Modulation of the activity and subcellular distribution of protein tyrosine kinases in human neutrophils by phorbol esters.

Abstract

Although several tyrosine kinases are present in human neutrophils, little is known regarding the biochemical basis for their activation. We have identified two tyrosine kinase activities in 0.1 and 1% Triton cell extracts of human neutrophils using a non-denaturing gel assay. The first protein tyrosine kinase activity of a faster mobility was associated exclusively with the 0.1% Triton cell extract. The second activity, of slower mobility, was mainly associated with the 0.1% Triton cell extract and to a lesser extent with the 1% Triton cell extract. A modulation of the activities and the distribution of these two tyrosine kinase activities was observed upon stimulation of neutrophils with PDBu (phorbol 12,13-dibutyrate), a direct PKC (protein kinase C) activator. The addition of 1 microM PDBu induced a time-dependent decrease of both tyrosine kinases in the 0.1% Triton cell extract. Although the fast mobility tyrosine kinase activity disappeared completely, the slow mobility tyrosine activity decreased only partially. Concomitantly, an increase in the latter activity was detected in the 1% Triton cell extract. The pattern of tyrosine phosphorylation upon PDBu stimulation was also examined and the results showed that the phorbol ester induced time-dependent increases in the level of phosphotyrosine-containing proteins in at least 10 distinct bands. Two lines of evidence indicated that the effects of PDBu were mediated by PKC: 1) The stereo-isomer of PDBu, 4 alpha-PDBu, did not affect the activities and distribution of the tyrosine kinases, and 2) The PKC inhibitor, RO 318220, prevented the redistribution of the tyrosine kinase activities and inhibited the stimulation of tyrosine phosphorylation induced by PDBu. These results show that the activity and distribution of at least two human neutrophil tyrosine kinases are modulated after the activation of PKC and that the low mobility tyrosine kinase activity is the most sensitive to PDBu. Based on previous studies, the fast mobility tyrosine kinase activity was likely to be a member of the pp60src tyrosine kinase family and the slower one may be related to the pp93fes. Furthermore, these results begin to define the nature of the relationships among the PKC- and the tyrosine kinase-signaling pathways.