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doi: 10.1242/10.1242/jcs.00174

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

Catherine S. Chew^*, Xunsheng Chen, John A. Parente, Jr, Shannan Tarrer, Curtis Okamoto and Hai-Yen Qin

Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912-3175, USA
Present address: Genzyme Corporation, One Mountain Road, Framingham, MA 01701, USA
Present address: Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA

^* Author for correspondence (e-mail: cchew{at}immagene.mcg.edu)

Accepted 15 September 2002

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 K_d 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 K_d and decreased the B_max 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.

Key words: Gastric parietal cell, Stomach, Rabbit, Protein phosphorylation, Cytoskeleton, cAMP-dependent protein kinase

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