Activation of the p21-activated protein kinases from neutrophils with an antibody that reacts with the N-terminal region of Pak 1.
Neutrophils contain two renaturable p21-activated protein kinases (Paks) with molecular masses of ca. 69 and 63 kDa that undergo rapid activation upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. We now report that these kinases undergo a massive, ATP-dependent activation in lysates of unstimulated neutrophils during immunoprecipitation with an antibody generated to residues 2-21 of the N-terminal region of Pak1. This activation was specific as it was completely blocked by a peptide that corresponds to residues 2-21 of Pak1 and was not observed with an antibody generated to the C-terminal region of Pak 1. The properties of the Paks activated with the antibody were virtually identical to those observed for these kinases from stimulated neutrophils, or activated in vitro with Rac-GTPgammaS plus ATP. These data indicate that perturbation of the N-terminal region of Pak can trigger activation of this enzyme, and that both the 69 and 63 kDa kinases may represent forms of Pak 1 that differ in their content of phosphate.
Lian
J P
JP
Boston Biomedical Research Institute, MA 02114, USA.
Badwey
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000469:Alkaline Phosphatase; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006168:Guinea Pigs; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D010529:Peritoneal Cavity; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins
D000906:Antibodies; D010750:Phosphoproteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D000469:Alkaline Phosphatase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins