SERINE THREONINE KINASE ACTIVATION IN HUMAN NEUTROPHILS - RELATIONSHIP TO TYROSINE PHOSPHORYLATION/

Tyrosine phosphorylation is among the earliest responses of neutrophil s 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 desi gned to test whether tyrosine phosphorylation is required for activati on of PK65 and PK72. Pretreatment of cells with the tyrosine kinase in hibitors erbstatin or genistein prevented both phosphotyrosine accumul ation and activation of PK65 and PK72. In nondenaturing lysates, PK65 and PK72 became spontaneously inactivated in parallel with rapid endog enous tyrosine dephosphorylation. Spontaneous dephosphorylation and in activation of PK65 and PK72 were prevented in denatured lysates. Under these conditions, dephosphorylation could be induced by exogenous pho sphotyrosine phosphatase 1B. PK65 and PK72 activation persisted despit e virtually complete tyrosine dephosphorylation. Moreover, im munoprec ipitation 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 lon ger required for maintenance of their kinase activity.