MODULATION OF THE ACTIVITY AND SUBCELLULAR-DISTRIBUTION OF PROTEIN-TYROSINE KINASES IN HUMAN NEUTROPHILS BY PHORBOL ESTERS

Although several tyrosine kinases are present in human neutrophils, li ttle is known regarding the biochemical basis for their activation. We have identified two tyrosine kinase activities in 0.1 and 1% Triton c ell extracts of human neutrophils using a non-denaturing gel assay. Th e first protein tyrosine kinase activity of a faster mobility was asso ciated exclusively with the 0.1% Triton cell extract. The second activ ity, of slower mobility, was mainly associated with the 0.1% Triton ce ll extract and to a lesser extent with the 1% Triton cell extract. A m odulation of the activities and the distribution of these two tyrosine kinase activities was observed upon stimulation of neutrophils with P DBu (phorbol 12,13-dibutyrate), a direct PKC (protein kinase C) activa tor. The addition of 1 muM PDBu induced a time-dependent decrease of b oth tyrosine kinases in the 0.1% Triton cell extract. Although the fas t mobility tyrosine kinase activity disappeared completely, the slow m obility tyrosine activity decreased only partially. Concomitantly, an increase in the latter activity was detected in the 1% Triton cell ext ract. The pattern of tyrosine phosphorylation upon PDBu stimulation wa s also examined and the results showed that the phorbol ester induced time-dependent increases in the level of phosphotyrosine-containing pr oteins 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, 4alpha-PDBu, did not affect the activities and distribution of the tyrosine kinases, and 2) The PKC inhibitor, RO 318220, prevented t he 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 neutrop hil tyrosine kinases are modulated after the activation of PKC and tha t the low mobility tyrosine kinase activity is the most sensitive to P DBu. Based on previous studies, the fast mobility tyrosine kinase acti vity was likely to be a member of the pp60src tyrosine kinase family a nd the slower one may be related to the pp93fes. Furthermore, these re sults begin to define the nature of the relationships among the PKC- a nd the tyrosine kinase-signaling pathways.