Actin binding of human LIM and SH3 protein is regulated by cGMP- and cAMP-dependent protein kinase phosphorylation on serine 146.
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.
Butt
Elke
E
Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany. butt@klin-biochem.uni-wuerzburg.de
Gambaryan
Stepan
S
Göttfert
Nina
N
Galler
Annette
A
Marcus
Katrin
K
Meyer
Helmut E
HE
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000502:physiology*; D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D018909:src Homology Domains; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D018398:Homeodomain Proteins; C096353:LASP1 protein, human; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases