Abstract
The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established. On the other hand, the link between the G protein-coupled receptors (to which the receptors for chemotactic factors belong) and the activation of tyrosine kinases is very poorly characterized. Based on previous observations indicating that the stimulation of tyrosine phosphorylation was sensitive to inhibition by the phosphatidylinositol 3-kinase inhibitor wortmannin and the recent description of pleckstrin homology domain-containing tyrosine kinases (the Tec family), we have examined the potential implication of the latter in the responses of human neutrophils to chemotactic factors. The results obtained indicate firstly that several members of the Tec family of tyrosine kinases are expressed in human neutrophils, including Tec, Btk, and Bmx. Stimulation of the cells with fMet-Leu-Phe led to a rapid activation of Tec as indicated by its translocation to a membrane fraction and to increases in its in situ level of tyrosine phosphorylation and its capacity to tyrosine phosphorylate itself or an exogenous substrate (SAM68-GST) in in vitro kinase assays. The activation of Tec was inhibited by pertussis toxin as well as by wortmannin. The results of this study provide direct evidence for the implication of Tec family kinases in the responses of human neutrophils to chemotactic factors. They also suggest that one of the links between G protein-coupled receptors and tyrosine kinases depends on the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol 3,4,5-trisphosphate.
Highlights
The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established
Activation of Tec Family Members by Chemotactic Factors in Human Neutrophils—One of the characteristic features of the activation of Tec family tyrosine kinases is their stimulated translocation to membrane fractions, an event that is thought to result from high affinity interactions of their PH domains with PtdIns[3,4,5]P3 [27, 28, 50, 51]
We had shown previously that the stimulation of human neutrophils by chemotactic factors was associated with the translocation of p110␥ to a crude membrane fraction [24]
Summary
The importance of the tyrosine phosphorylation cascades in the initiation and regulation of the functional responsiveness of human neutrophils is well established. The results of this study provide direct evidence for the implication of Tec family kinases in the responses of human neutrophils to chemotactic factors They suggest that one of the links between G protein-coupled receptors and tyrosine kinases depends on the activation of phosphatidylinositol 3-kinase and the generation of phosphatidylinositol 3,4,5-trisphosphate. The results of a previous study have provided evidence that the stimulation of tyrosine phosphorylation by chemoattractants in human neutrophils was sensitive to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, wortmannin [20] This observation was originally difficult to reconcile with the characteristics of PI 3-kinases known at the time, which were themselves controlled by the tyrosine phosphorylation of their regulatory p85 subunit [21]. Its presence and activation in human neutrophils, in preference over that of p85/p110, by chemoattractants has been reported [24]
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