Effects of the Protein Phosphatase Inhibitors, Tautomycin and Calyculin-A, on Protein Phosphorylation and Cytoskeleton of Human Platelets.

Abstract

Key words: protein phosphatase/protein phosphorylation/cytoskeleton/myosin light chain ABSTRACT. Effects of the protein phosphatase inhibitors, tautomycin and calyculin A on protein phosphorylation and cytoskeleton of human platelets. It has been discovered recently that manycytotoxic compoundsisolated from a variety of sources are potent phosphatase inhibitors. Two of these, tautomycin (TM) and calyculin-A (CL-A) were applied to human platelets to investigate the role of protein phosphorylation on cytoskeletal structure and function. Exposure to 10 pM TMor 0.1 pM CL-A induced marked morphological changes. The granules were centralized and surrounded by actin filaments, but there was no evidence of granule release. Myosin became more centralized, was occluded from the granulomere, but was not confined to the micro filament ring. These changes occurred without an increase in cytosolic Ca2+ concentrations, as determined by measurements with fura-2. TMand CL-Ainduced an overall increase in protein phosphorylation. Phosphorylation of the 20,000 dalton light chain of myosin increased markedly and multiple phosphorylation sites were indicated. Cytoskeletons were prepared from control, thrombin-and TM-treated platelets, the latter prepared in the absence of external calcium. The major difference in protein composition was the increased content of myosin associated with the cytoskeleton from TM-treated platelets where the dominant phosphoprotein was the 20,000 dalton light chain. These results suggest that myosin phosphorylation drives the initial shape changes, and via a contractile process results in the formation of the micro filament ring and centralization of granules.

Thrombin-Induced Activation of RhoA in Platelet Shape Change

“…In platelets, Rho-kinase can phosphorylate the myosin binding subunit, decreasing the myosin phosphatase activity (6). In addition, an inhibition of the myosin phosphatase in platelets, using selective phosphatase inhibitors such as tautomycin as shown in this study, can induce a significant increase in MLC 20 phosphorylation as well as platelet shape change (25). In permeabilized platelets, thrombin (as well as GTP␥S) induced RhoA activation should therefore result in Ca 2ϩ -independent effects on MLC-P and shape change.…”

Actin cytoskeleton is responsible for the change of cytoplasmic organization in root hair cells induced by a protein phosphatase inhibitor, calyculin A

“…The presence of myosin(s) responsible for the wound-induced cytoplasmic contraction has been suggested in the green alga Ernodesmis verticillata (La Claire 1991). In animal cells such as 3T3 cells (Chartier et al 1991, Kimura et al 1996 and platelets (Kurisaki et al 1995), the phosphorylation level of light chains in myosin II has been revealed to increase by CA treatment. Therefore, from these results and evidence, it is assumed that myosin(s), whose activities are regulated by the phosphorylation and dephosphorylation processes, is involved in the organization of the actin cytoskeleton in root hair cells.…”

Actin‐based centripetal flow: Phosphatase inhibition by calyculin‐A alters flow pattern, actin organization, and actomyosin distribution

“…In addition, CalyA-induced contractile responses are evident in another coelomocyte cell type present in the cell isolates that contains stress fiber-like structures. It is important to note that numerous published studies report that CalyA treatment induces contractile responses in many different cell types [Chartier et al, 1991;Hosoya et al, 1993;Yano et al, 1995;Kurisaki et al, 1995;Essler et al, 2000;Parizi et al, 2000], including sea urchin eggs and embryos [Tosuji et al, 1992;Asano and Mabuchi, 2001].…”

“…We reasoned that phosphatase inhibition should increase actomyosin contraction forces in the cell center through increased phosphorylation of the myosin regulatory light chain (MRLC). Previous studies on several different cell types have shown that CalyA elevates phosphorylation of MRLC and can lead to contractile responses [Chartier et al, 1991;Tosuji et al, 1992;Hosoya et al, 1993;Yano et al, 1995;Kurisaki et al, 1995;Parizi et al, 2000]. Importantly, Asano and Mabuchi [2001] have recently shown that CalyA increases MRLC phosphorylation in sea urchin eggs and that this induces actomyosin-dependent cortical contractions.…”