latrunculin-a and fasudil

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

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of (125)I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10-20 microM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of (125)I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Actins; Animals; Bombesin; Bridged Bicyclo Compounds, Heterocyclic; Cytochalasin D; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; GTP-Binding Proteins; Iodine Radioisotopes; Kinetics; Mice; Nucleic Acid Synthesis Inhibitors; Phosphorylation; Receptors, Bombesin; Receptors, Endothelin; Signal Transduction; Thiazoles; Thiazolidines