ZYMOSAN-TRIGGERED ASSOCIATION OF TYROSINE PHOSPHOPROTEINS AND LYN KINASE WITH CYTOSKELETON IN HUMAN MONOCYTES

Phagocytosis of pathogens and inert particles such as zymosan by macro phages, and related secretory functions require the combination of sev eral intracellular signals and the reorganization of cytoskeleton. We recently reported that zymosan stimulated the tyrosine phosphorylation s of several endogenous substrates in human monocytes. In this work, t he relationship between zymosan-stimulated tyrosine phosphoproteins an d detergent-insoluble material considered as cytoskeleton was investig ated. Triton X-100-insoluble fraction contained two proteins of 53 and 56 kDa that were tyrosine phosphorylated after only 5 min of stimulat ion with zymosan and remained labeled for 30 min. Because 53- and 56-k Da phosphoproteins migrated, as did some components of the src tyrosin e kinase family, namely p53-56(lyn), we wondered if 53- and 56-kDa pho sphoproteins were related to lyn kinase. First, the amount of immunore active p53-56(lyn) increased in Triton X-100-insoluble fraction as did zymosan-stimulated tyrosine phosphoproteins. This property of p53-56( lyn) was unique, as no other member of the src family was found in thi s fraction. Second, when the immunoblots were reprobed with anti-phosp hotyrosine mAb, the m.w. of p53-56(lyn) and tyrosine-phosphorylated pr oteins were identical in apparent size. Third, p53-56(lyn) was probabl y activated after cell stimulation with zymosan, because the phosphory lation levels of a synthetic copolymer of glutamine-tyrosine were incr eased in Triton X-100-insoluble fraction. In addition, we studied the distribution of lyn kinase and tyrosine phosphoproteins in phagocytozi ng monocytes. By using immunofluorescence, we showed that lyn kinase w as located preferentially in the periphagosomal region in a specific m anner, as an src tyrosine kinase such as p59(hck), which was not assoc iated with cytoskeleton, was not concentrated around the vacuoles. Mor eover, periphagosomal phosphoproteins were also detected and found to be colocalized with polymerized actin. Because zymosan interacts with human monocytes via beta 2 integrins, which are known to be cytoskelet on-associated, we suggest that p53-56(lyn) provides the molecular link between zymosan receptors and cytoskeleton, and directs the cytoskele tal reorganization in the periphagosomal area.