Dual Palmitoylation of Psd-95 Mediates Its Vesiculotubular Sorting, Postsynaptic Targeting, and Ion Channel Clustering

El-Husseini, Alaa; Craven, Sarah E.; Chetkovich, Dane M.; Firestein, Bonnie L.; Schnell, Eric; Aoki, Chiye; Bredt, David S.

doi:10.1083/jcb.148.1.159

Cited by 272 publications

(302 citation statements)

References 37 publications

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“…35 This pseudo-palmitoylation is able to restore intracellular trafficking of the cytosol protein PSD-95 even in the presence of the palmitoylation inhibitor 2-BrPA. 34,36 Overexpression of pseudo-palmitoylated JNK3 Parlm was able to completely abolish the effects of Wnt7a, indicating the involvement of JNK3 palmitoylation in Wnt7a signalling pathways for axonal development. Interestingly, Jnk3 À/À mice 4,5 and neurons overexpressing JNK3 WT (Figure 2) show normal axonal development.…”

Section: Discussionmentioning

confidence: 99%

“…Next, we fused the prenylated and dual palmitoylated motif of paralemmin to the COOH-terminus of JNK3 CS to mimic a constant pseudo-palmitoylation on JNK3 (JNK3 Parlm). [34][35][36] The effects of Wnt7a on axonal branching were completely blocked in neurons overexpressing JNK3 Parlm (secondary, 2.8±0.2, Po0.01; tertiary, 0.5±0.1, Po0.01; higher, 0.0 ± 0.0, Po0.01; total, 4.3 ± 0.3, Po0.01) compared with WT treated with Wnt7a (Figures 4c and d), suggesting that it is in fact JNK3 palmitoylation that modulates Wnt7a-induced axonal branching. Furthermore, overexpression of JNK3 Parlm was found to reduce axonal branching and total axon length in untreated neurons, compared with controls ( Figure 4e).…”

Section: Resultsmentioning

confidence: 99%

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Isoform-specific palmitoylation of JNK regulates axonal development

et al. 2011

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The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1-3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching. The c-jun N-terminal kinase (JNK) family consists of JNK1, JNK2 and JNK3 subgroups, which participate in diverse biological and pathological processes. 1,2 At least 10 JNK isoforms of varied sizes, with most between 46 and 54 kDa, are produced from the Jnk1-3 genes, giving rise to JNK isoforms. 1 In the nervous system, JNKs (particularly isoform JNK3) have been extensively studied as the key players in apoptosis and neurodegeneration. [3][4][5][6] However, accumulating evidence supports a physiological role of JNK in regulating neurite formation and morphogenesis. 7-12 Pharmacological inhibition of JNK activity blocks axogenesis in hippocampal neurons, arguing for an essential role of JNK in neurite development. 13 Growth factors and signalling molecules, including secreted proteins of the Wnt family, have also been found to activate JNK for remodelling dendrites and axons, a process that relies on cytoskeletal rearrangement. 11,[14][15][16] This has led to the identification of several actin-or microtubule-associated proteins as JNK substrates that modulate different aspects of cytoskeletal activity. 17 However, all the 10 JNK isoforms share the same kinase domain and activation mechanism. 17 The differential roles of JNK isoforms in neurite development and the mechanisms underlying isoform-specific regulation are poorly understood.Analysis of animals null for particular JNK isoforms provides the first evidence to support isoform-specific roles of JNKs in the brain. Mice with Jnk1 À/À show abnormalities in neurite development, 7 whereas mice null for Jnk1 and Jnk2 show embryonic lethality due to severe neuro...

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Section: Discussionmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Isoform-specific palmitoylation of JNK regulates axonal development

et al. 2011

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“…The second PDZ domain of postsynaptic protein PSD-95 is known to interact with a subunit (NR2) of NMDA receptors in dendrites (29) and is involved in memory and learning (39). The ligand preferences of this PDZ domain have been investigated with individually synthesized peptides, revealing a strong preference for S/TxV-COOH and, more rarely, leucine or isoleucine at the 0 position (30).…”

Section: Discussionmentioning

confidence: 99%

Intracellular protein interaction mapping with FRET hybrids

You

Nguyen

Jabaiah

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

100

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A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.cell sorting ͉ protein-ligand interactions T he ability to identify and quantitatively characterize proteinprotein interactions in living cells is essential for developing a detailed, system-level understanding of cellular function. The yeast two-hybrid (Y2H) system has served as the primary genetic tool to discover potential biological interaction partners for an enormous number of proteins (1, 2). Application of Y2H assays to each of the nearly 6,000 yeast ORFs enabled construction of the first large-scale protein interaction network in yeast (3, 4), and similar interaction studies were performed subsequently in Drosophila melanogaster and Caenorhabditis elegans (5, 6). Human-protein interaction networks are less well characterized and present difficult challenges to interaction mapping approaches. In fact, only a small fraction of an estimated 10 5 to 10 6 human-proteome interactions (7) have been unambiguously identified, primarily by using Y2H and mass spectrometry approaches (7,8). Unfortunately, high-throughput Y2H assays are typically prone to a high frequency of false positives that complicate the interpretation of interaction data. In some studies, it has been estimated that Ͼ50% of putative interactions identified are false positives (9).Several important challenges remain in the elucidation of protein-protein interaction networks and their influence on cell function (10). Large-scale interaction maps can portray basic network structure but lack quantitative features needed to understand network function. A predictive understanding of network function likely will require the elucidation of equilibrium and kinetic properties within a network, including individual equilibrium-binding constants. Furthermore, existing highthroughput approaches (e.g., Y2H) are not well suited to investigate real-time dynamics in protein networks essential for understanding cell function (11) because detection, in these cases, is ba...

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“…Generation of GW1 PSD-95, PSD-95 (C3,5S), fused to GFP and mutations within the palmitoylation motifs of GAP-43, paralemmin and PSD-95 were described previously (Craven et al, 1999;El-Husseini et al, 2000). The addition of the GAP-43, NSP, and ␤2A N-terminal palmitoylation motif to PSD-95 were constructed with oligomers encoding the appropriate wild-type or mutated motifs and restriction sites that were annealed and subcloned into GW1 PSD-95-GFP at a HindIII site upstream of the starter methionine and a silent KpnI site at amino acid 13 of PSD-95.…”

Section: Cdna Cloning and Mutagenesismentioning

confidence: 99%

“…Studies on the neuronal proteins GAP-43 and paralemmin show that palmitoylation is important for localizing these proteins to filopodia (Zuber et al, 1989a(Zuber et al, , 1989bKutzleb et al, 1998). In contrast, dual palmitoylation of PSD-95 is necessary for appropriate postsynaptic localization to dendritic spines (Craven et al, 1999;El-Husseini et al, 2000;El-Husseini and Bredt, 2002). Overexpression studies showed that the palmitoylated motif of GAP-43 not only targets heterologous proteins to filopodia in neuron-like cells but can also induce filopodia formation (Strittmatter et al, 1994a(Strittmatter et al, , 1994b.…”

Section: Introductionmentioning

confidence: 99%

Regulation of Dendritic Branching and Filopodia Formation in Hippocampal Neurons by Specific Acylated Protein Motifs

Gauthier-Campbell

Bredt

Murphy

et al. 2004

MBoC

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Although neuronal axons and dendrites with their associated filopodia and spines exhibit a profound cell polarity, the mechanism by which they develop is largely unknown. Here, we demonstrate that specific palmitoylated protein motifs, characterized by two adjacent cysteines and nearby basic residues, are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and the branching of dendrites and axons in neurons. Such motifs are present at the N-terminus of GAP-43 and the C-terminus of paralemmin, two neuronal proteins implicated in cytoskeletal organization and filopodial outgrowth. Filopodia induction is blocked by mutations of the palmitoylated sites or by treatment with 2-bromopalmitate, an agent that inhibits protein palmitoylation. Moreover, overexpression of a constitutively active form of ARF6, a GTPase that regulates membrane cycling and dendritic branching reversed the effects of the acylated protein motifs. Filopodia induction by the specific palmitoylated motifs was also reduced upon overexpression of a dominant negative form of the GTPase cdc42. These results demonstrate that select dually lipidated protein motifs trigger changes in the development and growth of neuronal processes. INTRODUCTIONNeurons possess an elaborate plasma membrane architecture that includes axons, dendrites, and synaptic sites (Da Silva and Dotti, 2002). Modulation of the plasma membrane shape and composition regulate processes outgrowth, axonal development, dendritic branching, and the construction of synapses (Jontes and Smith, 2000). These changes are regulated by interactions between the constituents of the plasma membrane, the exo/endocytic vesicles, and the cytoskeleton (Wood and Martin, 2002). In nonneuronal cells, differential modulation of membrane flow can result in the formation of processes such as microspikes, lamellopodia, and filopodia (Wood and Martin, 2002). However, factors that regulate the formation and maintenance of specific classes of membranous processes in neuronal cells remain poorly understood.Dendritic filopodia, cell surface extensions filled with tight parallel bundles of actin filaments, are thought to be precursors for developing synapses (Dailey and Smith, 1996;Small, 1988). Filopodia are typically 5-35 m in length and rapidly alter their position and shape. In developing axons, filopodia occur at the tips of growth cones and may aid the axon in finding its appropriate target. In dendrites, filopodia act as precursors to spines, short bulbous protrusions of the dendrite that form excitatory postsynaptic contacts on many neurons (Jontes and Smith, 2000). Thus, changes in membrane structure that result in the formation of filopodia are likely important in axonal guidance and spine formation. In contrast, dendritic branching is thought to be mediated via an actin and/or microtubule-dependent mechanisms (Jan and Jan, 2003). Recent studies showed that in early neuronal development dendrites originate from lamellopodia and short neurites (Jan and Jan, ...

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Dual Palmitoylation of Psd-95 Mediates Its Vesiculotubular Sorting, Postsynaptic Targeting, and Ion Channel Clustering

Cited by 272 publications

References 37 publications

Isoform-specific palmitoylation of JNK regulates axonal development

Isoform-specific palmitoylation of JNK regulates axonal development

Intracellular protein interaction mapping with FRET hybrids

Regulation of Dendritic Branching and Filopodia Formation in Hippocampal Neurons by Specific Acylated Protein Motifs

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