Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

Abstract

Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins,nebulin, nebulette and N-RAP.In this study, we show that lasp-1 binds to non-muscle filamentous (F)actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent Kd of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 μM with a saturation stoichiometry of ∼1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the Kd and decreased the Bmax for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S99 and S146. BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S99 and S146, into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC,these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.

Overexpression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation

“…Interestingly, in these cells, zyxin could still be detected along the actin stress fibres, indicating the potential existence of another zyxin-recruiting protein along actin stress fibres since earlier results detected LASP-1 only in the focal adhesion plaques (Chew et al, 2002;Butt et al, 2003).…”

“…LASP-1 has previously been shown to localise to sites of cell adhesion and to interact with zyxin and actin (Chew et al, 2002;Li et al, 2004). To assess whether silencing of LASP-1 affects these binding partners, siRNA-treated SKOV-3 cells were stained with phalloidin green against actin or mouse anti-zyxin hybridoma supernatant.…”

“…The actin-binding domains in the core of the LASP-1 protein mediate an interaction between LASP-1 and actin at cell membrane extensions, but not along the actin stress fibres (Schreiber et al, 1998;Chew et al, 2002;Butt et al, 2003;Keicher et al 2004;Nakagawa et al, 2006). The exact cellular function of LASP-1 is still not known, but the protein has previously been reported to localise within multiple sites of dynamic actin assembly such as focal contacts, focal adhesions, lamellipodia, membrane ruffles and pseudopodia (Tomasetto et al, 1995a;Chew et al, 1998Chew et al, , 2000Chew et al, , 2002Lin et al, 2004).…”

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

“…Western blots were generated and analyzed with enhanced chemiluminescence detection as previously described (8). Cells or immunoprecipitates were electrophoresed on SDS-PAGE gels (7%) or on isoelectric focusing strips (pH 3-10, nonlinear; Amersham-Pharmacia) (8) then transferred to PVDF membranes (Amersham-Pharmacia).…”

Nuclear localisation of LASP-1 correlates with poor long-term survival in female breast cancer

“…It became the first member of a newly defined LIM-protein subfamily of the nebulin group characterised by the combined presence of LIM and SH3 domains (Grunewald and Butt, 2008). LASP-1 is localised within multiple sites of dynamic F-actin assembly such as focal contacts, lamellipodia and membrane ruffles it binds to the specific shuttle proteins Zyxin and lipoma preferred partner (LPP) and is involved in cell migration and proliferation (Schreiber et al, 1998;Chew et al, 2002;Butt et al, 2003;Nakagawa et al, 2006). Silencing of LASP-1 by RNA-interference in various cancer cell lines resulted in strong inhibition of proliferation and migration with cell cycle arrest in G2/M-phase (Grunewald et al, 2006(Grunewald et al, , 2007b.…”