Investigation of the role intracelluar MST/Krs protein

星形孢子素及其类似物(K-2S2a，RK-286C)在没有干预有丝分裂的大鼠二倍体成纤维细胞3Y1细胞中抑制蛋白激酶，并引起与细胞松弛素B相同的DNA重新复制，产生四倍体细胞。我们发现，这些药物通过抑制细胞分裂而诱导K562细胞体积增大，而不是通过抑制细胞核分裂来诱导细胞增大。这种现象可能与一种蛋白激酶的关系有关，我们发现MST1/Krs2和MST2/Kps1与细胞分裂对细胞增殖的影响有关，可能是这些药物的靶向因子之一。为了研究MST/KRS在细胞分裂中的重要性，我们研究了激酶失活的MST/KRS在细胞增殖过程中的作用。K562细胞中MST/KRS的表达抑制了细胞的生长，细胞体积增大是由于细胞内核数的增加所致。此外，在人宫颈上皮样癌HeLa细胞中，当激酶失活的MST/KRS过表达时，我们进行了Hoechst 33258和罗丹明鬼臼蛋白的双重染色。在表达激酶失活的MST/KRS的细胞中，我们观察到细胞核数量的增加和肌动蛋白细丝的粘连。这些结果表明，MST/KRS是这些药物的靶向因子之一，并通过与细胞骨架蛋白如肌动蛋白的稳定性有关而在细胞分裂中发挥重要作用。

Staurosporine and its analogues (K-2S2a, RK-286C) are known to inhibit the protein kinases and also to cause DNA re-replication the same as cytochalasin B in rat diploid fibroblast 3Y1 cells without an intervening mitosis, producing tetraploid cells. We found that these drugs induced the increase of cell size by the inhibition of cell division but not nucleus division in human chronic myelogenous leukemia K562 cells. This phenomenon was expected to the relation of one protein kinase and we found that the human protein kinases, MST1/Krs2 and MST2/Kpsl, related to the cell division on the cell proliferation and might be one of the target factors of those drugs. To examine the importance of MST/Krs in the cell division, we investigated the effect of kinase-inactive MST/Krs expression during the cell proliferation. The expression of kinase-inactive MST/Krs in K562 cells induced the inhibition of cell growth and increase of the cell size was caused by the increase of nucleus count in the cells. Moreover, we performed the double staining with Hoechst 33258 and rhodamine phalloidin, when the kinase-inactive MST/KrS was overexpressed in human cervix epithelioid carcinoma HeLa cells. On the cells expressed kinase-inactive MST/Krs, we observed the increase of the nucleus count and the siassembly of actin filament. These results suggest that MST/Krs is one of the target factors of those drugs and plays the important role on the cell division by the relation to the stability of cytoskeleton protein such as actin.