Effects of tautomycin, a protein phosphatase inhibitor, on recycling of mammalian cell surface molecules.

Abstract

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.

Total Synthesis of the Serine/Threonine-Specific Protein Phosphatase Inhibitor Tautomycin1

“…Unfortunately, complete desilylation of the bis-silyl ester 29b (from bis(2-(trimethylsilyl)ethyl)acetylenedicarboxylate) 45 under many reaction conditions proved unmanagable due to the robust 2-(trimethylsilyl)ethyl esters. The commonly used reagent TBAF could remove these protecting groups, but the basicity associated with “dry” sources of fluoride-induced acyl migration followed by β-elimination at C22a known degradative process of tautomycin. 9b, Faced with no obvious alternatives, we were forced to retrace our steps with the bis-benzyl ester in place of the bis-TMSE ester (Schemes 6 and 7)…”

“…This molecular “on-off switch” is responsible for regulating such diverse and important processes as memory, cell growth, neurotransmission, glycogen metabolism, muscle contraction, and many others . Much of the current knowledge of the serine/threonine-specific protein phosphatases has disseminated from studies of their inhibition by a strikingly diverse collection of natural products known as the okadaic acid class of inhibitor, which is comprised of three structural types: (1) cyclic peptides such as the microcystins and nodularins; (2) terpenoids including cantharidin and thyrsiferyl 23-acetate; and (3) polyketides such as the calyculins, tautomycin (TM), okadaic acid (OA), and the okadaic acid congeners acanthafolicin and the dinophysistoxins . Many of these structurally dissimilar inhibitors have been exploited as small molecule probes in molecular biology because they all selectively inhibit two of the four major types of serine/threonine PPs (PP1 and PP2A over PP2B (calcineurin) and PP2C).…”

“…Among other targets, we chose tautomycin because it is the only small molecule that is selective for PP1 over PP2A (albeit only slightly: IC 50 = 22 and 32 nM, respectively). TM was discovered in 1987, and its structure was elucidated in 1993. 9a, Two groups have reported total syntheses of TM, and others have published synthetic progress toward the molecule . We have independently developed a convergent and efficient synthetic route that will allow us to pursue the goal of discovering PP1-selective inhibitors.…”

Myosin Phosphatase and Cofilin Mediate cAMP/cAMP-dependent Protein Kinase-induced Decline in Endothelial Cell Isometric Tension and Myosin II Regulatory Light Chain Phosphorylation

“…The PP1 phosphatase inhibitor, tautomycin (51,52), was used to determine whether inhibition of myosin phosphatase activation prevented the decrease in isometric tension and RLC phosphorylation elicited by cAMP. At 10 nM concentration, tautomycin is a specific inhibitor of PP1 but not PP2 phosphatases.…”