Cytoskeletal reorganization of human platelets induced by the protein phosphatase 1/2 A inhibitors okadaic acid and calyculin A

Abstract

Okadaic acid (OA) and calyculin A (CLA), which are potent and specific inhibitors of serine/threonine protein phosphatases type 1 and 2A, have been shown to induce drastic changes in platelet morphology. The aim of this study was to analyse the molecular mechanisms of OA- or CLA-induced cytoskeletal reorganization, with a specific focus on microtubules and actin filaments. Confocal fluorescence microscopy revealed that OA or CLA altered the distribution of microtubules from marginal band arrangements to homogeneous patterns, consistent with the transmission-electron-microscopic finding that microtubules were fragmented and redistributed into pseudopod-like processes. In thrombin-activated platelets, OA or CLA induced extremely long pseudopods containing an array of microtubules and actin filaments, and a condensed mass of actin filaments in the centre of platelets. OA or CLA induced the constriction of actin filaments without an increase in filamentous (F)-actin, and also rather significantly inhibited actin polymerization in thrombin-activated platelets. Furthermore, neither OA or CLA enhanced phosphorylation of myosin light chain (MLC). By immunoprecipitation of platelet lysate with anti-alpha-tubulin antibody, a 90 kDa protein was co-precipitated with tubulin and was predominantly phosphorylated in the presence of OA. As the time-dependent phosphorylation of 90 kDa protein correlated well with the reorganization of microtubules, these data suggest that phosphorylation and dephosphorylation of this protein might play a role in the regulation of microtubule organization. These findings indicate that OA or CLA induces reorganization of microtubules and actin filaments via the phosphorylation of a microtubule-associated 90 kDa protein and an MLC-phosphorylation-independent mechanism. mechanism.

A Role for the Actin Cytoskeleton in the Initiation and Maintenance of Store-mediated Calcium Entry in Human Platelets

“…As shown previously in many cell types, including platelets, calyculin A at concentrations Յ 100 nM induced reorganization of the actin cytoskeleton (11,24,25). Actin filaments appear tightly condensed at the PM, without induction of polymerization, and thrombin-induced actin polymerization is prevented (25). In agreement with these observations, our results show that calyculin A did not alter the F-actin content of unstimulated platelets but did abolish TG-induced actin polymerization.…”

“…A highly effective means to induce phosphorylation-dependent association of these proteins, and thus actin filaments with the PM, is by using serine/threonine phosphatase inhibitors like calyculin A, a very potent and highly specific inhibitor of protein phosphatases 1 and 2. As shown previously in many cell types, including platelets, calyculin A at concentrations Յ 100 nM induced reorganization of the actin cytoskeleton (11,24,25). Actin filaments appear tightly condensed at the PM, without induction of polymerization, and thrombin-induced actin polymerization is prevented (25).…”

“…To induce phosphorylation-dependent association of the actinbinding proteins with the PM we treated human platelets with calyculin A, a serine/threonine phosphatase inhibitor that inhibits protein phosphatases 1 and 2 (11,23). In many cells, including platelets, treatment with calyculin A results in a condensation of actin filaments at the PM (11,24,25). Treatment of human platelets for 2 min at 37°C with calyculin A caused a concentration-dependent reduction of Ca 2ϩ entry induced by TG (Fig.…”

Calcium Influx Factor from Cytochrome P-450 Metabolism and Secretion-like Coupling Mechanisms for Capacitative Calcium Entry in Corneal Endothelial Cells

“…CalyA enhances association of ezrin, radixin, and moesin proteins, powerful mediators of actin cross-linking, with the PM (54,55). After treatment of corneal endothelial cells with 200 nM CalyA for 1 h, cells lost their hexagonal appearance, becoming thickened on the periphery (not shown), as observed in many other cell types (19,21,56,57). The base-line fluorescence ratios of Me 2 SO-and CalyA-treated cells were not significantly different (not shown), indicating that CalyA did not alter the basic Ca 2ϩ homeostasis inside the cell.…”

Lymphocyte Migration Through Monolayers of Endothelial Cell Lines Involves VCAM-1 Signaling Via Endothelial Cell NADPH Oxidase

“…Phosphatase activity in neutrophils and Fao hepatoma cells is inhibited by exogenous H 2 O 2 or activation of NADPH oxidase (67,70). Phosphatases regulate many cell functions, including cytoskeletal structure, cell adhesion to extracellular matrix, and cell-cell junctions in fibroblasts, neutrophils, platelets, and endothelial cells (71)(72)(73)(74)(75)(76)(77)(78)(79). For example, cell constriction and reorganization of actin and microtubules in HUVECs are induced by inhibitors of phosphatases PTP1 and PTP2A (80), and cell-cell junction separation occurs when phosphatase PTP1B activity is blocked (81).…”