Experimental evidence for the limiting role of enzymatic reactions in chemoattractant-induced pseudopod extension in human neutrophils.

David Chodniewicz, Abdullatif M Alteraifi, Doncho V Zhelev

Journal: The Journal of biological chemistry 2004;279(23):24460-6

PMID: 15051729

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Abstract

Chemoattractant-stimulated pseudopod growth in human neutrophils was used as a model system to study the rate-limiting mechanism of cytoskeleton rearrangement induced by activated G-protein-coupled receptors. Cells were activated with N-formyl-Met-Leu-Phe, and the temperature dependence of the rate of pseudopod extension was measured in the presence of pharmacological inhibitors with known mechanisms of action. Three groups of inhibitors were used: (i) inhibitors sequestering substrates involved in F-actin polymerization (latrunculin A for G-actin and cytochalasin D for actin filament-free barbed ends) or sequestering secondary messengers (PIP-binding peptide for phosphoinositide lipids); (ii) competitively binding inhibitors (Akt-inhibitor for Akt/protein kinase B); and (iii) inhibitors that reduce enzyme activity (wortmannin for phosphoinositide 3-kinase and chelerythrine for protein kinase C). The experimental data are consistent with a model in which the relative involvement of a given pathway of F-actin polymerization to the measured rate of pseudopod extension is limited by a slowest (bottleneck) reaction in the cascade of reactions involved in the overall signaling pathway. The approach we developed was used to demonstrate that chemoattractant-induced pseudopod growth and mechanically stimulated cytoskeleton rearrangement are controlled by distinct pathways of F-actin polymerization.

Address: Department of Mechanical Engineering and Materials Science, Duke University, Durham, NC 27708-0300, USA.

DOI: 10.1074/jbc.M312764200

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MeSH Terms: Actins, Alkaloids, Androstadienes, Benzophenanthridines, Bridged Bicyclo Compounds, Heterocyclic, Cytochalasin D, Cytoskeleton, Enzyme Inhibitors, Enzymes, Humans, Kinetics, Lipids, Models, Chemical, N-Formylmethionine Leucyl-Phenylalanine, Neutrophils, Peptides, Phenanthridines, Phosphatidylinositol 3-Kinases, Protein Binding, Protein Kinase C, Receptors, G-Protein-Coupled, Temperature, Thiazoles, Thiazolidines, Time Factors, Wortmannin