Okadaic acid: a new probe for the study of cellular regulation

Cohen, Philip; Holmes, Charles F.B.; Tsukitani, Yasumasa

doi:10.1016/0968-0004(90)90192-e

Cited by 1,318 publications

(771 citation statements)

References 34 publications

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“…Okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A, but not of tyrosine phosphatases or alkaline or acid phosphatases (Cohen et al 1990). As with staurosporine, if the dorsal thorax is cultured in okadaic acid in pupae 36–41 h of age, the rate of bristle elongation was identical to thoraces cultured without okadaic acid (Fig.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles

Tilney

Connelly

Vranich

et al. 2000

The Journal of Cell Biology

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Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker–specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.

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Section: Resultsmentioning

confidence: 99%

Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles

Tilney

Connelly

Vranich

et al. 2000

The Journal of Cell Biology

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“…to study phosphorylation as a possible mechanism for coupling of FPR to the membrane skeleton, we used the potent serine/threonine phosphatase inhibitor okadaic acid [ 13]. Okadaic acid has profound effects on protein phosphorylation in neutrophils [23].…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, we studied the effect of energy depletion on immobilization of FPR to the membrane skeleton. In addition, the effects of the nonspecific protein kinase C inhibitor staurosporine [12] and the serine/threonine phosphatase inhibitor okadaic acid [13] on FPR-membrane skeleton-coupling were tested because ATP depletion might also affect desensitization pathways that involve phosphorylation.…”

mentioning

confidence: 99%

The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent

Klotz¹,

Jesaitis²

1994

Cellular Signalling

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Abstract-Desensitization of N-fonnyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process~ however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeletonwas studied. Human neutrophils were desensitized using the photoreactive agonist N-formy1-met-leu-phe-1ys-N'-[1251]2(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[ 125 l]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by Sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear tobe related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases bad no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy,dependent process which does not appear to require modification of the receptor protein by phosphorylation.

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“…The compartment ablation approach has also been applied to a functional analysis of GLUT4 trafficking in the presence and absence of okadaic acid, an inhibitor of protein phosphatases [47]. It is well established that okadaic acid treatment of adipocytes inhibits insulin-stimulated glucose transport and GLUT4 translocation [36].…”

Section: Okadaic Acid On Deglc Transport and Glut4 Ablation In 3t3-l1mentioning

confidence: 99%

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

Livingstone

James

Rice

et al. 1996

Biochemical Journal

138

159

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The translocation of a unique facilitative glucose transporter isoform (GLUT4) from an intracellular site to the plasma membrane accounts for the large insulin-dependent increase in glucose transport observed in muscle and adipose tissue. The intracellular location of GLUT4 in the basal state and the pathway by which it reaches the cell surface upon insulin stimulation are unclear. Here, we have examined the colocalization of GLUT4 with the transferrin receptor, a protein which is known to recycle through the endosomal system. Using an anti-GLUT4 monoclonal antibody we immunoisolated a vesicular fraction from an intracellular membrane fraction of 3T3-L1 adipocytes that contained 90 % of the immunoreactive GLUT4 found in this fraction, but only 40 % of the transferrin receptor (TfR). These results suggest only a limited degree of colocalization of these proteins. Using a technique to cross-link and render insoluble (' ablate ') intracellular compartments containing the TfR by means of a transferrin-horseradish peroxidase conjugate (Tf-HRP), we further examined the relationship between the endosomal recycling pathway and the intracellular compartment containing GLUT4 in these cells. Incubation of non-stimulated cells with Tf-HRP for 3 h at 37 mC resulted in quantitative ablation of the intracellular TfR, GLUT1 and mannose-6-phosphate receptor and a shift in the density of Rab5-positive membranes. In contrast, only 40 % of intracellular GLUT4 was ablated under the same conditions. Ablation was specific for the endosomal system as there was no significant

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Okadaic acid: a new probe for the study of cellular regulation

Cited by 1,318 publications

References 34 publications

Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles

Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles

The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

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