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.

Key word: tautomycin/protein phosphatase/actinfilament/cytoskeleton ABSTRACT. A protein phosphatase inhibitor, tautomycin induces blebs on the surface of human myeloid leukemia K562 cells within 10 min. In this paper we examined the cytoskeleton of tautomycin-treated cells. In the presence of tautomycin, cells with blebs turned into segmented forms with smooth surfaces after 15 min and into smooth round shapes without microprotuberance after 60 min. Observation with fluorescence microscopy showed that F-actin detached from the plasma membraneat the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Underelectron microscopy, an electron dense ring-structure was detected at the segmentation center. Tautomycin did not induce major changes of the microtubule network although F-actin accumulated near the microtubule organizing center. The amount of F-actin increased in tautomycin-treated cells. These results indicate that the morphological changes are induced by reorganization of actinfilaments.

The effects of tautomycin, a protein phosphatase inhibitor, on recycling of cell surface molecules were studied with transferrin receptor (TFR) of human myeloid leukemia K562 cells and with CD4 of murine thymocytes. which was induced by PDBu.The results suggest that certain inhibitors of protein phosphatases preferentially inhibit endocytosis of cell surface molecules.Tautomycin is an antifungal antibiotic isolated from Streptomyces spiroverticillatuslf2). Tautomycin induced blebs on the surface of human myeloid leukemia K562cells3). The morphological change, named bleb formation, was also induced by phorbol dibutylate (PDBu)3), cytochalasin D (CyD) and okadaic acid (OA)4) suggesting that modulation of actin filament network and/or protein phosphorylation is involved in tautomycin-induced bleb formation4). Further studies on tautomycin revealed that tautomycin is a novel inhibitor of protein phosphatases and that the specificity of tautomycin to the enzymes was distinct from known inhibitors of protein phosphatases5'6*. Cytoskeleton and protein phosphorylation are also involved in recycling of cell surface receptors for transferrin and epidermal growth factor (EGF)7'8). In fact, phorbol esters are knownto affect expression of a numberof important regulatory molecules on cell surface including the receptors for EGF8) and transferring and CD410). In the present paper, we studied the effects of tautomycin on phorbol ester-induced down-regulation of transferrin receptor (TFR), on K562 cells, and the effect of CD4on murine thymocytes. Materials and Methods ChemicalsTautomycin was isolated as described by Cheng et al.l) Staurosporine was also isolated from the culture broth of an actinomycetes. Okadaic acid and dinophysistoxin-1 were kindly donated by Dr. H. Fujiki (National Cancer Center Research Institute, Japan). Cytochalasin D and PDBu were purchased from Sigma Chem. H-7, H-8 and W-7 were purchased from Seikagaku Kogyo Co., Ltd. Japan. K252a and KT5926 were kindly gifted by Dr. H. Kase (Kyowa Hakko Kogyo Co., Ltd.). Cell CultureCells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 0.05him 2-mercaptoethanol, 50 mg/liter kanamycin and 8 mg/liter tylosin at 37°C in 5% CO2 atmosphere.

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