Analysis of hematopoietic cell specific protein, M-DOCK

Full length cDNA of M-DOCK was isolated at Kazusa DNA Institute and its sequence has been deposited as KIAA0209. The cDNA sequence of M-DOCK contains a 5490 bp open reading frame that encodes a 1830-aa protein with a calculated molecular mass of 212 kDa. The amino acid sequence of M-DOCK has 62.3% identity with DOCK180, excluding the carboxyl terminal variable regions. DOCK180, which was originally identified as a major protein bound to Crk oncogene product, is an archetype of a family of proteins including Ced-5 of C. elegans and Mbc of D. melanogaster. DOCK180 is localized at focal adhesions and regulates cell morphology through the activation of Rac. Expression of DOCK180 is ubiquitous except in hematopoietic cells. Here, the author reports on the characterization of M-DOCK protein, which is closely related to DOCK180 but expressed only in the hematopoietic cells. We immunized rabbits with bacterially-expressed GST-M-DOCK protein, and obtained an anti-serum specific to M-DOCK. Tissue distribution of M-DOCK was examined by Northern blotting and Western blotting. M-DOCK was expressed abundantly in peripheral blood leukocytes. M-DOCK expression was detected also in the cell lines derived from T-cells, B-cells, and monocytes, but never in the adherent cells. Most of the lymphocytes and macrophages in human organs expressed M-DOCK diffusely in the cytoplasm, which was analyzed by immunohistochemistry using an anti-M-DOCK antibody. We previously reported that membrane-targeted form of DOCK180 induced spreading of NIH 3T3 cells. By contrast, overexpression of M-DOCK rounded up flat NRK cells. This suggests that M-DOCK may regulate cell detachment of adherent cells. These results demonstrate that M-DOCK is a new member of DOCK180-family proteins and regulates cell shape in suspension cells.