STIMULATION OF NEUTROPHILS WITH A CHEMOATTRACTANT ACTIVATES SEVERAL NOVEL PROTEIN-KINASES THAT CAN CATALYZE THE PHOSPHORYLATION OF PEPTIDESDERIVED FROM THE 47-KDA PROTEIN-COMPONENT OF THE PHAGOCYTE OXIDASE AND MYRISTOYLATED ALANINE-RICH C-KINASE SUBSTRATE

Novel protein kinases that may participate in the signal transduction pathways of neutrophils were sought by a procedure based on the abilit y of these enzymes to undergo renaturation and catalyze the phosphoryl ation of a peptide substrate fixed in a gel. We report that neutrophil s contain four uncharacterized protein kinases with molecular masses o f about 69, 63, 49, and 40 kDa, which are rapidly activated upon stimu lation of these cells with the chemoattractant fMet-Leu-Phe. These kin ases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH oxidase system (p 47-phox). A peptide that corresponds to residues 153-178 of the human myristolyated alanine-rich C kinase substrate (MARCKS) protein was als o a substrate for the 69- and 63-kDa kinases. The time course for the activation of these enzymes was similar to the phosphorylation of p47- phox and MARCKS in intact neutrophils. In contrast, stimulation of the se cells with 4beta-phorbol 12-myristate 13-acetate, the calcium ionop hore A23187, or the combination of these agonists did not activate the se enzymes. Activation of the 63- and 40-kDa protein kinases was block ed by pertussis toxin, calyculin A, and staurosporine. Several other u nidentified protein kinases were also active with these peptides but d id not exhibit enhanced activity after cell stimulation with this meth od.