RECRUITMENT OF THE LIM PROTEIN HIC-5 TO FOCAL CONTACTS OF HUMAN PLATELETS

Platelets are anuclear, membrane-bounded fragments derived from megaka ryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts, The focal contacts resemble those o f tissue culture cells. However, they lack paxillin, a conspicuous com ponent of these organelles. We found that instead of paxillin, platele ts contain a related protein with a molecular mass of 55 kDa that cros sreacts with a monoclonal antibody against paxillin, The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned o ut to be identical to a recently discovered gene encoding hic-5, Like paxillin, hic-5 is a cytoskeletal protein containing four carboxytermi nal LIM domains and LD motifs in the aminoterminal half. The LIM domai ns of both hic-5 and paxillin are capable of targetting green fluoresc ent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. On ly trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell l ine, and in megakaryocytes cultured from CD34(+) cells obtained from u mbilical cord blood, However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers s pecific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 ex pression must occur late in the maturation of megakaryocytes into plat elets.