Immunocytochemical localization of protein kinases Yes and Src in amoeboid microglia in culture: association of Yes kinase with vimentin intermediate filaments.

Amoeboid microglia isolated from primary cultures of neonatal rat brain correspond to a transient form of activated microglia, a resident population of macrophage-like cells. In order to understand the molecular aspects of microglial activation, we have studied amoeboid microglia in primary culture for the presence of Yes and Src protein tyrosine kinases, 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. Preadsorption of anti-c-yes antibody with an excess of antigenic peptide inhibited anti-c-yes immunofluorescence, while vimentin immunofluorescence remained unchanged. Double immunofluorescence images analyzed with the two-dimensional intensity distribution program (2-D scattered histograms) on Zeiss confocal scanning laser microscope demonstrate the colocalization of c-yes with vimentin. The extent of colocalization was more prominent after exposure of intact cultured microglia to dibutyryl cyclic AMP (dBcAMP), or to phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) or to okadaic acid, an inhibitor of protein phosphatases. The findings suggest that vimentin might serve as molecular support for Yes kinase and, since previous studies have shown that vimentin in amoeboid microglia is one of the major protein substrates of serine/threonine protein kinases, this function could be regulated by phosphorylation.