Abstract
Sam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction regulation of cell growth and proliferation, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in B lymphocyte leukemia DT40 cell line. By using gene targeting method, we isolated a mutation of deleted proline rich domain from Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. The results have demonstrated that mutant cells grew at a slower rate (2.1 hours growth elongation). Cell cycle pattern was observed in mutant cells by flow-cytometric analysis, the retardation of cell growth was mainly due to the elongation of S phase (1.7 hours elongation). When the cell cycle was analyzed at different serum concentration conditions, we found that the effect of Sam68 mutant on the S phase was further supported by the serum depletion experiment. In light of evidence that RNA binding activity is essential for functions, Sam68 is implicated in cell growth control probably by modulating the function of mRNAs in S phase or earlier. Larger amounts of dead cells was observed in the culture of mutant cells, and serum depletion enhanced the ratio of dead cells in both culture conditions. Since the putative control of cell cycle by Sam68 appeared to be related to signal transduction, we next assessed the involvement of Sam68 in B cell signal transduction pathway. After cross-linking of B cell receptor on the DT40 cells by IgM antibody, phosphorylation of cellular proteins was analyzed by protein blotting with anti-phosphotyrosine antibody. The result demonstrated that the overall pattern of B cell receptor in Sam68 mutant cells was similar to that in the wild type DT40 cells. However, the maximal level of tyrosine phosphorylation on BLNK, a prominent signal molecule in B cell, has been observed no significantly changes in the mutant cells. Since the expression levels of B cell receptor on the Sam68 mutation clones were essentially same as that of parental DT40 cells, it was suggested that deletion of partial of proline region in Sam68 resulted in the decrease in B cell signal transduction. We have concluded that the proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.
Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint
