Characterization of the CATS Protein Which Interacts with the Leukemogenic Fusion Protein CALM/AF10 Using Mono-Clonal Antibodies and Fluroescent Protein Tags.

Abstract

The CATS gene was first identified in a yeast two hybrid screen as CALM interacting protein expressed in thymus and spleen. The CATS interaction region of CALM is contained in the leukemogenic fusion protein CALM/AF10, which is found in acute myeloid leukemia, malignant lymphoma and acute lymphoblastic leukemia. We performed co-immunoprecipitation experiments to confirm CALM-CATS interaction found in the yeast system. RT PCR analysis showed CATS expression in patient cells and cell line carrying the CALM/AF10 rearrangement. Further expression analysis showed CATS expression in different cell subpopulation from the murine thymus (CD4+, CD8+ and CD4+/CD8+) and bone marrow (cKit+, Sca 1+/CKit+, Ter119, Gr1, CD8+, Mac1) but nearly absent in B220 positive cells. Interestingly, there was a high CATS expression in CALM/AF10 expressing B220+ leukemic cells in a CALM/AF10 murine bone marrow transplant model. Several monoclonal antibodies against the C-terminus of human CATS were generated. These antibodies recognize both the human and the murine CATS protein. Using these antibodies we could show a high expression of CATS in different human leukemia, lymphoma and solid tumor cell lines, as well as in normal proliferating cell lines (HEK293 and WI38), but not in T-cell lines (TYRF8 and JB4). Using protein lysates from cell cycle synchronized cells (Hela and U2OS) a clear cell cycle dependent regulation of CATS protein levels was demonstrated. Moreover, Western Blot analysis shows that serum stimulation after serum starvation leads to increased expression of CATS in the glioblastoma cell line T98G. Immunofluorescence shows that CATS is localized mainly to the nucleus. Coexpression of CFP-CATS with YFP tagged CALM or CALM/AF10 was able to markedly increase the nuclear localization of both CALM and the CALM/AF10 fusion protein. This effect of CATS is stronger on the YFP-CALM/AF10 fusion protein than on the CALM protein. When fused to a GAL4 DNA binding domain, CATS acts as a strong repressor of transcription in reporter gene assays. Our results indicate that the subcellular localization of CALM and CALM/AF10 could depend in part on the presence of CATS with a greater fraction of CALM or CALM/AF10 being present in the nucleus in cells with high CATS expression (e.g. lymphoid cells). The CALM-CATS interaction might thus play an important role in CALM/AF10 mediated leukemogenesis.