THE RENATURABLE 69-KDA 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) ar e known to exhibit rapid activation of four protein kinases with molec ular masses of similar to 69, similar to 63, similar to 49, and simila r to 40-kDa. Activation of these kinases is blocked by antagonists of phosphatidylinositol 3-kinase and type 1 and/or type 2A protein phosph atases. These enzymes can be detected by their ability to undergo rena turation and catalyze the phosphorylation of a peptide substrate that corresponds to amino acid residues 297-331 of the 47-kDa subunit of th e NADPH-oxidase complex fixed within a gel. In this report, we demonst rate that an antibody generated to a fusion protein containing amino a cid residues 175-306 of p21-activated protein kinase 1 (Pak1) reacts w ith three proteins in guinea pig neutrophils with molecular masses in the 60-70-kDa range during Western blotting. This antibody immunopreci pitates both the 69- and 63-kDa renaturable kinases from lysates of st imulated cells along with a minor 60-kDa kinase. No activities were ob served for any of these enzymes in immunoprecipitates from unstimulate d neutrophils. However, addition of ATP and activated Rac 1 or Cdc42 t o immunoprecipitates from unstimulated cells resulted in the stimulati on of two renaturable kinases with molecular masses in the 69- and 63- kDa range. These immunoprecipitates also contained two novel protein k inases with masses of similar to 49 and 40 kDa that were selectively a ctivated by Cdc42. In contrast, the 69- and 63-kDa kinases were not im munoprecipitated from lysates of stimulated neutrophils with an antibo dy to Pak2 or with nonimmune serum. These data indicate that the renat urable 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.