calyculin-a and fasudil

Nerve growth factor, retinoic acid, dibutyryl cAMP, ganglioside G(Q1b), and botulinum C3 exoenzyme were evaluated for their neural differentiating potential on human neuroblastoma GOTO cells. C3 exoenzyme is an ADP-ribosyltransferase inactivating Rho protein (a small GTP-binding protein). C3 exoenzyme caused the fastest differentiation of GOTO cells into neural cells and induced the strongest network of the cells. Fasudil, an inhibitor of Rho-kinase, induced outgrowth of the neurites in the GOTO cells. Calyculin A, an inhibitor of phosphatases including myosin phosphatase, counteracted C3 exoenzyme-induced neurite outgrowth of the cells. These findings suggest that differentiation of GOTO cells triggered by C3 exoenzyme is attained via inactivation of Rho protein, inhibition of Rho-kinase, and activation of myosin phosphatase. Because of the strong differentiating potential of C3 exoenzyme, the transduction pathway consisting of Rho protein, Rho-kinase, and myosin phosphatase seems to be main stream in the neural differentiation of GOTO cells. A single GOTO cell was observed continuously after treatment with C3 exoenzyme. The cell started to change shape from its original morphology only 15 min after treatment with C3 exoenzyme, and it was completely spherical within 60 min. Neurites appeared on the membrane of the cell 2 hr after the treatment and then gradual outgrowth began. These observations are fundamental information in elucidating the mechanism of neural differentiation, especially at an early stage.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; ADP Ribose Transferases; Botulinum Toxins; Cell Differentiation; Cell Line, Tumor; Humans; Intracellular Signaling Peptides and Proteins; Marine Toxins; Neuroblastoma; Oxazoles; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction

Treatment of intact Swiss 3T3 cells with calyculin-A, an inhibitor of myosin light chain (MLC) phosphatase, induces tyrosine phosphorylation of p125(Fak) in a sharply concentration- and time-dependent manner. Maximal stimulation was 4.2 +/- 2.1-fold (n = 14). The stimulatory effect of calyculin-A was observed at low nanomolar concentrations (<10 nM); at higher concentrations (>10 nM) tyrosine phosphorylation of p125(Fak) was strikingly decreased. Calyculin-A induced tyrosine phosphorylation of p125(Fak) through a protein kinase C- and Ca(2+)-independent pathway. Exposure to either cytochalasin-D or latrunculin-A, which disrupt actin organization by different mechanisms, abolished tyrosine phosphorylation of p125(Fak) in response to calyculin-A. Treatment with high concentrations of platelet-derived growth factor (20 ng/ml) which also disrupt actin stress fibers, completely inhibited tyrosine phosphorylation of p125(Fak) in response to calyculin-A. This agent also induced tyrosine phosphorylation of the focal adhesion-associated proteins p130(Cas) and paxillin. These tyrosine phosphorylation events were associated with a striking increase in the assembly of focal adhesions. The Rho kinase (ROK) inhibitor HA1077 that blocked focal adhesion formation by bombesin, had no effect on the focal adhesion assembly induced by calyculin-A. Thus, calyculin-A induces transient focal adhesion assembly and tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin, acting downstream of ROK.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Animals; Bombesin; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Crk-Associated Substrate Protein; Cytochalasin D; Cytoskeletal Proteins; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Immunoblotting; Indoles; Maleimides; Marine Toxins; Mice; Microscopy, Fluorescence; Myosin-Light-Chain Phosphatase; Nucleic Acid Synthesis Inhibitors; Oxazoles; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Proteins; Retinoblastoma-Like Protein p130; Thiazoles; Thiazolidines; Time Factors