cytochalasin-d and tautomycin

Human enhancer of filamentation 1 (HEF1), a multifunctional docking protein of the Cas family, participates in integrin and growth factor signaling pathways that regulate global cellular processes including growth, motility and apoptosis. HEF1 consists of two isoforms, p105 and p115, the larger molecular weight form resulting from Ser/Thr phosphorylation of p105HEF1. The molecular mechanisms that regulate the interconversion of the two HEF1 species as well as the function of HEF1 Ser/Thr phosphorylation are unknown. Our study reveals that cell adhesion and detachment regulate the interconversion of the two HEF1 isoforms. Experiments using various inhibitors of cytoskeletal organization indicated that disruption of actin microfilaments but not intermediate filaments or microtubules resulted in a complete conversion of p115HEF1 to p105HEF1. The conversion of p115HEF1 to p105HEF1 was prevented by inhibition of protein phosphatase 2A (PP2A), suggesting that cytoskeletal regulation of PP2A activity controlled the dephosphorylation of p115HEF1. Degradation of endogenous HEF1 was dependent on proteasomes with the p115 species of HEF1 being preferentially targeted for turnover. Dephosphorylation of HEF1 by suspending cells or disrupting actin filaments protected HEF1 from degradation. These results suggest that the adhesion-dependent actin organization regulates proteasomal turnover of HEF1 through the activity of PP2A.

Topics: Actin Cytoskeleton; Actins; Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Cell Adhesion; Cell Line; Colchicine; Cytochalasin D; Enzyme Inhibitors; Humans; Intermediate Filaments; Marine Toxins; Microtubules; Nocodazole; Nucleic Acid Synthesis Inhibitors; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Isoforms; Protein Phosphatase 2; Pyrans; Serine; Signal Transduction; Spiro Compounds; Threonine; Tubulin Modulators

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

Topics: Actins; Animals; Antifungal Agents; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Calcineurin; Calcium; Calcium Signaling; Cells, Cultured; Cloning, Molecular; Cyclosporine; Cytochalasin D; Demecolcine; Depsipeptides; Embryonic Development; Enzyme Inhibitors; Female; gamma-Aminobutyric Acid; GAP-43 Protein; Gene Expression Regulation, Developmental; Growth Cones; Molecular Sequence Data; Neurites; Neurons; Nucleic Acid Synthesis Inhibitors; Okadaic Acid; Paclitaxel; Peptides, Cyclic; Phosphorylation; Pyrans; Spinal Cord; Spiro Compounds; Xenopus laevis

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 membrane at the site of the blebs. Further treatment with tautomycin induced the accumulation of F-actin at the segmentation centers. Under electron 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.

Topics: Actin Cytoskeleton; Actins; Antifungal Agents; Cell Membrane; Cytochalasin D; Humans; Intermediate Filaments; Leukemia, Myeloid; Microscopy, Electron; Microscopy, Fluorescence; Microtubules; Phosphoprotein Phosphatases; Pyrans; Spiro Compounds; Tumor Cells, Cultured

A novel antibiotic tautomycin induced many blebs on the surface of K562 human chronic myeloid leukemia cells, similar to the morphological changes induced by phorbol esters. However, tautomycin did not induce nitroblue tetrazolium reducing activity, when HL60 human promyelocytic leukemia cells were caused to differentiate by quinomycin into mature granulocytes. It did not induce spread of HL60 cells, one of the phenotypes of mature macrophages. In addition, it did not compete with phorbol dibutyrate to bind to the cell surface of K562 cells. However, tautomycin significantly activated protein kinase C (PKC) extracted from K562 cells. These results indicate that tautomycin is a new activator of PKC, distinct from phorbol esters.

Topics: Anti-Bacterial Agents; Antifungal Agents; Cell Differentiation; Cytochalasin D; Cytochalasins; Enzyme Activation; Humans; Leukemia, Myeloid; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Pyrans; Spiro Compounds; Tumor Cells, Cultured