Phosphorylation of the activation loop of gamma p21-activated kinase (gamma-Pak) and related kinases (MSTs) in normal and stressed neutrophils.
Neutrophils stimulated with a variety of chemoattractants exhibit a rapid activation of two p21-activated kinases (Paks) with molecular masses of approximately 63 and 69 kDa (gamma- and alpha-Pak). A number of in vitro studies suggest that modification of Thr(402) in the activation loop (AL) of gamma-Pak can play a critical role in the regulation of this kinase under certain circumstances. A phosphospecific Ab was generated to this region of Pak (pPak(AL)Ab). This Ab reacted with activated gamma- and alpha-Pak from fMLP-stimulated neutrophils that contain the sequence KRXT(P)XXGTP in their ALS: The rapid but transient activation of Paks in normal stimulated neutrophils coincided with phosphorylation and dephosphorylation at the ALs of these enzymes. In contrast, stressed cells exhibited a prolonged phosphorylation at Thr(402) in both intact gamma-Pak and a proteolytic fragment of this kinase. The pPak(AL)Ab also reacted with the mammalian sterile twenty-like kinases (MSTs) (members of the Pak family) in osmotically stressed neutrophils and neutrophils treated with certain apoptotic agents (i.e., tumor promoters that inhibit type 1 and 2A protein phosphatases) but not in normal fMLP-stimulated cells. Thus, our results indicate that the AL of gamma-Pak undergoes transient phosphorylation during normal neutrophil stimulation and chronic phosphorylation in stressed cells. In addition, we demonstrate that a number of MSTs are present in neutrophils and also undergo phosphorylation during stressful circumstances.
Lian
J P
JP
Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Toker
A
A
Badwey
J A
JA
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D006168:Guinea Pigs; D007106:Immune Sera / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008387:Marine Toxins; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism*; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D010748:Phosphopeptides / Q000276:immunology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin; D054462:p21-Activated Kinases
D000730:Androstadienes; D007106:Immune Sera; D008387:Marine Toxins; D010080:Oxazoles; D010446:Peptide Fragments; D010748:Phosphopeptides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin