Abstract
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.
Highlights
Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa
1 The abbreviations used are: AMPA, ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; combinatorial molecular phenotypes (CMPs), combinatorial molecular phenotype; GLUR, glutamate receptor; Myc-binding domain (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; TSC2, tuberosclerosis complex 2; E1, ubiquitinactivating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; C-PAM, C terminus of PAM
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, and dynamic changes such as enhanced trafficking of synaptic vesicles [2,3,4,5,6]. 24 h after formalin injection long term synaptic alterations took place that are based on gene expression changes [1, 2]
Summary
Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. 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 ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane [3, 4], transcription- and translation-dependent changes in protein expression [2, 5], and ubiquitylation-mediated protein degradation [6]. 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
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