PACSIN 1 forms tetramers via its N‐terminal F‐BAR domain

Abstract

In eukaryotic cells, complex regulatory mechanisms involving numerous proteins must operate to ensure the temporal and spatial specificity of intracellular membrane-trafficking pathways. We and others have identified three members of the protein kinase C and casein kinase 2 substrate in neurons (PACSIN) protein family, also named syndapin and focal adhesion protein 52 (FAP52), which participate in rearrangements of actin networks during endocytosis [1][2][3][4][5]. In contrast to the neuron-specific PACSIN 1, other members of the PACSIN protein family show a broader tissue distribution [2,4,6]. Via their C-terminal Src homology 3 (SH3) domains, PACSIN proteins bind to proline-rich domains of dynamin, synapsin and synaptojanin, three proteins also involved in vesicle endocytosis, as well as to neural Wiskott-Aldrich syndrome protein The ability of protein kinase C and casein kinase 2 substrate in neurons (PACSIN) ⁄ syndapin proteins to self-polymerize is crucial for the simultaneous interactions with more than one Src homology 3 domain-binding partner or with lipid membranes. The assembly of this network has profound effects on the neural Wiskott-Aldrich syndrome protein-mediated attachment of the actin polymerization machinery to vesicle membranes as well as on the movement of the corresponding vesicles. Also, the sensing of vesicle membranes and ⁄ or the induction of membrane curvature are more easily facilitated in the presence of larger PACSIN complexes. The N-terminal Fes-CIP homology and Bin-Amphiphysin-Rvs (F-BAR) domains of several PACSIN-related proteins have been shown to mediate self-interactions, whereas studies using deletion mutants derived from closely related proteins led to the view that oligomerization depends on the formation of a trimeric complex via a coiled-coil region present in these molecules. To address whether the model of trimeric complex formation is applicable to PACSIN 1, the protein was recombinantly expressed and tested in four different assays for homologous interactions. The results showed that PACSIN 1 forms tetramers of about 240 kDa, with the self-interaction having a K D of 6.4 · 10 )8 m. Ultrastructural analysis of these oligomers after negative staining showed that laterally arranged PACSIN molecules bind to each other via a large globular domain and form a barrel-like structure. Together, these results demonstrate that the N-terminal F-BAR domain of PACSIN 1 forms the contact site for a tetrameric structure, which is able to simultanously interact with multiple Src homology 3 binding partners.

Gas7b (Growth Arrest Specific Protein 7b) Regulates Neuronal Cell Morphology by Enhancing Microtubule and Actin Filament Assembly

“…F-BAR proteins were shown to form oligomers, and Gas7b was revealed to form a tetramer or a higher order oligomer in solution because PACSIN 1 forms tetramers via its F-BAR domain (26). The oligomeric formation presumably enables a single Gas7b molecule to bind multiple MTs and F-actin by formation of symmetric and multiple binding sites for these cytoskeletons (Fig.…”

Setting the F-BAR: Functions and regulation of the F-BAR protein family

“…In one study, globular conformations of PACSIN were observed by electron microscopy. 39 We were able to image full length Cdc15 by electron microscopy and detected elongated, oligomeric forms; 25 however, heterogeneity of the protein prohibited averaging for high-resolution structures. Full-length syndapin I was used for X-ray crystallography studies, which showed that one SH3 domain binds to the F-BAR domain, 12 however sequences connecting the F-BAR and SH3 domains could not be visualized in this analysis, possibly due to inherent flexibility, nor could the other SH3 domain.…”

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

“…The C-terminal SH3 domain of PACSIN 2 regulates interactions with dynamin1, synapsin 1, mSos and N-WASP. The N-terminal F-BAR domain of PACSIN 2 is required for homo-ligomerization and association with curved membranes [29][30][31] and mediates several cytoskeletal rearrangements required for cytokinesis in S. pombe. 32 We examined further the domains of PACSIN 2 required for cyclin D1 binding using C-terminal truncation mutants.…”