okadaic-acid and mastoparan

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.

Topics: Animals; Axons; Calcium; Chick Embryo; Cyclin-Dependent Kinases; Enzyme Inhibitors; Growth Cones; Heterotrimeric GTP-Binding Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Okadaic Acid; Peptides; Pertussis Toxin; Retina; Superior Colliculi; tau Proteins; Virulence Factors, Bordetella; Wasp Venoms

We describe the presence in bovine retinal rod outer segments of a phosphatase which dephosphorylates phosphoopsin with an efficiency similar to that of PP2A, and which is stimulated by submicromolar levels of Ca2+ (half-maximal activation, 0.4-0.5 microM). This enzyme is designated CA2+ -activated opsin phosphatase (CAOP). CAOP has a molecular mass of 70-75 kDa as determined by gel filtration on Superose 12 and exhibits reversible Ca2+ -dependent oligomerization. An unidentified protein of approximately 25 kDa is necessary for full activity of CAOP and for cooperative binding of Ca2+ (h > 2). CAOP does not require Mg2+ and is inhibited by okadaic acid (median inhibitory concentration > 25 microM), which suggests that it is related to the PP1/2A/2b class of protein phosphatases. Like PP2B, CAOP is inhibited by trifluoperazine (median inhibitory concentration 40 microM), but calmodulin has no effect on CAOP activity, and CAOP is inhibited by mastoparan at much higher concentrations than PP2b. This combination of properties suggests that CAOP is not identical to any characterized protein phosphatase. Since the cytoplasmic concentration of Ca2+ -sensitive opsin phosphatase activity suggests that light-dependent Ca2+ levels may control rhodopsin dephosphorylation.

Topics: Animals; Antiemetics; Calcineurin; Calcium; Calmodulin-Binding Proteins; Cattle; Ethers, Cyclic; Eye Proteins; Intercellular Signaling Peptides and Proteins; Nickel; Okadaic Acid; Peptides; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Rod Cell Outer Segment; Rod Opsins; Trifluoperazine; Wasp Venoms

Previous studies demonstrated that both protein kinase C (PKC) and arachidonic acid (AA) are required for IgG-mediated phagocytosis by human monocytes. We have characterized a calcium-independent "phagocytic" phospholipase A2 (designated pPL) that mediates arachidonic acid release. The present studies were designed to order PKC and pPL in the phagocytic signaling pathway. The PKC inhibitors staurosporine and calphostin C caused a coordinated decrease in phagocytosis of IgG-opsonized erythrocytes and arachidonic acid release. The PLA2 activators mastoparan and melittin restored phagocytosis to PKC-inhibited cells, but were ineffective in monocytes pretreated with the pPL inhibitor bromoenol lactone. Similarly, PKC activation with PMA and diacylglycerol enhanced phagocytosis in the absence, but not in the presence, of bromoenol lactone. These results indicate that pPL may be regulated by an upstream phosphorylation event. Thus, we examined the effects of Ab-opsonized glass bead ingestion, okadaic acid-mediated inhibition of phosphatases, and PMA treatment on the activity of pPL and on its distribution between the cytosolic and membrane-associated compartments. IgG-opsonized erythrocytes and okadaic acid caused an overall increase in pPL activity, with a twofold increase in membrane-associated pPL. PMA treatment caused a 1.8-fold increase in membrane-associated pPL activity. Okadaic acid and PMA mimic IgG-opsonized erythrocytes with respect to membrane activation of pPL, suggesting that pPL activity may be regulated by PKC. Collectively, these results indicate that pPL activity is modulated by PKC during IgG-mediated phagocytosis, and that the PKC requirement can be bypassed by direct activation of pPL.

Topics: Arachidonic Acid; Cells, Cultured; Enzyme Activation; Ethers, Cyclic; Humans; Immunoglobulin G; Intercellular Signaling Peptides and Proteins; Melitten; Monocytes; Okadaic Acid; Peptides; Phagocytosis; Phospholipases A; Phospholipases A2; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate; Time Factors; Wasp Venoms

The activation of phospholipase D (PLD) induced by formyl peptides (fMLP), as evaluated by production of tritiated phosphatidylethanol (PEt) and phosphatidic acid (PA) in polymorphonuclear leukocytes (PMN), was markedly enhanced (50-125%) by low okadaic acid concentrations (0.25-0.5 microM) but inhibited by higher concentrations (2-3 microM), although the drug caused protein hyperphosphorylation. Both effects of okadaic acid were amplified when PLD activation was primed with cytochalasin B. Stimulation of PMN with mastoparan, a wasp venom toxin that activates Pertussis toxin(PTX)-sensitive G proteins, resulted in a weak calcium-dependent production of PEt which was respectively enhanced and inhibited by okadaic acid (1-2 microM) in unprimed and cytochalasin-primed PMN. The results show that low okadaic acid concentrations primed fMLP-mediated activation of PLD, in keeping with a down-regulatory role of protein phosphatases. The contrasting effects of okadaic acid in mastoparan-stimulated PMN further suggest that protein phosphatases may regulate the generation of second messengers through alteration of major signaling events at/or downstream of PTX-sensitive G proteins (Gi).

Topics: Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Ethers, Cyclic; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kinetics; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Okadaic Acid; Peptides; Phospholipase D; Phospholipids; Phosphoprotein Phosphatases; Phosphoproteins; Wasp Venoms

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angiotensin II and vasopressin. Mastoparan, a peptide toxin from wasp venom that mimics receptors by activating G-proteins, also stimulates the accumulation of inositol phosphates in hepatocytes. Interestingly, this action of mastoparan was also inhibited by okadaic acid. Our data indicate that okadaic acid inhibits the phosphoinositide turnover signal transduction system in hepatocytes at a level distal to the receptors.

Topics: Animals; Cells, Cultured; Ethers, Cyclic; Female; Hormones; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Liver; Okadaic Acid; Peptides; Phosphatidylinositols; Phosphoprotein Phosphatases; Rats; Rats, Inbred Strains; Signal Transduction; Wasp Venoms