Abstract

Various drugs that elevate cGMP levels and activate cGMP-dependent protein kinase (cGK) inhibit agonist-induced platelet activation. In the present study we identified the LIM and SH3 domain protein (LASP) that was recently cloned from human breast cancer cells (Tomasetto, C., Regnier, C., Moog-Lutz, C., Mattei, M. G., Chenard, M. P., Liderau, R., Basset, P., and Rio, M. C. (1995) Genomics 28, 367-376) as a novel substrate of cGK in human platelets. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Cotransfection of PtK-2 cells with LASP and cGK confirmed phosphorylation of LASP in vivo. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo. LASP is an actin-binding protein, and the phospho-LASP-mimicking mutant S146D showed reduced binding affinity for F-actin in cosedimentation experiments. Immunofluorescence of transfected PtK2 cells demonstrated the localization of LASP in the tips of cell membrane extensions and at cell-cell contacts. Expression of the human LASP mutant S146D resulted in nearly complete relocalization to the cytosol and reduced migration of the cells. Taken together, these data suggest that phosphorylation of LASP by cGK and cAK may be involved in cytoskeletal organization and cell motility.

Highlights

  • The activation process of human platelets and vessel wallplatelet interactions is tightly regulated under physiological conditions and is often impaired in thrombosis, arteriosclerosis, hypertension, and diabetes

  • Phosphorylation of LIM and SH3 domain protein (LASP) in Intact Human Platelets Treated with the cGK-specific Stimulus 8pCPT-cGMP—To identify substrates of cGK in intact human platelets, cells were labeled with [32P]orthophosphate, stimulated with 500 ␮M of the specific cGMP-dependent protein kinase activator 8pCPT-cGMP, and proteins of the resulting platelet lysate were separated by two-dimensional gel electrophoresis

  • Phosphorylation of rap 1b has been observed in nitric oxide-stimulated human platelets through stimulation of guanylyl cyclase and activation of the cGMP-dependent protein kinase [5]

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Summary

Introduction

The activation process of human platelets and vessel wallplatelet interactions is tightly regulated under physiological conditions and is often impaired in thrombosis, arteriosclerosis, hypertension, and diabetes. Recombinant human LASP was phosphorylated by cGMP- and cAMP-dependent protein kinase (cAK) in vitro. Studies with human LASP mutants identified serine 146 as a specific phosphorylation site for cGK and cAK in vivo.

Results
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