Serine/threonine kinase activation in human neutrophils: relationship to tyrosine phosphorylation.
Tyrosine phosphorylation is among the earliest responses of neutrophils to chemotactic peptides. Tyrosine phosphorylated proteins comigrate with serine/threonine kinases of 65 and 72 kDa (PK65 and PK72), which are activated concomitantly by the chemoattractants. Studies were designed to test whether tyrosine phosphorylation is required for activation of PK65 and PK72. Pretreatment of cells with the tyrosine kinase inhibitors erbstatin or genistein prevented both phosphotyrosine accumulation and activation of PK65 and PK72. In nondenaturing lysates, PK65 and PK72 became spontaneously inactivated in parallel with rapid endogenous tyrosine dephosphorylation. Spontaneous dephosphorylation and inactivation of PK65 and PK72 were prevented in denatured lysates. Under these conditions, dephosphorylation could be induced by exogenous phosphotyrosine phosphatase 1B. PK65 and PK72 activation persisted despite virtually complete tyrosine dephosphorylation. Moreover, immunoprecipitation experiments indicated that PK65 and PK72 are not themselves tyrosine phosphorylated. We concluded that tyrosine phosphorylation is a necessary upstream event in the activation of the serine/threonine kinases. However, once the posttranslational modification that renders PK65 and PK72 active has occurred, tyrosine phosphorylation is no longer required for maintenance of their kinase activity.
Brumell
J H
JH
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Grinstein
S
S
D004789:Enzyme Activation; D006801:Humans; D008387:Marine Toxins; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D014443:Tyrosine / Q000378:metabolism*
D008387:Marine Toxins; D010080:Oxazoles; D014443:Tyrosine; C059041:calyculin A; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases