P 147 Immunolocalization of the myristoylated alanine-rich C kinase substrate (MARCKS) in human corneal keratocytes

m. MARCKS is a ubiquitous protein kinase C substrate which interacts with cahncdulh~ and cross-links the actin filaments Its cellular function is probably related to the regulation of these two proteins. MARCKS is also a physiological substrate for prolio&irected kinase.s like cyclindependent kinasea (cdks) and MAP kinases. The expression of MARCKS is high in quiescent fibroblasts. with a raoid down-rermlation occunininp after se&m stiinulation. Altogether, these data suggest a f&tion of the protein in the regulation of the cell cwle. MARCKS is localized at the olasma membrane and reversibly tramlocates to the cytosolic and lysosomal cbmpamnents after phosphorylation by PKC in moose embryo tibroblasts. M&~Q&. We have used cell fractionation, immonoblotting, and immtmofluomacence on intact cells and nuclear preparations to investigate the expression and localization of MARCKS in cultured human comeal keratocytes before or after serum or PMA stimulation. m. ‘Ihe expression of MARCKS in human keratocytes was followed by immtmoblotting of crude cellular extracts Maximal expression was observed in quiescent (serum-deprived) cells, and a clear decrease occurred after 16 hours of serum stimulation. This corresoonds to the beginning of the S phase in this cell type. Immunoblotdng of &clear and non-nuclear (cvto0lasm and membranes) extracts revealed the existence of a nuclear popula& kf the protein lxXh h1’3T3 fibroblasts and keratocytes. The nuclear population seems to be constant after serum 01 PMA stimulation. hnmunofluorexence of intact cells confirms the membrane localization in keratocytes, and the cytoplasmic redistribution after PMA stimulation. In nuclezu preparations, the protein is clearly present in the nuclei, but with a different dishibution in 3T3 fibroblasts and in keratwytes. In this cell type, a pmtctate distribution was observed, that is not changed after PM.4 stimulation. The nature of these nuclear shwtmw remains to be established. Conelusions We demonstrate the regulated expression of MARCKS during the cell’cycle in keratocytes, and describe the. existence of a nuclear population of the protein in specific nuclear stmctarez not yet identified.