Consequently, Mirk siRNA treatment increased growth rate of the human cancer cells which could be blocked by U0126, suggesting knockdown Mirk induced cell cycle alteration may be involving MAPK/ERK pathway. Mirk modulates cell survival associated with activation of MAPK/ERK To further determine Gemcitabine mechanism the effects of MAPK/ERK pathway involved in Mirk modulating cancer cell survival, the H292 cells were treated with 20 nM siRNAs with/with out U0126 in gradient for 72 h followed by western blot and flow cytometry analyses, respevtively. As shown in Figure 4A, U0126 blocked knockdown Mirk induced ERK1/2 activation, as well as alteration of downstream signals p27kip1 and cyclin D1 in H292 cells. Interest ingly, Mirk siRNA combined with U0126 led to in creased cell apoptosis evidenced by PARP cleavage and positive cells with active caspase 3.

Taken together, these results suggest that MAPK/ERK may be a novel pathway with which Mirk interacts to serves as an antiapoptotic factor in the human cancer cells. Discussion Mirk/Dyrk1B is a member of a conserved family of serine/threonine kinases that are actived by intramolecu lar tyrosine phosphorylation which mediate maturation in defferent tissues Mirk in skeletal muscle, Dyrk1A in brain, etc. Previous studies show that Dyrk1A activ ity was increased by binding to 14 3 3 protein, and is mediated by autophosphorylation at a C terminal serine, which is not conserved in Mirk. The possible YxY activation domain of Mirk within the conserved kinase region has been known to be intramolecularly phosphorylated only during translation, and the mature members of Mirk family have only serine/threonine kinase activity.

Whereas, in this study we uti lized a phosphoproteomics screen to identify pepti des corresponding to the tyrosine autophosphorylation site of Mirk/Dyrk1B in the human cancer cells and demonstrated a positive correlation between the expres sion of Mirk protein and the phosphotyrosine abun dance of Mirk/Dyrk1B. This suggests that activation of Mirk/Dyrk1B kinase in the deregulated cancer cells may be mediated by tyrosine autophosphorylation, although further study is required. Mirk/Dyrk1B is also an arginine directed serine/threo nine kinase which has limited expression in normal tissue with highest expression seen in skeletal muscle, heart, tests, and brain. Initially, most of the studies of Mirk/Dyrk1B have been conducted using myogenesis as a model system. It has been known that Mirk/ Dyrk1B functions as a transcription factor activator in muscle differentiation. In cultured myoblasts, mitogen Cilengitide deprivation increased Mirk/Dyrk1B protein levels pre dominantly via transcriptional mechanisms regulated by RhoA and Cdc42, and to a lesser extent by Rac1.