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Originally published In Press as doi:10.1074/mcp.M800201-MCP200 on August 26, 2008.
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Molecular & Cellular Proteomics 7:2475-2485, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Toponomics Analysis of Functional Interactions of the Ubiquitin Ligase PAM (Protein Associated with Myc) during Spinal Nociceptive Processing*,S

Sandra Pierre{ddagger}, Christian Maeurer{ddagger}, Ovidiu Coste{ddagger}, Wiebke Becker{ddagger}, Achim Schmidtko{ddagger}, Sabrina Holland{ddagger}, Claus Wittpoth§, Gerd Geisslinger{ddagger} and Klaus Scholich{ddagger},

From the {ddagger} pharmazentrum frankfurt, ZAFES, Institute of Clinical Pharmacology, Klinikum der Goethe-Universität, 60590 Frankfurt, Germany and § MelTec GmbH, 39120 Magdeburg, Germany


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

Synapses are dynamic structures that expand, retract, and remodel to accomplish activity-dependent modification of neuronal circuits. During peripheral inflammation, the synaptic contacts between primary sensory neurons and dorsal horn neurons are modified in a way that the responsiveness of the system to subsequent stimuli is increased, resulting in hypersensitivity to noxious stimuli (1, 2). These activity-dependent changes in synaptic morphology and strength are based on many different mechanisms including alterations in ion channel and receptor activities due to phosphorylation by protein kinases, the translocation of {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)1 receptors to the postsynaptic membrane (3, 4), transcription- and translation-dependent changes in protein expression (2, 5), and ubiquitylation-mediated protein degradation (6). Often these changes occur only at a few synapses, making the analysis by common proteomics techniques extremely difficult. Multiepitope ligand cartography (MELC) is a novel imaging technology that utilizes biomathematical tools to identify and quantify protein complexes after consecutive immunohistochemical visualization of up to 100 proteins on the same sample (7–10). The data are processed to analyze colocalization patterns as "combinatorial molecular phenotypes (CMPs)" that are used for statistical evaluation. Based on these properties it is possible to correlate cellular localization of proteins with their function and to describe changes in the composition and distribution of protein complexes. The usefulness of this method to describe immunological changes in inflammatory skin and bowel disease as well as colorectal cancer has already been shown (9–11). Here we tested this method for its use to identify and characterize rare and intermittent activity-dependent changes of specific proteins during synaptic processes.

The E3 ubiquitin ligase PAM (protein associated with Myc) is a giant protein of 510 kDa. It was originally identified by its ability to bind specifically to the N terminus of Myc (12). Although PAM mRNA was found to be expressed at low levels in nearly all human tissues tested so far, its expression is exceptionally high in peripheral and central neurons (12–14). Loss-of-function mutations in Caenorhabditis elegans, Drosophila melanogaster, zebrafish, and mice demonstrated a crucial role of PAM in presynaptic terminal organization, regulation of synaptic growth, synaptogenesis, and neurite growth (15–19). These findings led to the realization that PAM is a negative regulator of synaptic growth during development. In addition to its role in neuronal development, PAM also modulates activity-dependent synaptic changes (14, 20). The intracellular signaling pathways that are controlled by PAM include cyclic AMP (cAMP)-, tuberin/mammalian target of rapamycin (mTOR)-, p38 mitogen-activated protein kinase (MAPK)-, and bone morphogenetic protein-signaling pathways (21–25). Most of its interacting partners are thought to be targeted for degradation through polyubiquitylation, although PAM-mediated ubiquitylation might be reversible, indicating monoubiquitylation of its substrates (26).

Here we used the formalin test, a common model for acute and inflammatory pain that depends on sensitization processes in the spinal cord, to study activity-dependent changes of PAM-containing protein complexes. MELC immunofluorescence microscopy with 37 fluorescence-labeled tags recognizing neuronal and cytoskeletal proteins was performed on spinal cords slices. The analysis led to the identification of 1390 PAM-related CMPs that describe differences between untreated and formalin-treated spinal cords. The majority of these motifs related to various ubiquitin-dependent processes and an interaction of PAM with the actin cytoskeleton. By verifying the previous unknown PAM/actin interaction using biochemical and cellular assays, we show that the MELC technology is a useful tool to describe synaptic processes and to identify novel protein/protein interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—
Sprague-Dawley rats (250–300 g) were supplied by Charles River Wiga GmbH (Sulzfeld, Germany). In all experiments the ethics guidelines for investigations in conscious animals were obeyed, and the procedures were approved by the local Ethics Committee.

Sample Preparation—
50 µl of a 5% formaldehyde solution (formalin) were injected subcutaneously into the dorsal surface of one hind paw of adult rats, and lumbar spinal cords (L4-L5) were excised after 45 min or 24 h. Spinal cords were snap frozen in liquid nitrogen and stored at –80 °C. After embedding the spinal cords in Tissue-Tek (Sakura Finetek, Leiden, Belgium), cryosections of 10-µm thickness were sliced using the cryotome Frigotom 2800 (–30 °C; Leica, Wetzlar, Germany) and applied on silane-coated coverslips. After fixing the tissue with acetone for 10 s at room temperature, the coverslips were stored at –20 °C. In preparation for the MELC procedure, the tissue was fixed once again with acetone for 10 min at –20 °C. Afterward the sample was rehydrated with Dulbecco's PBS (PAA Laboratories, Pasching, Austria), and nonspecific signals were blocked with normal goat serum (PAA Laboratories; diluted 1:15 with PBS) for 30 min at room temperature. After rinsing the sample five times with PBS, analysis of the tissue with the MELC technique was started.

MELC Library—
We used a MELC library of 37 fluorescence tags comprising antibodies, lectins, phalloidin, DNase, and propidium iodide (Table I and supplemental Table S1). The appropriate working dilutions, incubation time (15 min), and positions within the MELC run had been established and validated in the course of systematic experiments based on conventional immunohistochemistry and MELC calibration runs (7, 8).


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  		TABLE I MELC antibody and fluorescence tag library

Ehnert et al. refers to Ref. 14; Schmidtko et al. (47). TRITC, tetramethylrhodamine isothiocyanate; CamKII, calmodulin kinase II; PKB, protein kinase B; GFAP, glial fibrillary acidic protein; Syn. Systems, Synaptic Systems; Santa Cruz, Santa Cruz Biotechnology; Cell Signaling, Cell Signaling Technology; -, not applicable; CGRP, calcitonin gene-related peptide; Neu N, neuronal nuclei.

 
Data Acquisition by Toponome Imaging Cycler Multiepitope Readout—
The MELC technology has been described previously (7, 8). Briefly a slide with the spinal cord slice was placed on the stage of an inverted wide field fluorescence microscope (Leica DM IRE2; x63 oil lens; numerical aperture, 1.32). By a robotic process, first the slices were incubated for 15 min with predetermined fluorescence tags and rinsed with wash solution. Afterward the phase contrast and fluorescence signals were imaged by a cooled charge-coupled device camera (Apogee KX4, Apogee Instruments, Roseville, CA; 2048 x 2048 pixels; 2x binning results in images of 1024 x 1024 pixels; final pixel size was 286 x 286 nm). To delete the specific signal of the given tag before addition of the next, a bleaching step was performed. A postbleaching image was recorded and subtracted from the following fluorescence tag image during the data analysis. Performance of the first three MELC cycles with PBS and fluorochrome-labeled mouse IgG intrinsic fluorescence and unspecific tag binding was controlled. Pipetting, recording all image data, and coordinating all system components were controlled by software developed by MelTec GmbH (Magdeburg, Germany). Three visual fields were recorded simultaneously in each MELC run.

Data Analysis—
Using the corresponding phase-contrast images, fluorescence images produced by each tag were aligned pixelwise. The alignment reached a resolution of ±1 pixel. Images were corrected for illumination faults using flat field correction. Postbleaching images were subtracted from the following fluorescence tag images. Finally cases of section artifacts were excluded as invalid by a mask-setting process. Preprocessed image data were subjected to binarization. The thresholds automatically generated by the system were validated and adjusted manually for each fluorescence signal. The expression of a protein was set to the value of 0 for a signal below the threshold and to 1 for a signal above the threshold in projection to a pixel. Superimposed binarized images composed a matrix of CMPs that represented a binary (yes/no) code of n epitope expression in relation to each pixel (286 x 268-nm2 area) of a visual field (1024 x 1024 pixels). Thus, this MELC approach detected a theoretical maximum of as high as 2n different CMPs. Further analysis dealt with CMP motifs characterizing corresponding pixels. These CMP motifs are defined as pixel-related code of 1/0/wildcard ciphering. We used TopoMiner software packages (MelTec GmbH) to search for CMP motifs, whose overall frequency differs significantly in two different sample groups using the Wilcoxon rank-sum test. In detail, TopoMiner calculated the relative frequency of CMP motifs in relation to the number of all valid pixels of the observed visual field or to the frequency of predefined CMP motifs (base motifs). The search through the space of motifs was performed in a sequentially ordered strategy: all single epitopes and combinations of two, three, four, and so on (n = epitopes) were searched. Because of the large number of possible motifs and because of restricted computational time, the search depth was limited to n = 5. TopoMiner analysis was used to compare untreated animals and animals 45 min and 24 h after formalin injection.

RT-PCR—
2 µg of total RNA from rat tissue, PC12 cells, or HeLa cells was annealed with 10 ng of oligo(dT) primer and reverse transcribed using reverse transcriptase (Promega, Madison, WI) for 60 min at 37 °C. Amplification of the Myc-binding domain (MBD) domains was performed with the equivalent of 130 ng of reversed RNA and the following primers: MBD1, TTC TGT GAG AGT GAT GAA; MBD2, TGA CCT CAA CTT TGC AGC; and MBD3, TGG ACG CCA GGG GCT ATT.

Expression and Purification of PAM and Its Domains—
Full-length PAM was purified as described previously (14). The C terminus of human PAM was cloned from a cDNA fragment (bp 9191–13923) that was generated by RT-PCR from HeLa cells, inserted in pCR-XL-TOPO (Invitrogen), and subcloned with EcoRI (bp 12903–13923) in pTrcHisB (Invitrogen). The splice variants of the MBD of PAM were cloned from an RT-PCR (primers MBD2 and MBD5 (ATT GAA ATA GAT GCT GGC CTT)) from rat or human mRNA, inserted in pCR4-blunt-TOPO (Invitrogen), and subcloned with KpnI and XhoI in pTrcHisA. BL21 bacteria expressing the C terminus of PAM (C-PAM) were lysed for 20 min in 50 mM Tris, pH 8.0, 300 mM NaCl, 10 mM imidazole, 0.1% Triton. After sonication and centrifugation (18,000 x g for 15 min at 4 °C), the supernatant was incubated with 1 ml of nickel-nitrilotriacetic acid (Qiagen, Monheim, Germany) for 1 h and washed three times with lysis buffer and once with 50 mM Tris, pH 8.0. Proteins were eluted with 50 mM Tris, pH 8.0, 150 mM imidazole and concentrated with Vivaspin 30 columns (Millipore, Bedford, MA). If necessary the proteins were loaded on a Mono Q column (Amersham Biosciences) and eluted with an NaCl gradient (150–500 mM).

F-actin Cosedimentation Assay—
The assays were performed as described previously (27). Briefly 20 µg of rabbit skeletal muscle actin (Tebu-Bio, Offenbach, Germany) were polymerized for 30 min at room temperature in polymerization buffer (2 mM MgCl2, 1.2 mM ATP, 50 mM KCl, 0.2 mM CaCl2, 0.5 mM dithiothreitol, 5 mM Tris, pH 8.0) in a total volume of 100 µl. Then 3.5 µg of the indicated proteins were added, and the reaction was diluted with polymerization buffer to a final volume of 400 µl. The reactions were centrifuged for 1 h at 100,000 x g, and the pellets were washed three times with PBS, resuspended in gel loading buffer, and analyzed with 12% SDS-PAGE.

In Vitro Ubiquitylation—
The assay was performed in 40 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM ATP, 0.5 mM DTT, 50 nM rabbit E1 (Calbiochem), 500 nM E2 proteins (Boston Biochem, Cambridge MA), 0.15 µg/µl His-ubiquitin (K48R) (Calbiochem). Actin (200 ng) was incubated in the absence or presence of full-length PAM (600 ng) for 90 min at 27 °C. The reaction was terminated with gel loading buffer. G- and F-actin were generated by incubating 200 ng of actin in 10 µl of depolymerization buffer (5 mM Tris, pH 7.4, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT) on ice or polymerization buffer (5 mM Tris, pH 7.4, 0,2 mM CaCl2, 1.2 mM ATP, 0.5 mM DTT, 2 mM MgCl2, 50 mM KCl) at room temperature for 1 h.

Immunocytochemistry—
Primary spinal cord cultures from rat embryos were prepared as described previously (28). HeLa and PC12 cells (German Resource Centre for Biological Material, DSMZ, Braunschweig, Germany) were grown in RPMI 1640 medium with 10% FCS (all from Invitrogen). To monitor distribution of PAM and F- or G-actin, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were blocked for 1 h in 3% BSA in PBS and then incubated for 1 h with the anti-PAM antibody followed by incubation with FITC-labeled goat anti-rabbit antibody (Sigma). Finally Texas Red-labeled phalloidin was applied for 1 h.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Given the complex changes involved in the activity-dependent regulation of synaptic functions and the fact that these changes often occur only at a limited number of synapses, we aimed to evaluate the MELC technology for its use to study these synaptic processes. Therefore, we first established a library of 37 fluorescence tags that recognize proteins involved in synaptic processes as well as cell and tissue markers in several independent MELC runs (Fig. 1, Table I, and supplemental Table S1). We used the formalin test, a commonly used pain model that depends on sensitization processes in the spinal cord, and compared the spinal cords from untreated rats with spinal cords that were removed 45 min or 24 h after formalin injection in one hind paw. Notably 45 min after formalin injection sensitization processes in the spinal cord are based on posttranslational modifications such as phosphorylation and ubiquitylation events, the recruitment of proteins to the synapses (e.g. translocation of AMPA receptors), and dynamic changes such as enhanced trafficking of synaptic vesicles (2–6). 24 h after formalin injection long term synaptic alterations took place that are based on gene expression changes (1, 2).


Figure 1
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  		FIG. 1. Visualization of protein expression and localization in the lumbar spinal cord using MELC technology. Single MELC images of six exemplary fluorescence tags on the same spinal cord slice and the merged image of the six indicated proteins in false color are shown (IB4, purple; NeuN, green; glial fibrillary acidic protein (GFAP), yellow; PAM-N, red; propidium iodide (PI), blue; mono/polyubiquitin (ubi), white). Laminas I–III are depicted in the merged image. Lamina II is separated into the outer (IIo) and the inner (IIi) part. The white bar represents 50 µm.

 
The expression of the 37 fluorescence tags was mapped in three animals per group (untreated and 45 min and 24 h after formalin injection) using two to four slices from each lumbar spinal cord. We identified 1667 CMP motifs that were distinct between animals without treatment and animals undergoing the short term formalin treatment (45 min) on a significance level of p < 0.005. 2557 CMP motifs distinguished the spinal cords of controls and long term treatment (24 h). Interestingly both formalin-treated groups were distinguished by 4042 CMP motifs, which reflects the different nature of the activity-dependent changes that occur at these two time points (examples are shown in supplemental Tables S2 and S3).

Previously it was demonstrated that in different MELC runs single marker-labeled cells or tissues exhibit comparable fluorescent counts, resulting in a stability index of 0.97 and 0.94, respectively (8, 9). To confirm these observations for spinal cord tissue, we compared exemplarily the relative frequencies of signals from antibody against the N terminus of PAM (PAM-N) and PAM-C. Only small non-significant variations were seen in the relative frequencies between the three groups (Fig. 2A). However, the similar relative frequencies for PAM-C and PAM-N in the three groups suggested no change of the PAM protein levels; this was confirmed by Western blot analysis (Fig. 2B).


Figure 2
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  		FIG. 2. Stability and reproducibility of signals from PAM antibodies. A, the relative frequency of the fluorescent signals from C-PAM and PAM-N antibodies is shown for spinal cords from untreated animals (control) and from the two treatment groups, 45 min or 24 h after formalin injection in the hind paw. The data are shown as average of 8–10 MELC runs. The data are shown as average of 8–10 MELC runs ± S.E.M. B, Western blot for PAM expression with spinal cord lysates (30 µg) from untreated animals and spinal cord lysates 45 min or 24 h after formalin injection. HSP70 was used as loading control.

 
To evaluate the applicability of the MELC technology for the investigation of synaptic ubiquitylation events, we first tested whether known ubiquitylation processes can be visualized with this method. NMDA receptor density has been described to be regulated by ubiquitylation after synaptic stimulation, and the ubiquitylation of the NMDA receptor subunits NR2A and NR2B is well established (29, 30). The relative frequencies of NMDA receptor subunits NR2A and NR2B together (Fig. 3A) or alone (data not shown) were not altered in spinal cords after formalin injection into one hind paw suggesting no overall change in the protein expression levels. However, the colocalization of NR2A and NR2B with ubiquitin and the synaptic marker syntaxin1A increased significantly after formalin treatment (Fig. 3B). As expected this increase was more pronounced 45 min after formalin injection (acute phase) in contrast to 24 h after treatment (late phase) where gene expression changes dominate. The colocalization of NR2A, NR2B, and ubiquitin seemed to be restricted to synapses as indicated by the finding that only in the absence of the synaptic marker was no increase in the NR2A/NR2B/ubiquitin colocalization observed (Fig. 3C). The same finding was seen with an antibody recognizing mono- and polyubiquitylated proteins instead of a ubiquitin antibody indicating that this colocalization represents an ubiquitylation of NR2A and/or NR2B (Fig. 3D). To verify the pain-related nature of this interaction, we studied whether or not the colocalization of NR2A, NRB, syntaxin1A, and ubiquitin is localized in the superficial laminae of the spinal cord. Indeed the CMP motif was found in the superficial laminae of the spinal cord as shown by covisualization with the lectin Griffonia simplicifolia IB4 that recognizes small diameter, non-myelinated dorsal root ganglia neurons whose central axons terminate in inner lamina II of the spinal cord (31) (Fig. 3E).


Figure 3
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  		FIG. 3. Toponome analysis of NR2A and NR2B colocalization with ubiquitin and syntaxin1A in the spinal cord. A, box plot analysis of NR2A and NR2B colocalization shows no changes in control and the two treatment groups (45 min and 24 h after formalin injection into a hind paw). B, box plot analysis of NR2A and NR2B colocalization with syntaxin1A and ubiquitin shows an intermittent increase in spinal cord 45 min after formalin injection. Statistical analyses were performed using the Wilcoxon rank-sum test. The respective significance levels are depicted within the graphs. C, box plot analysis of NR2A and NR2B colocalization with ubiquitin in the absence of syntaxin1A shows no increase. D, box plot analysis of NR2A and NR2B colocalization with syntaxin1A and the FK1 antibody against mono- and polyubiquitinated proteins shows an intermittent increase in spinal cord 45 min after formalin injection. E, localization of the motif representing the colocalization of NR2A, NR2B, syntaxin1A, and mono- and polyubiquitylated proteins (red) in a spinal cord 45 min after formalin injection. Lamina II is separated into the outer (IIo) and the inner (IIi) part. IB4 staining for lamina IIi is shown in yellow. The white bar represents 10 µm.

 
After we confirmed that ubiquitylation events are detectable with the MELC technology, we analyzed the data concerning PAM-related CMP motifs in the spinal cord. With 687 (41%) and 703 (27%) motifs 45 min and 24 h after formalin injection, respectively, a high number of the motifs were connected with PAM indicating a central role of PAM in spinal nociceptive processing (Table II). 37% of the PAM-related CMP motifs that distinguish spinal cords of untreated animals with short term formalin-treatment were connected to ubiquitin or mono- and polyubiquitylated proteins (Table II). This number decreased to 9% when spinal cords from untreated animals were compared with spinal cords 24 h after the formalin injection, indicating that PAM executes its function as a ubiquitin ligase predominantly at the early stages of central sensitization.


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  		TABLE II Numeric distribution of different CMP expression in spinal cords of controls and 45 min or 24 h after formalin injectiona

Lumbar spinal cords were taken from untreated animals and 45 min or 24 h after formalin injection. The given values are the total numbers of significantly up- or down-regulated expression of combinatorial molecular phenotypes within the three groups (p < 0.005).

 
Then we selected motifs containing PAM and ubiquitin that showed a significant increase in their relative frequencies after formalin treatment. CMP motifs that retained significance in the absence of PAM were excluded from the further analyses. Finally three motifs were obtained. The first motif contained PAM, ubiquitin, F-actin (visualized by phalloidin staining), and tuberosclerosis complex 2 (TSC2), a known substrate for the ubiquitin ligase activity of PAM. The motif showed a 5-fold increase in the relative frequency between controls and the short term formalin treatment (Fig. 4A). The second motif contained PAM, ubiquitin, F-actin, and the metabotropic glutamate receptor 5 (mGluR5) and showed an 8.8-fold increase in the relative frequency between controls and the short term formalin treatment (Fig. 4B). The third motif contained PAM, ubiquitin, F-actin, and the AMPA receptor GLUR1 and showed after short term formalin treatment a 4.4-fold increase in its relative frequency (Fig. 4C). Importantly in all cases significance was not reached if PAM was not included in the motif (data not shown). As seen for NR2A and NR2B (Fig. 3) the interaction was most evident in spinal cords 45 min after formalin injection in one hind paw. The relevance of the findings is underlined by the fact that TSC2 is a known substrate for PAM and that both receptors, GLUR1 and mGluR5, are known substrates for ubiquitin ligases (32, 33). Furthermore other glutamate receptors such as the AMPA receptors GLUR2 and GLUR3, the kainite receptor GLUR5, and the NMDA receptor subunit NR1 showed no significant motifs connecting them with PAM and the ubiquitin system. Finally several adenylyl cyclase isoforms are well documented to be inhibited by PAM (21, 22, 34), and the relevance of this interaction for the formalin test was shown recently (14). However, previous reports also suggest that the inhibition of adenylyl cyclases is independent of the ubiquitin ligase activity of PAM (21, 22, 34). In support of these reports, no significant changes for CMP motifs that contained PAM, adenylyl cyclase type 1, and ubiquitin were seen between the treatment groups (Fig. 4E).


Figure 4
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  		FIG. 4. PAM colocalization with TSC2, GLUR1, mGluR5, and adenylyl cyclase type 1. A and E, box plot analysis of relative frequencies for CMP motifs containing PAM, TSC2, and ubiquitin in the presence (A) and absence (E) of F-actin. Statistical analyses were performed using the Wilcoxon rank-sum test. The respective significance levels are depicted within the graphs. B and F, box plot analysis of relative frequencies for CMP motifs containing PAM, mGluR5, and ubiquitin in the presence (A) and absence (F) of F-actin. C and G, box plot analysis of relative frequencies for CMP motifs containing PAM, GLUR1, and ubiquitin in the presence (C) and absence (G) of F-actin. D and H, box plot analysis of relative frequencies for CMP motifs containing PAM, adenylyl cyclase type 1 (AC1), and ubiquitin in the presence (D) and absence (H) of F-actin.

 
Interestingly significance for the colocalization of PAM and ubiquitin with TSC2, GLUR5, or mGluR5 was only reached when F-actin was also included in the motif (Fig. 4, E–H). Moreover we found a significant increase in the colocalization of PAM, ubiquitin, and F-actin alone in spinal cords 45 min after formalin injection (Fig. 5A). Motifs containing only ubiquitin and PAM or ubiquitin and F-actin did not show a significant change in their frequencies (Fig. 5B and data not shown). Moreover 45 min after formalin injection the colocalization of PAM and F-actin was significantly increased in the spinal cords (Fig. 5B). Taken together the data suggest an important role of F-actin for the ubiquitin ligase activity of PAM.


Figure 5
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  		FIG. 5. PAM binds to F-actin. A, box plot analysis of relative frequencies for CMP motifs containing PAM, F-actin, and ubiquitin shows an intermittent increase in spinal cord 45 min after formalin injection. B, box plot analysis of relative frequencies for the indicated CMP motifs in untreated rats and 45 min after formalin injection. Statistical analyses were performed using the Wilcoxon rank-sum test. The respective significance levels are depicted within the graphs. C, Western blot (WB) analysis of cosedimentation experiments using purified F-actin, PAM, and BSA. D, immunocytochemical analysis of primary spinal cord neurons for PAM (green), phalloidin (red), and together as the merged image (merge). con., control; ns, not significant.

 
Because so far the data suggest that nociceptive stimulation increases the colocalization of PAM and F-actin and that this colocalization is a necessity for the ubiquitylation of PAM substrates, we aimed to verify the interaction between PAM and F-actin. In cosedimentation assays with purified human PAM (supplemental Data 1A) and F-actin, we were able to show cosedimentation of PAM and F-actin suggesting a direct interaction of both proteins (Fig. 5C). Finally immunocytochemical analysis of primary spinal cord neurons showed a colocalization of PAM with the phalloidin staining for F-actin in neurites and in parts of growth cones (Fig. 5D). Taken together the data strongly suggest that PAM and actin are directly interacting with each other.

To locate the actin binding site of PAM, we searched for PAM domains that are linked to cytoskeletal functions. We found a filamin/ABP 280 repeat profile (PROSITE number PS50194) that is observed in many actin-cross-linking proteins. In close proximity to this profile is the domain that originally was found to bind Myc (MBD) (12). We amplified the MBD of PAM using RNA from rat and human origin and received multiple PCR products representing alternative splice products (Fig. 6, A and B) (36). The corresponding proteins were expressed in bacteria and then tested in cosedimentation assays for their ability to bind to F-actin. Interestingly the splice variants behaved very differently in the cosedimentation assay. The highest rate of cosedimentation was seen with a splice variant lacking exons 53 and 54 ({Delta}2MBD; Fig. 6, A and C). The amount of the MBD variants cosedimenting with F-actin decreased reciprocally with an increase in their exon number (Fig. 6, A and C) suggesting that the affinity of PAM toward F-actin differs strongly between its splice forms. The C-PAM that harbors the RING finger domain was also cloned, expressed, and purified (supplemental Fig. 1B). C-PAM was catalytically active (supplemental Fig. 1C) but did not cosediment with F-actin (Fig. 6C). Notably the {Delta}2MBD splice variant that shows the best binding capacity to F-actin in the cosedimentation assay is one of two predominantly expressed splice variants found in the spinal cord (Fig. 6B). Next we studied the functional consequences of the F-actin/PAM interaction for both proteins. Full-length PAM purified from HeLa cells did not alter actin polymerization or F-actin depolymerization in in vitro assays independently of the absence or presence of HeLa lysates (supplemental Fig. 2). Also a ubiquitylation of actin by PAM was not observed (Fig. 6D) suggesting that PAM does not alter the biochemical properties of monomeric or filamentous actin. However, we found that F-actin, but not G-actin, attenuated the ubiquitin ligase activity of PAM (Fig. 6E). Unfortunately we were unable to purify sufficient amounts of full-length PAM from rat spinal cords for an investigation of the effects of F-actin on PAM splice variants from this tissue.


Figure 6
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  		FIG. 6. PAM splice variants in the MBD region differ in their affinity to F-actin. A, schematic showing the splice variants found in the human and rat MBD region of PAM. The exons are numbered according to the GenBankTM sequence NM207215. The location of the primers that were used for amplification is indicated (MBD1, MBD2, and MBD3). B, RT-PCR products from the MBD region in rat spinal cord (SC), rat brain (B), and PC12 and HeLa cells. The primer pairs used for amplification are indicated. C, Western blot analysis of cosedimentation experiments using purified F-actin and the indicated PAM domains. D, ubiquitylation assays with purified PAM and G-actin. The K48R mutant of His-ubiquitin was used to permit better visualization of only monoubiquitylation. The arrows indicate the position of actin and the expected position of monoubiquitinated actin. E, ubiquitylation assays with purified PAM. Samples were analyzed by Western blotting for ubiquitylation activity of PAM using anti-His antibody to detect ubiquitylated proteins. Depol., depolymerizing; Pol., polymerizing.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Activity-dependent changes at synaptic terminals are difficult to investigate because these changes are restricted to specific synapses of low numbers of cells. This makes qualitative and quantitative studies of proteomic changes difficult because of the small number of changes that occur as compared with the total protein content. Additionally the activity-dependent alterations at synapses are often temporary posttranslational modifications and changes in the subcellular localization of proteins and protein complexes. The MELC technology offers a platform to investigate changes in the composition of protein complexes and their location in restricted areas while keeping the three-dimensional organization of the sample intact. Therefore we investigated the usefulness of the MELC technology for the investigation of activity-dependent changes at synapses.

We used the formalin test, a commonly used pain model for acute pain to induce activity-dependent changes at synapses in spinal cord neurons. 20–60 min after the formalin injection, sensitization processes in the spinal cord cause an increase in the nociceptive behavior of the animals. The underlying mechanisms are based on long term changes in nociceptive processing of neurons of the central nervous system (central sensitization). This central sensitization comprises at the early stages posttranslational modifications such as phosphorylation, mono-, and polyubiquitylation as well as dynamic changes such as the recruitment of proteins to the synaptic endings (e.g. translocation of AMPA receptors) and the enhanced trafficking of synaptic vesicles (1–4). Transcription-dependent processes are then responsible for the maintenance of central sensitization in dorsal horn neurons. The transcriptional changes take several hours to manifest and last for prolonged periods after the end of the initial stimuli (1, 2). The different nature of the synaptic changes that occur after short term (45-min) and long term (24-h) treatment was reflected by the high number of CMPs that distinguished both time points.

PAM has been described earlier to modulate spinal nociceptive processing during the early phases of the formalin test (14). Here we investigated whether or not we could find events that are related to two known targets of PAM, adenylyl cyclase type 1 and TSC2 (22, 24). The importance of cAMP signaling is an important feature of nociceptive plasticity in the formalin test and has been known for a long time, but a potential role of TSC2 in nociception has not been investigated so far. However, because TSC2 is an important inhibitor of mTOR signaling (37) and mTOR activation mediates nociceptive processing in the formalin test (38), a role for TSC2 in nociceptive processes is also conceivable. We showed that known activity-dependent events such as the ubiquitylation of NMDA receptor subunits can be detected by using MELC technology and present for the first time evidence that PAM may ubiquitylate TSC2 in the spinal cord after nociceptive stimulation. Because it has already been shown that PAM can mediate the ubiquitylation of TSC2 in certain neurons (39), it is tempting to speculate that one option by which PAM regulates synaptic activity is through activation of translational processes by the TSC2/mTOR signaling pathway.

Previously we demonstrated that PAM inhibits adenylyl cyclase activity in the spinal cord and that PAM modulates nociceptive behavior in the formalin test (14). The role of the cAMP-dependent signaling pathways in regulation of neuronal excitability and the pronociceptive effects of increased spinal cAMP concentrations are well established. It has been demonstrated that intrathecally applied cAMP analogues as well as the adenylyl cyclase activator forskolin produce hyperalgesia (40), and inhibitors of cAMP phosphodiesterases prolong hyperalgesia (40, 41). Moreover agents that decrease cAMP levels, such as opioids and adenosine, reduce hyperalgesia (42, 43). In accordance with these findings, studies show that adenylyl cyclase knock-out mice have reduced behavioral responses in several pain models (44, 45) as well as a reduced response to opioids (35, 46). Because PAM is a strong inhibitor of adenylyl cyclase type 1 (22) and we found that adenylyl cyclase type 1 is expressed in the spinal cord neurons, we tested whether we could find evidence for the ubiquitylation of this enzyme by PAM. However, in contrast to our findings concerning TSC2 there was no evidence for a ubiquitylation of adenylyl cyclase type 1 by PAM. This observation is in accordance to previous findings that show that inhibition of adenylyl cyclases by PAM is independent of its ubiquitin ligase activity (22, 34).

We obtained over 1000 PAM-related CMPs describing a multitude of possible interactions and interaction partners for PAM. Of this variety only the interaction of PAM with actin was subjected to further validation because this interaction was found to be an important element in many other CMPs containing PAM. Other CMP motifs such as the examples listed in supplemental Data 1 or the interaction between PAM and GLUR1 or mGluR5 await verification by biochemical and cell biological methods. However, the here identified PAM-dependent ubiquitylation events depended strongly on the presence of F-actin in the respective CMPs. There are several possibilities regarding how PAM functions might be influenced by F-actin. Because we showed previously that PAM activation can cause its translocation (21), the actin cytoskeleton might play a role in bringing PAM and its targets together. In this case the differential splicing could direct the different splice variants to distinctive cellular locations, depending on their affinity toward the actin cytoskeleton. On the other hand we found that F-actin can inhibit the ubiquitin ligase activity of PAM. This surprising finding seems to contradict the results from the MELC analysis that suggest an enhanced ubiquitin ligase activity by PAM in the presence of F-actin. However, our findings are limited by the fact that the PAM splice variants in HeLa cells differ from the splice variants found in rat spinal cords. For example, the variants with high affinity for F-actin were only seen in the spinal cord samples, and the question of what effect the different splice variant combinations have on PAM activity is open to speculation. Similarly it should be noted that the finding that neither actin polymerization nor depolymerization of F-actin were altered in vitro by PAM is restricted to the splice variants found in HeLa cells. Only the detailed analysis of the F-actin binding capabilities of the different PAM splice variants in comparison with the effects of F-actin on the ubiquitin ligase activity of the splice variants will give a conclusive picture about the roles that the PAM/F-actin interactions fulfill.

The main limitations of the MELC system can be deduced from the technologies on which the MELC system is based on. For example the spatial resolution (here 265 x 286 nm/pixel) underlies the general limitations of fluorescence microscopy. Other limits are set by the stability of the tissue and the quality of the fluorescence tags (e.g. antibodies). Although 100 MELC cycles have been performed using skin biopsies (8), we found in exploratory runs that spinal cord slices started to show the first signs of disintegration due to the physical stress after 45 cycles. More cycles may be possible by reducing the run time using one instead of three fields of vision. To minimize the run time in the MELC system fluorescence tags are normally incubated for 15–30 min with the sample before the wash step is started. This relatively short incubation time excludes the use of antibodies with weak epitope affinities because only strong fluorescence signals are able to generate the necessary signal-to-noise ratios that allow the binarization of the images. However, the data presented here show that the MELC technology provides a useful tool to unravel novel protein interactions and to investigate rare proteomic changes as they occur during spinal nociceptive processing.


  FOOTNOTES
 
Received, May 6, 2008, and in revised form, August 11, 2008.

Published, MCP Papers in Press, August 26, 2008, DOI 10.1074/mcp.M800201-MCP200

1 The abbreviations used are: AMPA, {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CMP, combinatorial molecular phenotype; GLUR, glutamate receptor; MBD, Myc binding domain; MELC, multiepitope ligand cartography; mGluR5, metabotropic glutamate receptor 5; mTOR, mammalian target of rapamycin; NMDA, N-methyl-D-aspartate; PAM, protein associated with Myc (alternate names, MYCBP2 and PHR1); TSC2, tuberosclerosis complex 2; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; C-PAM, C terminus of PAM. Back

* The work was supported by Deutsche Forschungsgemeinschaft Grants SCHO817-1 and -2. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed: Inst. für Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. Tel.: 49-69-6301-83103; Fax: 49-69-6301-83378; E-mail: Scholich{at}em.uni-frankfurt.de


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