IMMUNOCYTOCHEMICAL LOCALIZATION OF PROTEIN-KINASES YES AND SRC IN AMEBOID MICROGLIA IN CULTURE - ASSOCIATION OF YES KINASE WITH VIMENTIN INTERMEDIATE FILAMENTS

Amoeboid microglia isolated from primary cultures of neonatal rat brai n correspond to a transient form of activated microglia, a resident po pulation of macrophage-like cells. In order to understand the molecula r aspects of microglial activation, we have studied amoeboid microglia in primary culture for the presence of Yes and Src protein tyrosine k inases, two kinases which have been implicated in signal transduction process during monocyte/macrophage activation. Immunofluorescence with an antibody raised against the peptide from unique N-terminal domains of Yes and Src demonstrated that Yes and Src kinases are enriched in perinuclear areas of amoeboid microglia, In addition, the antibody to c-yes peptide had a cytoplasmic distribution which coincided with the distribution of vimentin-containing intermediate filaments. Preadsorpt ion of anti-c-yes antibody with an excess of antigenic peptide inhibit ed anti-c-yes immunofluorescence, while vimentin immunofluorescence re mained unchanged. Double immunofluorescence images analyzed with the t wo-dimensional intensity distribution program (2-D scattered histogram s) on Zeiss confocal scanning laser microscope demonstrate the colocal ization of c-yes with vimentin. The extent of colocalization was more prominent after exposure of intact cultured microglia to dibutyryl cyc lic AMP (dBcAMP), or to phorbol ester TPA (12-O-tetradecanoylphorbol-1 3-acetate) or to okadaic acid, an inhibitor of protein phosphatases. T he findings suggest that vimentin might serve as molecular support for Yes kinase and, since previous studies have shown that vimentin in am oeboid microglia is one of the major protein substrates of serine/thre onine protein kinases, this function could be regulated by phosphoryla tion.