MEK1, a gene product that regulates cell growth and differentiation, also plays an important role in Golgi breakdown during the cell cycle. We have recently shown that polo-like kinase (Plk3) is Golgi localized and involved in Golgi dynamics during the cell cycle. To study the mode of action of Plk3 in the Golgi fragmentation cascade, we examined functional as well as physical interactions between Plk3 and MEK1/ERKs. In HeLa cells, although a significant amount of Plk3 signals dispersed in a manner similar to those of Golgi during mitosis concentrated Plk3 was detected at spindle poles, which colocalized with phospho-MEKs and phospho-ERKs. Pull-down assays showed that Plk3 physically interacted with MEK1 and ERK2. Nocodazole activated Plk3 and its activation was blocked by MEK-specific inhibitors (PD98059 or U0126). Moreover, transfection of activated MEK1 resulted in an enhanced kinase activity of Plk3; Plk3-induced fragmentation of Golgi stacks was significantly reduced after treatment with MEK inhibitors. Consistently, ectopic expression of activated MEK1, but not kinase-dead MEK1(K97R), stimulated Plk3 to induce Golgi breakdown and the stimulation was not observed in cells expressing Plk3(K52R). Furthermore, PLK3(-/-) murine embryonic fibroblast cells exhibited a significantly less fragmentation of the Golgi complex than that in wild-type cells after exposed to nocodazole. Thus, our studies strongly suggest that Plk3 may be a key protein kinase mediating MEK1 function in the Golgi fragmentation pathway during cell division.