A cell-free binding assay maps the LSP1 cytoskeletal binding site to the COOH-terminal 30 amino acids

Abstract

The leukocyte specific protein 1 or LSP1 is a multi functional protein involved in such divers biological processes as the regulation of neutrophil motility, chemotaxis, adhesion and membrane immunoglobulin M (mIgM) mediated apoptosis of B-lymphocytes. The 330-amino-acid mouse LSP1 protein contains a high-affinity F-actin binding site and in intact cells localizes to the F-actin filament containing cytoskeleton. Here we use a high-speed F-actin co sedimentation assay and transfection experiments in the LSP1- T-lymphoma cell line BW5147 to show that LSP1 interacts with F-actin and the cytoskeleton through residues downstream of amino acid residue 230. We then designed a novel cell-free cytoskeleton binding assay in which a set of GST-LSP1 fusion proteins are allowed to bind to the cytoskeleton in NP-40 soluble lysates of BW5147 cells and are recovered in the low-speed detergent insoluble pellet. Using this assay the cytoskeleton binding site of mouse LSP1 maps to the 300-330 interval. These results will allow the design of LSP1 mutants that do not bind to the cytoskeleton to determine the importance of LSP1 cytoskeleton binding for the diverse functions of LSP1.

LSP1-myosin1e bi-molecular complex regulates focal adhesion dynamics and cell migration

“…Although it is known that LSP1 interacts with actin filaments (Klein et al, 1990;Wong et al, 2003;Zhang et al, 2001), the mechanisms underlying LSP1 function in the regulation of actin-driven processes are, as yet, poorly defined. Before discussing potential scenarios describing LSP1 function, our previous study (Maxeiner et al, 2015) and the findings described here clearly indicate that one possible mechanism for LSP1 function requires its interaction with myosin1e.…”

“…The amino-terminal half of LSP1 incorporates Ca 2+ -binding sites and a coiled-coil region (Jongstra et al, 1988;Klein et al, 1989), suggesting that Ca 2+ signalling and dimerization could regulate LSP1 function. The carboxy-terminal half incorporates a caldesmon-like region having a weaker F-actin-binding activity Zhang et al, 2001) and two villin headpiece-like sequences, which primarily mediate the interaction of LSP1 with F-actin (Klein et al, 1990;Wong et al, 2003;Zhang et al, 2001). We have demonstrated that the carboxy-terminal half of LSP1 directly interacts with the SH3 domain of the molecular motor myosin1e through the noncanonical SH3-binding site AGDMSKKS (Maxeiner et al, 2015).…”

“…Since LSP1 directly interacts with F-actin (Wong et al, 2003) and regulates the dynamics of the actin cytoskeleton (Maxeiner et al, 2015), we hypothesised that LSP1 could control cell migration. To this end, we focused on macrophages because LSP1 is essential for another actin-dependent process in this cell type, namely Fcg receptormediated phagocytosis (Maxeiner et al, 2015).…”

Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to the proteasome in dendritic cells

“…The basic C-terminal domain contains amino acid sequences homologous to two known F-actin binding proteins, caldesmon and the villin headpiece (36,37). Although LSP1 is an F-actin binding protein, it is also a very important regulator of microfi lamentous cytoskeleton dynamics (34).…”

“…LSP1 is an F-actin binding protein involved in cell motility, cell adhesion, and IgM internalization (34)(35)(36)(37). The caldesmonlike (CI and CII) and villin-like (VI and VII) are two domains on LSP1 that bind F-actin to facilitate neutrophil motility (38).…”