Jiabing Ding

Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex. Evidence for a novel cycle of phosphorylation and dephosphorylation

Abstract Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4β-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2–) release. Immunofluorescent studies revealed that cofilin was present thouroughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2–/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

The Renaturable 69- and 63-kDa Protein Kinases That Undergo Rapid Activation in Chemoattractant-stimulated Guinea Pig Neutrophils Are p21-Activated Kinases

Neutrophils stimulated with the chemoattractant fMet-Leu-Phe (fMLP) are known to exhibit rapid activation of four protein kinases with molecular masses of ~69, ~63, ~49, and ~40-kDa. Activation of these kinases is blocked by antagonists of phosphatidylinositol 3-kinase and type 1 and/or type 2A protein phosphatases. These enzymes can be detected by their ability to undergo renaturation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297-331 of the 47-kDa subunit of the NADPH-oxidase complex fixed within a gel. In this report, we demonstrate that an antibody generated to a fusion protein containing amino acid residues 175-306 of p21-activated protein kinase 1 (Pak1) reacts with three proteins in guinea pig neutrophils with molecular masses in the 60-70-kDa range during Western blotting. This antibody immunoprecipitates both the 69- and 63-kDa renaturable kinases from lysates of stimulated cells along with a minor 60-kDa kinase. No activities were observed for any of these enzymes in immunoprecipitates from unstimulated neutrophils. However, addition of ATP and activated Rac 1 or Cdc42 to immunoprecipitates from unstimulated cells resulted in the stimulation of two renaturable kinases with molecular masses in the 69- and 63-kDa range. These immunoprecipitates also contained two novel protein kinases with masses of ~49 and 40 kDa that were selectively activated by Cdc42. In contrast, the 69- and 63-kDa kinases were not immunoprecipitated from lysates of stimulated neutrophils with an antibody to Pak2 or with nonimmune serum. These data indicate that the renaturable 69- and 63-kDa kinases are Paks and reveal some of the upstream events that are necessary for the rapid activation of this family of protein kinases in neutrophils.

Products of Inflammatory Cells Synergistically Enhance Superoxide Production by Phagocytic Leukocytes

Superoxide (O 2 − ) is a major component of the oxygen-dependent antimicrobial and cytocidal arsenal of neutrophils 1, 2. The oxidase system that generates this substance is dormant and disassembled in unstimulated cells and consists of both membrane-bound and soluble (“cytosolic factors”) components3, 4. The known membrane-components are a low-potential, heterodimeric b-cytochrome5, 6 and a ras-related GTP-binding protein7. The most thoroughly characterized cytosolic factors are proteins with molecular masses of 47 (p47) and 67kDa8–11. Upon stimulation of neutrophils, there is a translocation of the soluble components to the plasmalemma where the oxidase is assembled12, 13 (Figure 1). This assembly requires the presence of the b-cytochrome12, 14 and is associated with and/or organized by cytoskeletal proteins15. The intact system produces O 2 − according to the following stoichiometry:

Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils

{\rm{NAPDH + 2}}{{\rm{0}}_{\rm{2}}} \to {\rm{2}}{{\rm{0}}_{\rm{2}}}^{\rm{ - }}{\rm{ + NAD}}{{\rm{P}}^{\rm{ + }}}{\rm{ + 2}}{{\rm{H}}^{\rm{ + }}}{\rm{.}}

Farnesyl thiotriazole, a potent neutrophil agonist and structurally novel activator of protein kinase C

Immobilon-bound phosphoproteins labeled with 32P were utilized as substrates to study the enzymes in neutrophils that are active against the major products of protein kinase C. The labeled proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to immobilon-P membranes. Both particulate and soluble phosphatases were found to be active against the blotted phosphoproteins. Reactions were followed by autoradiography as the loss of 32P from individual protein bands. The tumor promoter okadaic acid and the hepatoxin microcystin-LR inhibited these reactions in a manner consistent with the enzymes being type 1 and/or 2A protein phosphatases.