultimately facilitates synaptic connections in neurons.6 Neuronal regeneration depends on the formation of neurites to repair injured or lost connections. These neuronal growth events require appropriate signals and promoter molecules. Among them, neurotrophins such as NGF, induce neurite outgrowth and a variety of metabolic changes in pheochromocytoma (PC12) cells, making them a very useful model of sympathetic neurons.7 This action is initiated by NGF binding to its primary receptor TrkA,8 which, in turn, leads to the activation of several signaling pathways, including ras, rap-1 and the cdc42-rac/rho family, as well as pathways regulated * To whom correspondence should be addressed. Senena Corbala´n-Garcı´a and Roman A. Zubarev. † These authors contributed equally to this manuscript. ‡ Karolinska Institutet. § Universidad de Murcia. | Uppsala University. 10.1021/pr100742r
2011 American Chemical Society
by MAP kinase (Mitogen Activated Protein Kinase), PI3-kinase (phosphatidylinositol 3-kinase)/Akt (protein kinase B) and phospholipase-C-γ/PKC.9,10 PC12 cells also express a wide variety of purinergic receptors11 and extracellular ATP plays an important role as neurotransmitter by signaling through P2X ionotropic and P2Y metabotropic receptors.12-16 ATP itself does not affect the differentiation of PC12 cells but enhances neurotrophininduced neurite outgrowth.17,18 In a previous study, we demonstrated that ATP cooperates with NGF during the neural differentiation of PC12 cells by increasing the length of the neurites;19 an effect that was inhibited when the cells were incubated in the presence of bisindolylmaleimide XI, a specific inhibitor of PKCR19 or by knocking-down PKCR synthesis by siRNA,20 suggesting a possible role for this isoenzyme in the neural differentiation process exerted by NGF and ATP. In addition, we have demonstrated that the phosphoinositide, PtdIns(4,5)P2, specifically binds to the polybasic region in the C2 domain of PKCR, serving as a target to anchor the enzyme in the plasma membrane in a Ca2+-dependent manner.19-26 However, exactly how ATP transduces signals downstream of PKCR in these cells remains unsolved. In the present study, we use a proteomics-based approach to explore specific PKCR signaling simultaneously stimulated by NGF and ATP. For this, PC12 cells were grown under three different conditions and overexpressed PKCR was immunoprecipitated; the interacting partners of each condition were Journal of Proteome Research 2011, 10, 529–540 529 Published on Web 10/25/2010
research articles identified by mass spectrometry and compared, allowing us to define a series of putative signaling partners. Plectin, fascin, drebrin, gelsolin, filamin A, peripherin and β-actin were found to be the major PKCR-interacting proteins when PC12 cells were differentiated with NGF and ATP. Double-immunofluorescence was used to determine the endogenous localization of these proteins, clearly showing that PKCR colocalized with several of them at different subcellular compartments when the cells were treated with NGF and ATP. Furthermore, depletion of PKCR resulted in two important events: first, impairment of the intermediate filament assembly of peripherin and the cross-linker protein plectin; second, impeding cortical β-actin polymerization.
Experimental Section cDNA Constructions. cDNAs encoding PKCR and mutants were cloned as described previously.22,27 All constructs were confirmed by DNA sequencing. The stability and viability of the mutated proteins were studied by reference to specific activity measurements.22,28 Cell Culture and Transfections. PC12 cells (ECACC, European Collection of Cell Cultures) were cultured on poly ornithine-coated culture coverslips in DMEM supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum. To promote cell differentiation, the medium was changed to DMEM supplemented with 5% heat-inactivated horse serum, 2.5% fetal bovine serum and 100 ng/mL of mouse submaxillary NGF (Alomone laboratories Ltd., Israel) and/or 100 µM ATP, as will be indicated in each case. The cells were kept under differentiating conditions for 2-3 days and the differentiation medium was replaced every 24 h. Cells were transfected with 4 µg DNA and Lipofectamine-2000 (Invitrogen, Carlsbad, CA) in 9 cm plates following the instructions provided by the manufacturer. PKCr-siRNA. siRNA transfections were carried out using 25mer PKCR-siRNAs (PRKCA Stealth RNAi, Invitrogen, Life Technologies, Paisley, GB), predesigned to target the mRNA of rat PKCR (GenBank accession number XM_001081588). The sense sequence of siRNA (PKCR-siRNA) was: 5′-CCCAAUCUUGCAAA GUGCAGUAUGA-3′. The nonspecific control, siRNA, was provided by the manufacturer. The cells were transfected by electroporation using a Gene Pulser Xcell (BioRad, Hercules, CA), in square wave mode, 900 V, 2 pulses of 4 ms and 5 s interval. After 16 h, the medium was changed to differentiation medium, as described above. Coimmunoprecipitation, Subcellular Fractionation, and Western Blot. For coimmunoprecipitation experiments, 5 × 106 differentiated PC12 cells were lysed with 50 mM Tris pH 7.5, 10% glycerol, 1% NP-40, 150 mM NaCl, complemented with a complete protease inhibitor cocktail (Roche, Indianapolis, IN) 4 µg/mL pepstatin A, 5 µM bestatin and phosphatase inhibitors cocktail I and II (Sigma). Cell lysates were precleared with agarose resin for 30 min. Then, the overexpressed HA-PKCR was pulled down by using an antibody which specifically recognizes the epitope (YPYDVPDYA) of the human HA-tag clone 3F10. This antibody was covalently bound to an agarose matrix (Roche, Indianapolis, IN) and the associated proteins were released by acidic elution with glycine buffer. Part of the sample was examined by Western blot using a polyclonal antiPKCR antibody. Detection and quantification were performed with chemiluminescence reagents (ECL Plus, GE Healthcare, Buckinghamshire, U.K.) by using a Typhoon 9410 scanner (GE 530
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Marı´n-Vicente et al. Healthcare, Buckinghamshire, UK). Subcellular fractionation assays were described in ref 27. Sample Preparation and Mass Spectrometry. The coimmunoprecipitated protein complexes were subjected to 1D10% SDS-PAGE and stained with colloidal Coomassie blue.29 Samples were run in different gels or some distance apart to minimize cross-contamination. The lanes were excised into 14 equally sized fractions, avoiding the disruption of bands. Each sample was in-gel reduced, alkylated and digested with modified sequence-grade trypsin (Promega, Madison, WI), as described previously in the literature.30 Finally, the samples were vacuum-centrifuged to remove all organic solvents and reconstituted prior to analysis in HPLC-grade water containing 0.1% TFA (Sigma, St. Louise, MO). All experiments were performed on a 7-T LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany), modified with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The high-performance liquid chromatography setup used in conjunction with the mass spectrometer (LC-MS) consisted of a solvent degasser, nanoflow pump, and thermostatted microautosampler (Agilent 1100 nanoflow system, Wilmington, DE). Chromatographic separation of peptides was achieved on a 15 cm fused silica emitter (75 µm inner diameter, Proxeon Biosystems) packed in-house with a methanol slurry of reversed-phase, fully end-capped Reprosil-Pur C18-AQ 3 µm resin (Dr. Maisch GmbH, Ammerbuch-Entrigen). Briefly, the tryptic peptides were autosampled onto the packed column at a flow rate of 500 nL/min and then eluted at a flow rate of 200 nL/min using a linear gradient of 4.5-40.5% acetonitrile in 0.5% acetic acid over 90 min and ionized by an applied voltage of 1.8 kV to the emitter. Analysis was performed using unattended data-dependent acquisition mode, in which the mass spectrometer automatically switches between a high-resolution survey scan (resolution ) 100 000 at m/z 400), followed by acquisition of both an electron capture dissociation (ECD)31 and collision activated dissociation (CAD) tandem mass spectra (resolution ) 25 000) of the two most abundant peptides eluting at this moment from the nano-LC column. Complementary fragmentation of the same peptide yielded different fragment ions, which increased the specificity of the sequence information. Using this complementary information32 for protein ID not only improves the confidence in protein identification performed by search engines, but, also for a fixed confidence level, identifies a larger number of peptides and proteins than when only one fragmentation technique is used.33 All data from the acquired MS/MS spectra was extracted into a set of peak list (dta-files) using TurboSEQUEST software (Thermo Electron; Waltham, MA). These dta- files contain the mass and the charge state of the precursor, as well as the m/z values and intensities of all the fragment ion peaks in the spectrum above a certain cutoff intensity value. An in-house written Java program was used for the extraction of complementary fragment masses before a database search. Briefly, two dta-files are present for each precursor ion, one generated by CAD and the other one by ECD fragmentation. The dta-files are deisotoped and charge-deconvoluted to the neutral state, as described by Zubarev and co-workers.33,34 The new dta-files containing complementary fragment masses were merged into a single file, which was searched using the Mascot Search Engine (Matrix Science, Boston, MA35) against the full NCBI mammalian database (version 3.15; downloaded February 2006) with carbamidomethyl cysteine as the fixed modification and
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PKCr Controls Neuronal Differentiation Table 1. Relative Quantification of PKCR Interacting-Proteins Found in Each Condition G.F.a
M.W.
protein
NGF/GMb
NGF+ATP/GM
NGF+ATP/NGF
c
518 145 292 725 193 187 40 052 33 196 50 437 52 058 42 066 114 132 78 223 103 466 86 413 96 232 89936 94795 77883 73985 72505 61021 66198 54349 46364 70317 55007 50344 38939 39691 16366
Plectin 2 gi40849888 Filamin A gi62667565 Clathrin, heavy polypeptide gi9506497 Slc25a3 gi47718004d Adenine Nucleotide Translocator gi400427 Elongation Factor 1R2 gi131092 Fascin 1 gi30023548 β-actin gi71620 ATPase R1, Na/K gi358959d Drebrin A gi297821 Actinin gi13591902 Gelsolin gi51260019 Elongation Factor 2 gi8393296d Valosin Cont. Prot. gi55217 Heat Shock Prot. 90 KDa gi194027 PKCR gi56914 Grp 75 gi1000439 Glucose Transporter 78 KDa gi121570 d Matricin gi347839 Keratin gi11935049d Peripherin gi2253157 DnaJ gi2281451 Traslation Initiation Factor gi55621482 Aromatic Laa Decarboxilase gi975309d Elongation Factor like protein gi16506251 Annexin A2 gi9845234 Aldolase A gi202837d Thy-1 protein precursor gi207310
0.7 2.5 1.5 1.4 2 1.5 17 24 1.4 4 2 0.4 1 0.5 0.6 0.7 2 2 1 0.6 1 1.5 0.8 1 0.8 2.6 0.8 2
20 13 3 1.0 1.5 1.8 72 384 1 83 1.5 3 0.5 0.2 1 1 0.3 0.8 0.5 0.5 8 0.35 0.4 0.7 0.4 3.3 0.7 5
20 5 1 0.8 0.7 0.3 4 16 0.8 23 0.6 4 0.5 0.5 0.3 1.4 0.1 0.2 0.5 0.8 17 0.2 0.5 0.6 0.6 1 0.8 2.4
1
3
4
5 6
7 8
10 12
a Gel fraction. b These ratios were corrected for the different levels of immunoprecipitated PKCR in each condition, as calculated by densitometry after Western blot. GM, growing media. c The average MW corresponding to each fraction in the SDS-PAGE were: F1, 250 kDa; F2, 200 kDa; F3, 150 kDa; F4, 100 kDa; F5, 75 kDa; F8, 50 kDa; F9, 45 kDa; F10, 37 kDa; F13, 25 kDa. d Internal standard for each fraction analyzed in the MS.
oxidized methionine as the variable modifications. Peptide searches were performed with an initial mass measurement tolerance of 10 ppm in MS mode, 0.6 Da in MS/MS mode and 1 missed cleavage. Peptides identified by MS/MS analysis were assigned to specific protein GI (GenInfo Identifier, National Center for Biotechnology Information [NCBI]) numbers. Only proteins identified by a minimum of two significant peptides with a mascot score higher than 30 were taken into consideration for this study (p < 0.05). Protein sequence redundancy was removed by comparing complete amino acid sequence of every identified GI entry with every other one; only entries differing by one or more peptides were considered independent proteins. Relative Quantification of the Identified Peptides. Proteins found by mass spectrometry in each condition with a protein score higher than 250 were chosen in order to be relatively quantified. We used two ways of quantifying our data: (1) most proteins were quantified using the data of Ion Intensity of each identified peptide, calculated by the open-source software, MSQuant (http://msquant.sourceforge.net); (2) when the proteins were only identified in one or two conditions by MS/MS, they were quantified by searching for the Relative Abundance of their three most abundant peptides in the full-MS spectrum. The abundances of common peptides (ion intensity or relative abundance) were compared between conditions for a specific protein by an X/Y dispersion analysis, passing through the origin and rendering a correlation factor considered as fold change. Ratios of 0.5-2 are considered not significant differences to 1, due to the variability intrinsic in the mass spectrometry assay. The similarity among the technical replicates was of 85 ( 15%,
considering the variability in the abundances of each protein in each replicate. The ratios obtained were also corrected for the different amounts of immunoprecipitated PKCR in each condition, as calculated by densitometry after Western blot. In addition, proteins with ratios close to 1 were used as internal standards for normalization among the different fractions analyzed independently in the MS. Fractions 1, 3, 4, 7, 10, and 12 (see Table 1), in which the relative quantification showed significant PKCR-interacting proteins were repeated two or three times (as biological replicates) and the results obtained were very similar. Immunofluorescence. Cells were fixed with 2% methanolfree formaldehyde for 10 min in phosphate-buffered saline (PBS) followed by an incubation with 50 mM ammonium cloride for 5 min. After permeabilizing with 0.1% Triton-X100 in PBS, cells were blocked with the product Image-iT-FX signal enhancer (Invitrogen, Oregon, USA) for 30 min at room temperature and then with 1% bovine serum albumin (BSA) for 30 min at room temperature. In the next step, cells were incubated with the corresponding primary antibodies in 1% BSA and 0.15% saponin for 3 h at room temperature. After blocking again with 1% BSA for 20 min, immunoreactivity was detected with the suitable fluorophore-conjugated secondary antibody (Alexa fluor 488 goat R-mouse and Alexa fluor 633 goat R-rabbit antibodies, Invitrogen, Eugene, OR) before mounting on slides with the Prolong Gold antifade reagent (Invitrogen, Eugene, OR). Antibodies used in these studies included antiplectin, anti-PKCR, antidrebrin, antiperipherin and antithy-1, all obtained from Abcam (Cambridge Science Park); Antifascin Journal of Proteome Research • Vol. 10, No. 2, 2011 531
research articles and antifilamin A obtained from Chemicon (Millipore Corporation, Bilerica, MA) and antigelsolin from BD Biosciencies (Belgium). Confocal Imaging and Data Analysis. Fixed and doubleimmunostained cells were examined with a Leica TCS SP2 AOBS confocal system (Leica, Heidelberg, Germany) using a Nikon HCX-PL-APO 63x/1.4-0.6 NA oil immersion objective. Co-localization analysis was performed with the Co-localization Finder plugin of ImageJ-NIH and showed images of the AlexaFluor 633 (red) merged with Alexa-Fluor 488 (green) secondary antibodies, and the colocalized pixels in orange (http:// rsb.info.nih.gov/ij/plugins/colocalization-finder.html). For each culture condition, at least five randomly chosen fields were scored from three different experiments. Images were collected in sequential mode to avoid bleed-through, at 512 × 512 resolution and pinhole at airy 1.0 width to provide a number of nm above the lateral resolution limit calculated by Abbe’s law.36 Taking into account that 13% of PKCR still remained after 72 h of treatment and to avoid a misinterpretation of the siRNA experiments, areas of the cell preparation, where endogenous PKCR was detected, were used to set the voltage of the photomultiplier. The rest of the cell preparation was then scanned to find down-regulated areas of PKCR where confocal images were collected without changing the voltage. The 3D reconstructions of PC12 cells were performed from confocal laser scanning images using Volume Viewer and 3D projection plug-ins (ImageJ-NIH) software. Statistical Analysis. Statistical significance was evaluated by two nonparametric tests: the Kruskal-Wallis test when three or more independent groups of sampled data were compared and the Mann-Whitney test when two groups of sampled data were compared. P < 0.05 was considered statistically significant. The statistical software program used was SPSS (Chicago, IL).
Results MS/MS Identification of Specific PKCr-Protein Complexes Established when PC12 Cells Are Differentiated into Neuronlike Cells with NGF and ATP. Previous results have suggested that NGF and ATP cooperate to increase the neuronal differentiation of PC12 cells17,19,37 and that PKCR is involved in the ATP-dependent process.17,19,26 To isolate and identify the specific PKCR-interacting proteins when PC12 cells are differentiated into neuron-like cells upon NGF and ATP treatment, we transiently transfected PC12 cells with a plasmid encoding HA-tagged PKCR. After transfection, the cells were incubated in three different media for 72 h: (1) Growing medium (GM), (2) Differentiation medium supplemented with 100 ng/mL NGF (NGF), and (3) Differentiation medium supplemented with 100 ng/mL NGF and 100 µM ATP (NGF+ATP). PKCR complexes formed under the three different conditions were isolated with immobilized anti-HA antibody, separated by 1D gel electrophoresis and digested with trypsin. The resulting peptides were analyzed by LC-MS/MS and assigned to specific proteins with the Mascot search engine. Thus, 139 proteins were identified in association with PKCR when PC12 cells were treated with growing media (GM), 210 when PC12 cells were differentiated with NGF and 202 when PC12 cells were differentiated with NGF+ATP. Evaluation of the entire data set to determine the intersections between the proteins identified in each condition, showed that 80 PKCR-associated proteins (40% of the total) were unique 532
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Figure 1. (A) Immunoblot analysis of the anti-HA immunoprecipitates derived from PC12 cells transfected with HA-PKCR and differentiated for 72 h with GM, NGF or NGF+ATP. Proteins were detected and quantified with chemiluminescence reagents, using a Typhoon 9410 scanner. (B) Venn diagram indicating overlapping and unique proteins. A comparison of the proteins identified by LC-MS/MS for each PKCR immunoprecipitate in the three different conditions used in this study is depicted. The GM condition includes 139 proteins, 47 unique to this condition, 11 common to the NGF+ATP condition and 19 common to the NGF stimulation. Differentiation with NGF includes 210 proteins, 80 unique for this condition, and 62 common to GM and NGF+ATP. NGF shares 49 proteins with NGF+ATP, while NGF+ATP stimulation presents 202 proteins, 80 unique to this condition. The number of common proteins among the three experiments was 62.
to PC12 cells differentiated with NGF+ATP, while another 80 (38% of the total) were unique to NGF differentiated cells (Figure 1). The cells treated with growing medium rendered 47 (34% of the total) unique PKCR-associated proteins. These three conditions share 62 common proteins. GM shares 11 proteins with NGF+ATP and 19 with NGF conditions. However, the NGF and NGF+ATP conditions have a greater number of common proteins between them (49 proteins, Figure 1) as should be expected. Gene ontology (Gene Ontology Consortium, htpp//www. geneontology.org) was used to classify the PKCR-interacting proteins identified for each neuronal differentiation condition using the Panther database.38,39 The unique interacting proteins of each condition were classified according to Biological Process, and in the case of PC12 cells differentiated with NGF+ATP most of the significant categories were related to cell structure and motility, and protein metabolism (Supporting Information, Table 1). However, the unique interacting proteins obtained under NGF conditions appeared in processes like carbohydrate metabolism and intracellular protein traffic. The proteins identified in GM conditions were assigned to the protein metabolism and modification category (Supporting Information, Table 1). The Molecular Function classification rendered very similar results, the unique proteins identified in NGF+ATP conditions were included into categories related to cytoskeletal and actin binding proteins (Supporting Information, Table 1), while the unique proteins identified under NGF conditions were related to oxidoreductases, dehydrogenases and mitochondrial carrier
PKCr Controls Neuronal Differentiation proteins (Supporting Information, Table 1). The unique proteins identified under GM conditions were included into translation elongation factor and chaperones categories (Supporting Information, Table 1). We were also interested in analyzing the group of 49 identified proteins shared by the NGF and NGF+ATP conditions. They were also classified according to the Molecular Function and Biological Process and, in both cases, the proteins were included in categories related with cell structure and motility, and cytoskeletal proteins (Supporting Information, Table 1). Taken together, these results showed that the immunoprecipitation of PKCR in PC12 cells differentiated either in NGF or NGF+ATP conditions, pulled-down a high number of interacting proteins related to cytoskeleton structure and function, especially in the case of NGF+ATP. However, the interacting proteins pulled-down in PC12 cells maintained under growing conditions (GM) showed a very different classification and were mainly related with protein metabolism and chaperones. This suggests that PKCR interacts with different partners, depending on the particular differential step the cells are undergoing, and that ATP+NGF induce a higher number of interactions with proteins related with the cytoskeleton than NGF alone. Relative Quantification of PKCr-Interacting Proteins Found in Each Condition. Another way to assess the relevance of the proteins interacting specifically with PKCR when PC12 cells are neurally differentiated with NGF+ATP is to perform a relative quantification of the immunoprecipitated proteins under each condition and compare the fold-changes of their relative abundances in the different cases (Table 1). It was observed that plectin, filamin A, fascin, β-actin, drebrin, gelsolin and peripherin exhibited higher abundances in the NGF+ATP than the NGF treatment (considering as significant, ratios higher than 2). Only filamin A40 and fascin41,42 have been previously described as PKCR-interacting proteins. At least 10 proteins appeared under-represented in the NGF+ATP compared with NGF (considering as significant, ratios lower than 0.5, Table 1), suggesting that ATP induces their dissociation from PKCR, thus leading to a higher degree of neural differentiation. Among the PKCR-interacting proteins determined when PC12 cells were differentiated with NGF+ATP, β-actin (42 kDa), and Fascin (55 kDa), appeared in fraction 1 of the electrophoresis gel, (higher molecular weights) probably due to the formation of multiple complexes that were pulled down by PKCR that were not dissociated with the SDS treatment. Biological Validation of PKCr-Interacting Proteins. We focused on the PKCR-interacting proteins that were relevant under NGF+ATP treatment of PC12 cells (listed in Table 1). For this, we explored the subcellular localization of the endogenous PKCR (red-AlexaFluor 633) and each potential interacting protein (green-AlexaFluor 488) by double-labeling immunofluorescence. Afterward, a colocalization analysis quantified the interaction between each pair of proteins analyzed (Figure 2 and Supporting Information, Table 2). The Pearson’s coefficients obtained in each case confirmed that plectin, filamin A, fascin 1, β-actin and peripherin colocalize better when PC12 cells are differentiated with NGF+ATP (Supporting Information, Table 2). Debrin exhibited an increase in colocalization with PKCR when the cells were differentiated with NGF, but
research articles however, no further colocalization increase was observed upon NGF+ATP treatment. PKCr Increases its Colocalization with Plectin when PC12 Cells Are Differentiated with NGF and ATP. To seek into the biological relevance of the PKCR-plectin interaction, we studied the subcellular localization of the endogenous proteins in PC12 cells by double-labeling immunofluorescence (plectingreen-AlexaFluor 488 and PKCR-red-AlexaFluor 633), after treating the cells for 72 h with growing medium (GM), NGF or NGF+ATP. When the cells were treated with GM, plectin was observed throughout the cytosol, distributed in small particles, around the nucleus, in the plasma membrane and in cell-cell contacts (Figure 2A). In contrast, PKCR was found homogeneously distributed through the cytosol in the same preparation (Figure 2A). When PC12 cells were differentiated with NGF, plectin was localized at the plasma membrane, around the nuclear envelope, probably due to its interaction with proteins located in these membranes, such as lamin B (inner) and nesprin-3 (outer),43,44 and in a filamentous staining pattern throughout the cytoplasm forming a network structure. This fiber-like distribution is due to the linking function that plectin exerts between the intermediary filaments and other members of the cytoskeleton architecture, such as microtubules and actin microfilaments.45,46 In this case, PKCR was localized in the cytosol, around the nuclear membrane and in some fibers in the cytoplasm, although it was more homogeneously distributed than plectin (Figure 2B). When PC12 cells were differentiated with NGF+ATP (Figure 2C), plectin was also observed at the plasma membrane and around the nucleus and showed a very noticeable filamentous staining. PKCR was also distributed in fibers and vesicles, particularly when the cells were differentiated with NGF alone. A colocalization analysis of the AlexaFluor488 and AlexaFluor633 images was performed using the Co-localization Finder plugin-ImageJ-NIH, as shown in Figure 2. There was a significant increase in colocalization when PC12 cells were differentiated with NGF (0.28 ( 0.01) or NGF+ATP (0.35 ( 0.02) rather than with GM (0.08 ( 0.01). Pixel correlation appeared mainly in some areas of the cytoskeletal-like structure, the nuclear membrane and in some areas of the plasma membrane (Figure 2B,C, merge and colocalization analysis). Due to the unexpected localization of PKCR in NGF+ATP differentiated cells, a subcellular fractionation of cell lysates was performed to quantify the proportion of protein localized in the cytosolic, Triton X-100 soluble and insoluble fractions. It was found that PKCR relocated from the cytosol to the insoluble fraction when the cells were differentiated with NGF+ATP (Figure 2D), suggesting that this treatment produces a higher accumulation of PKCR in the cytoskeletal fraction of PC12 cells. Down-Regulation of PKCr Severely Impairs the Localization of Plectin in the Cytoskeleton Network and Plasma Membrane. To further explore the role of the PKCR-plectin interaction, we induced the down-regulation of endogenous PKCR by transfecting PC12 cells with small interfering RNA (siRNA-PKCR) duplexes20 before inducing the neuronal differentiation process with NGF+ATP for 72 h. This siRNA treatment resulted in an approximately 87% down-regulation of the endogenous PKCR expression when determined by immunofluorescence or western-blot (Figure 3A). The cells were fixed and stained for immunofluorescence to detect plectin (green), PKCR (red) and nucleus (DAPI) (Figure 3B). As seen in a previous work,20 transfection of PKCR-siRNA duplexes Journal of Proteome Research • Vol. 10, No. 2, 2011 533
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Figure 2. Co-localization of PKCR and Plectin in PC12 cells differentiated by different treatments. PC12 cells were grown in growing media (GM) (A) or differentiated with NGF (B) or NGF+ATP (C), fixed with formaldehyde and processed for double-immunolabeling using monoclonal Ab to plectin (detected by using secondary Ab coupled to AlexaFluor 488, green images) and polyclonal Ab to PKCR (detected by using secondary Ab coupled to AlexaFluor 633, red images). RGB merged images including Alexa Fluor 488, Alexa Fluor 633 and DAPI (nuclei) are shown in the central panel (merge). The colocalization analysis was performed with the Co-localization Finder plugin of ImageJ-NIH. The left panel shows the scatter plot, where yellow represents the low intensity pixels and blue the high intensity pixels; the box in the diagram highlights the corresponding colocalized pixels shown in white in the overlaid image on the right panel and is also used to calculate the Pearson’s coefficient. (D) Subcellular fractionation of PC12 cells grown in GM, NGF and NGF+ATP media. The distribution of endogenous PKCR was analyzed by Western blot with anti- PKCR antibody. Lanes containing fractions corresponding to the cytosol, membrane and 1% Triton-X100 insoluble fraction are labeled as C, M and I, respectively. The graph represents the average of three different experiments ( SEM Black bars correspond to GM, light gray to NGF and dark gray to NGF+ATP conditions.
interfered in the neural differentiation process and a high percentage of PC12 cells did not develop neurites (Figure 3B), resembling cells treated with growing medium (compare Figures 3B and C). In the down-regulated PKCR cells, plectin formed a juxtanuclear aggregate and not the typical cytoskeletal fiber-like distribution appearing in NGF+ATP differentiated cells (Figure 3C, central panel). In addition, a significant decrease of plectin both in plasma membrane and cell-cell contacts was observed in the down-regulated PKCR cells (compare central panels in Figures 3B and C and the quantification bar chart in D). To explore plectin distribution in the depleted-PKCR cells as a whole, 3D reconstructions of plectin confocal images were obtained and compared with those of cells grown in GM (Figures 3E, F). Plectin was distributed around the nucleus and accumulated in high amounts at the cell-cell contacts in cells grown in GM (Figure 3E), the average 534
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height of the 3D reconstruction being 11.5 ( 2.0 µm (Figure 3G). However, in down-regulated PKCR cells grown in NGF+ATP, the 3D projections clearly showed that plectin accumulates in juxtanuclear aggregates (Figure 3F), while the perinuclear distribution disappeared, although the height of the 3D reconstruction was very similar to that obtained in the control cells (10.3 ( 2.5 µm, Figure 3G). No plectin accumulated at the cell-cell contacts at any cell level (Figure 3F), suggesting that PKCR controls the plectin function by impeding its localization in cell compartments that are critical for initiating neurite outgrowth. Down-Regulation of PKCr affects the Assembly of the Intermediate Filament Peripherin. Peripherin is a class III intermediate filament that is specifically expressed in neuronal tissue and at particularly high levels in PC12 cells differentiated with NGF.47-49 The results obtained in the coimmunoprecipi-
PKCr Controls Neuronal Differentiation
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Figure 3. Down-regulation of PKCR by siRNA affects the cytoskeletal assembly of plectin in NGF+ATP differentiated PC12 cells and its plasma membrane localization. (A) PC12 cells were transiently transfected with specific siRNA for PKCR, differentiated with NGF+ATP for 72 h and compared with cells treated under the same conditions in the absence of siRNA-PKCR. Three µg of each protein extract were prepared and endogenous PKCR expression was determined using Western blot and anti-PKCR antibody. Confocal micrographs showing PC12 cells transfected with 10 nM siRNA-PKCR and submitted to the differentiation process with NGF+ATP for 72 h (B) or grown in GM for 72 h (C). PKCR was detected by using secondary Ab coupled to AlexaFluor 633 (left), and plectin with a secondary Ab coupled to AlexaFluor 488 (central). The right panels show the overlay of differential interference contrast and Plectin staining. (D) Graph showing the percentage of cells exhibiting plectin at the plasma membrane of cells grown in GM and cells transfected with 10 nM siRNA-PKCR. 3D-reconstructed images of plectin staining in PC12 cells grown for 72 h in GM (E) or transfected with 10 nM siRNA-PKCR and grown with NGF+ATP (F). Volume Viewer (volume I) plug-in of ImageJ-NIH software was used to generate the 3D reconstructions from confocal laser images of 12.43 µm (E) and 13.6 µm (F). Images on the left show a top x-y view of the 3D reconstruction, yellow lines mark the different z-projections rendered in the 3D images on the right. Arrowheads in E point to cell-cell contact enrichment (top and bottom) and to the perinuclear plectin (bottom). In F, arrowheads point to the juxtanuclear aggregates (top) and to the cell-cell contact, in which plectin enrichment disappeared at any level of the cell (bottom). The color bar indicates the codes for minimum pixel intensity (0, black) and maximum pixel intensity (255, white). (G) Box plot representation of the heights calculated for plectin staining in the 3D reconstructions of undifferentiated cells and PKCR-down-regulated cells. The box for each condition represents the interquartile range (25-75th percentile) and, the black line within each box is the median value. Bottom and top bars of the whisker indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (closed circles). Each box represents the range of heights measured in the cells analyzed (n ) 29 and 23 for GM and siRNAPKCR, respectively). A Mann-Whitney test was performed to determine whether any statistically significance in height existed, P < 0.079 indicating that there was no significant difference. Journal of Proteome Research • Vol. 10, No. 2, 2011 535
research articles tation and the colocalization experiments suggest that PKCR interacts with peripherin, especially in NGF+ATP conditions (Table 1 and Supporting Information, Table 2). To explore this interaction, we performed a double-labeling immunofluorescence (peripherin-green-AlexaFluor 488 and PKCR-redAlexaFluor 633) of the endogenous proteins when PC12 cells were differentiated with NGF+ATP. In this case, the peripherin adopted a cellular distribution very similar to that show by plectin, of particular note being the filament network and the perinuclear localization (Figure 4A). The colocalization analysis of the images resulted in a Pearson’s coefficient of 0.22 ( 0.02, while the pixel correlation only appeared in some areas of the filament structure and the perinucleus (Figure 4A, merged), suggesting that PKCR and peripherin colocalize in restricted areas of these subcellular compartments. Peripherin was found forming juxtanuclear aggregates in down-regulated PKCR PC12 cells (Figure 4B) and did not reach the periphery of the cell membrane in 61 ( 22% of the cells analyzed (n ) 186 cells). This localization differed from that observed when PC12 cells were not differentiated and grown in GM (Figure 4C), when peripherin occupied the entire cell by forming a less intense juxtanuclear aggregate and small squiggles spread all over the cytosol to reach the periphery of the cell. Subcellular fractionation assays showed a higher amount of peripherin located in the insoluble fraction of downregulated PKCR cells than in undifferentiated cells (Figure 4D), probably due to the insolubility of the aggregates formed in the down-regulated PKCR cells. To gain further insight into the distribution of peripherin in these cells, we performed 3D reconstructions of peripherin confocal images (Figure 4F) and compared the protein distribution with that in cells maintained in GM (Figure 4E). In the latter, peripherin established a small reticular network that covered the whole volume of the cells, including the perinucleus (Figure 4E). However, in downregulated PKCR cells, peripherin accumulated in juxtanuclear aggregates that localize in the top-center of the cells, leading to an average height of only 6.5 ( 1.7 µm (Figure 4G) in the 3D reconstructions, while the undifferentiated cells were 12.2 ( 2.3 µm (Figure 4G) of height, suggesting that the lack of PKCR was affecting the initiation of the intermediate filament network organization, probably by altering the dynamic of the plectin-peripherin interaction. To test this hypothesis it was decided to investigate in which conditions plectin and peripherin colocalized better and similar results were found in the three differentiating conditions studied (Supporting Information, Figure 1). Figure 1A (Supporting Information) shows the confocal images obtained when the cells where differentiated with NGF+ATP and how both plectin and peripherin colocalize in the typical network they form (Pearson’s coefficient of 0.49 ( 0.03). We also explored the effect of down-regulating endogenous PKCR on plectin-peripherin localization and assembly in PC12 cells. As described above, both plectin and peripherin exhibited perinuclear localization and formed juxtanuclear aggregates (Supporting Information, Figure 1B). However, the Pearson’s coefficient calculated for these images was 0.39 ( 0.021, very similar to that obtained when the cells were grown with GM (0.45 ( 0.01, P < 0.026) and NGF (0.43 ( 0.07, P < 0.142). This, was still compatible with the colocalization of the two proteins in the juxtanuclear area (Supporting Information, Figure 1C), suggesting that although plectin and peripherin still interact when PKCR is down-regulated, this is not sufficient to initiate 536
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Marı´n-Vicente et al. the formation of the intermediate-filament network exhibited by PC12 cells differentiated with NGF and ATP. PKCr Interacts with β-Actin at the Neurite Extensions in NGF+ATP Differentiated PC12 Cells. The proteomics analysis also detected filamin A, fascin and β-actin as PKCR-interacting proteins, especially when PC12 cells were differentiated with NGF+ATP. Note that fascin and β-actin were identified in fraction 1 (Table 1), which contains proteins of higher molecular weight, suggesting that they were participating in a complex with the other proteins included in this fraction. The analysis showed that PKCR and β-actin colocalized at concrete areas of the plasma membrane (cortical actin) and in the extensions of the neurites (Supporting Information, Figure 2A). They gave a Pearson’s coefficient of 0.2 ( 0.01, which is significantly higher than the values obtained when the cells were differentiated with NGF or GM (0.09 ( 0.01 and 0.07 ( 0.01, respectively: see Supporting Information, Table 2), when no colocalization of the two proteins was detected. The effect of down-regulating PKCR on F-actin polymerization was also very significant, and F-actin appeared close to the plasma membrane in a very faint staining, reflecting a very low level of actin polymerization (Supporting Information, Figure 2B). This localization was also different from that of F-actin in nondifferentiated cells, where the protein was distributed homogenously through the cytosol (Supporting Information, Figure 2C). Taken together, these data imply that PKCR might also regulate actin polymerization dynamics during the neurite outgrowth induced by NGF+ATP stimulation, probably through the action of the two actin-binding proteins, fascin and filamin A.
Discussion In contrast to classical approaches to study signal transduction (based on the use of specific antibodies to identify signaling partners), proteomics offers the advantage of being able to identify unexpected partners, which could be very useful for defining new therapeutic targets in events such as neural regeneration. The present work has shed light onto the signaling pathways involved downstream of PKCR when PC12 cells are differentiated with NGF and ATP. The relative quantification of the PKCRinteracting proteins pointed to β-actin, and a collection of actinbinding-proteins such as plectin, peripherin, filamin A and fascin, all of which have been demonstrated to be involved in regulating different aspects of intermediate filaments and/or actin cytoskeleton organization and dynamics.45,50-53 PKCr Regulates the Cytoskeleton Assembly of the Intermediate Filament Peripherin, Mediated by Plectin. Plectin is an intermediate filament-associated cytolinker protein.45 In addition, there is increasing evidence that plectin serves an important function as platform for proteins involved in cellular signaling. Here, we demonstrate that plectin and the intermediate filament peripherin interact with PKCR in PC12 cells that are differentiated with NGF and ATP, and that both of them colocalize with the kinase in several subcellular compartments (Figure 2), such as plasma and nuclear membranes and the intermediate filament network. Given that PtdIns(4,5)P2 represents a substantial proportion of the phosphoinositides found in the plasma membrane of mammalian cells and that plectin and PKCR interact with the phosphoinositide,21,26,54,55 these lipid microdomains might serve as a target where plectin is phosphorylated by PKCR (Figure 5). Taking into account that such phosphorylation
PKCr Controls Neuronal Differentiation
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Figure 4. Influence of PKCR on peripherin cytoskeleton assembly. PC12 cells were transfected in the absence (A) or in the presence of 10 nM siRNA-PKCR (B), then submitted to differentiation with NGF+ATP for 72 h. Afterward, they were fixed with formaldehyde and processed for doubleimmunolabeling using monoclonal Ab to peripherin (detected by using secondary Ab coupled to AlexaFluor 488) and polyclonal Ab to PKCR (detected by using secondary Ab coupled to AlexaFluor 633). The nuclei were stained with DAPI to show the integrity of the cells. (C) Expression of endogenous PKCR and peripherin in PC12 cells treated with GM for 72 h. Immunofluorescence was performed as described above; white arrows point to the small squiggles formed by peripherin through the cytosol. (D) Subcellular fractionation of PC12 cells grown in GM, NGF and NGF+ATP media. The distribution of endogenous Peripherin was analyzed by Western blot with antiperipherin antibody. Grouped bars in the graph correspond to the cytosol, membrane and 1% Triton-X100 insoluble fractions quantified from three different experiments and the error bars are the SEM Black bars correspond to GM, light gray to NGF, dark gray to NGF+ATP and white bars to cells transfected with siRNA-PKCR grown in NGF+ATP. 3D-reconstructed images of peripherin staining in PC12 cells grown for 72 h in GM (E) or transfected with 10 nM siRNA-PKCR and grown with NGF+ATP (F). Volume Viewer (volume I) plug-in of ImageJ-NIH software was used to generate the 3D reconstructions from confocal laser images of 12 µm (E) and 8 µm (F). Gray images on top (E and F) represent a 3D projection of the Z stacks rendered by ImageJ, which overlapped with the differential contrast images (0 and 45° orientation). The left panel shows a top x-y view of the 3D reconstruction, yellow lines mark the different z-projections rendered in the 3D images on the right. Arrowheads in E point to the periphery of the cells (E). In F, arrowheads point to the juxtanuclear aggregates and to the cell-cell contact areas, in which Peripherin enrichment disappeared at any cell level. Color bar indicates the codes for minimum pixel intensity (0, black) and maximum pixel intensity (255, white). (G) Box plot representation of the heights calculated for peripherin staining in the 3D reconstructions of undifferentiated cells and PKCR-down-regulated cells. The box for each condition represents the interquartile range (25-75th percentile), and the black line within each box is the median value. Bottom and top bars of the whisker indicate the 10th and 90th percentiles, respectively. Outlier values are indicated (closed circles). Each box represents the range of heights measured in the cells analyzed (n ) 36 and 25 for GM and siRNA-PKCR, respectively). A Mann-Whitney test was performed to determine whether a difference in height existed, P < 0.001 indicating significant differences. Journal of Proteome Research • Vol. 10, No. 2, 2011 537
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Figure 5. Model for the signaling pathways activated downstream of PKCR upon ATP stimulation. A general scheme showing the different subcellular compartments and the wide variety of interactions of PKCR with the cytoskeleton components determined by LC-MS/MS and validated by specific double-immunolabeling. Note that the PtdIns(4,5)P2 molecules both in the plasma and nuclear membranes are represented in a bigger scale to facilitate the understanding of the scheme. Thus PKCR acts on the intermediate filament peripherin by controlling its phosphorylation and its interaction with plectin, which also cross-links F-actin and microtubules. At the leading edge, PKCR controls actin polymerization by phosphorylation of fascin and filamin A. It cannot be discarded that plectin and peripherin also play a role in the dynamics of actin polymerization at the leading edge in the neurite outgrowths.
decreases plectin interaction with intermediate filaments43 and favors plectin interaction with actin,54 this would be a way of accelerating cytoskeleton dynamics in areas close to the plasma membrane where neurite outgrowths are developing. Previous studies in our laboratory have demonstrated that the PtdIns(4,5)P2 pool of these cells does not hydrolyze completely when they are activated by ATP.19,20 This implies that in steady state conditions, IP3, DAG and PtdIns(4,5)P2 coexist, which would permit both plectin and PKCR to associate/dissociate from the plasma membrane, thus enabling plectin to exert its two functions by cross-linking the different elements of the cytoskeleton and by regulating actin dynamics. Regarding the nuclear colocalization of PKCR and plectin, it seems they might coincide at the inner nuclear membrane with lamin B (another intermediate filament), since it has been described that both proteins interact with the last one and that PKCR phosphorylates both plectin and lamin B.43,56 The carboxyl-terminus of lamin B binds to the V1 and part of the C2 domain of PKCR, which, in turn, phosphorylates the former in a phosphatidylserine/Ca2+ independent interaction.57 However, no information exists about the plectin domain interacting with lamin B or PKCR. One possibility is that PKCR localizes at the nuclear membrane to phosphorylate both proteins, one of which acts as the scaffolding. Pools of phosphoinositides have also been reported in the nucleus, but it is not known whether the cooperation of nuclear PtdIns(4,5)P2 is necessary. Our work also shows that plectin and PKCR colocalize at the cytoskeleton architecture of intermediate filaments, particularly when the cells are differentiated with NGF and ATP. It is known that plectin cross-links microtubules to intermediate filaments and to actin. In fact, peripherin, the major intermediate filament expressed in PC12 cells,58 was also identified in the immunoprecipitates analyzed, where they colocalized with both 538
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Marı´n-Vicente et al. PKCR and plectin when the cells were differentiated with NGF and ATP (Supporting Information, Table 2), suggesting that this interaction is also important in the development of the neural cytoarchitecture. However, no consensus phosphorylation sites for PKC have been described in peripherin to date. The only evidence that PKCs can interact with peripherin suggests that activated PKCε might be involved in the phosphorylation state of peripherin, although it is not known whether this effect is directly produced by PKCε-peripherin interaction or as a consequence of other PKCε-activated kinases.59 The fact that peripherin expression and intermediate-filament assembly are necessary for the shape transitions in neurons has been demonstrated by the use of peripherin genespecific small interfering RNA (siRNA).58 Under conditions of peripherin silencing, neurite extension and maintenance are completely inhibited, even though both microtubules and actin-filament arrays are normal. Taking into account that plectin and peripherin still colocalize in down-regulated PKCR cells, it is possible that the lack of plectin phosphorylation impedes the cytoskeleton dynamic remodeling needed to enhance and maintain this architecture. The 3D reconstructions of PC12 cells in which PKCR was down-regulated exhibited a different distribution of plectin and peripherin compared with their localization in undifferentiated cells (Figures 3 and 4 E,F), indicating that PKCR deficiency alters the distribution of these two proteins in the cells, thus promoting the formation of plectin and peripherin juxtanuclear aggregates, which although colocalized, do not contribute to the initiation of neurite outgrowth. Most of the localization defects observed in down-regulated PKCR cells are based on two observations: first, the insufficient presence of plectin and peripherin near the plasma membrane, which is probably one of the primary events needed to trigger the neurite outgrowth process; and second a lack of intermediate filaments in the cytoskeleton network. Both events are critical for the proper initiation, development and maintenance of the neuronal architecture.45,49,60 PKCr also Regulates the Actin Cytoskeleton. It is clear that PKC regulates the actin cytoskeleton in a wide range of cell types; a general impression obtained from the literature is that PKCR particularly has the capacity to alter the cytoskeleton in a promigratory manner.2 In this work, filamin A and fascin, two important actin-binding proteins, and β-actin itself were found to specifically interact with PKCR when PC12 cells were stimulated with NGF and ATP. Fascin is a 55 kDa actin-bundling protein involved in filopodia assembly and cancer invasion and the metastasis of multiple epithelial cancer types.51,61 Several works have shown that PKCR downregulates fascin function by phosphorylation at Ser39, which abrogates the activity of the amino-terminal actin-binding site.41,42 Such abrogation of this protein-protein interaction results in the increased formation of cell protrusions, the remodelling of focal adhesions and an increase in cell migration on fibronectin.42 In contrast, other works in colon carcinoma cells suggest that the maintenance of PKCRfascin complexes at the plasma membrane provides for a rapid cycling of fascin between the S39-phosphorylated and unphosphorylated states, which would facilitate the effective local positioning and remodeling of filopodia and protrusions for directional migration.62 Our results demonstrate that the activation of ATP receptors increases PKCR-fascin interaction, leading to a higher number and greater length of neurite outgrowths17,20 these are in accordance with the hypothesis
research articles
PKCr Controls Neuronal Differentiation proposed by Adams’s group, in which higher recycling of the PKCR-fascin complexes would facilitate remodeling of the leading edge to induce the initiation of neurite outgrowths and to increase their length by directional migration. It is well-known that phosphorylation of filamin A by PKCR influences the interaction of filamin with other ligands such as integrins or coreceptors, thereby regulating the association of the cortical actin with the plasma membrane and affecting cellular functions such as adhesion and migration.40,63 Our work reveals that ATP induces a higher number of PKCRfilamin A complexes in PC12 cells; taking into account that this interaction is regulated by Ca2+ and phospholipids, it is probable that the higher rate of Ca2+ pulses promoted by P2X and P2Y receptor stimulation with ATP produces a higher proportion of PKCR-filamin A interactions and, consequently, an acceleration of the actin polymerization that leads to the increase in neurite outgrowths observed in these cells. It is important to mention that a very recent study to determine the PtdIns(4,5)P2 interactome has identified PKCR, β-actin and filamin A as phosphoinositide interacting proteins, supporting the idea that they might be forming a functional complex.64 The huge decrease in β-actin polymerization when PC12 cells are depleted of PKCR (Supporting information, Figure 2B) clearly demonstrates the key role played by PKCR in orchestrating the complex processes that occurs during cortical actin polymerization. Observe that although some β-actin close to the plasma membrane is detected, this is not sufficient to induce the appearance of neurite outgrowths, probably due to the lack of phosphorylation of actin-regulating proteins that cannot be overcome by any other kinase, not even other isoforms of the same family.
Conclusions In conclusion, the results presented here have identified several target proteins of PKCR in the signaling pathways driven by the ATP-dependent neuronal differentiation of PC12 cells. It is demonstrated that PKCR is essential for establishing the cytoskeleton architecture of developing neural cells through its action at two levels: one, the assembly of the intermediate filament peripherin and its cytolinker plectin; second, the polymerization of cortical actin controlled mainly by fascin and filamin A at the leading edge, where neurite outgrowths and directional migration occur (Figure 5). The fact that plectin and peripherin are absent from the plasma membrane when the cells are depleted of PKCR also strengthen the idea that these two proteins are involved in initiating the neurite outgrowth process (Figure 5). Further studies are being undertaken to better understand the molecular mechanisms underlying this particular PKCR-dependent process.
Acknowledgment. This work was supported by Fundacio´n Me´dica Mutua Madrilen ˜ a, and Fundacio´n Se´neca, Regio´n de Murcia (grants FMM10195 and 08700/ PI/08 to SCG), and Ministerio de Ciencia e Innovacio´n-MICINN (grant BFU2008-01010/BMC to JCGF). R.Z. is supported by the Knut and Alice Wallenberg Foundation as well as the Swedish research council (grants 621-2004-4897 and 621-2003-4877 to RZ). C.M.V. holds a postdoctoral fellowship of the Ministerio de Ciencia e Innovacio´n-MICINN (Spain). Supporting Information Available: Supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.
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