Rabbit Anti-Human PTK2 (Phospho-Ser843) polyclonal antibody

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.

Caveolae have been hypothesized to be involved in Ca2+ signaling. By electron microscopy, caveolae were observed to be apposed to the endoplasmic reticulum (ER), which is a major intracellular Ca2+ pool. In the present study, we examined the relationship between caveolae and the ER when the distribution of the latter was changed by depolymerization of microtubules. Double immunofluorescence microscopy for detection of caveolin and the ER antigens, and time-lapse observation of green fluorescent protein (GFP)-tagged caveolin were employed. in normal human fibroblasts and PtK2 cells, the ER was seen as a network extending throughout the cytoplasm, and most caveolin occurred in patches along the cell edge. When microtubules were depolymerized by Colcemid or nocodazole, the ER became retracted from the cell periphery and aggregated around the nucleus; in the same cells, caveolin was not seen along the cell edge, but was aligned along the edge of the retracted ER. By time-lapse analysis, GFP-caveolin expressed in PtK2 cells was observed to move from the cell periphery toward the cell center in Colcemid-treated cells. The result shows that the apposition of caveolae and the ER is maintained even after the gross distributional change, and suggests a mechanical linkage between the two organelles.