Cude et al. noted that when primary cultures of human foreskin cells and smooth muscle cells, as well as transformed HeLa cells, were arrested at M phase of the cell cycle there was an electrophoretic mobility shift (activation) of the kinase ERK5, a member of the mitogen-activated protein kinase family. Further analysis in synchronized HeLa cells showed that ERK5 activity--as well as that of MEK5, the kinase that activates ERK5--increased during G2 and remained high through M phase. Expression of a constitutively active form of MEK5 was sufficient to increase the number of mitotic cells of an asynchronous HeLa cell population, and expression of a dominant-negative ERK5 reduced the number of mitotic cells. Experiments in synchronized cells in which ERK5 activity was inhibited specifically from G2 onward (to avoid interfering with any roles ERK5 may have in the G1 and S phases) confirmed that ERK5 activity was required at the G2 and M stages. Studies in transfected HEK293 cells suggested that after nocodazole treatment to arrest the cells in G2, the transcription factor NF-κB was activated (as measured in reporter gene assays and DNA binding assays), and this was blocked by dominant-negative ERK5. ERK5 did not apparently directly phosphorylate the inhibitor of NF-κB (IκB), nor was the inhibitor of NF-κB kinase (IKK) complex active during G2 and M. Instead, ERK5 colocalized in the nucleus and coimmunoprecipitated with the ribosomal S6 kinase RSK2 in nocodazole-treated HeLa cells arrested in G2-M. RSK2 isolated from nocodazole-treated cells exhibited greater kinase activity toward IκB than that from nonarrested cells. Pharmacological inhibitors of NF-κB were used to show that NF-κB activity was required for progression through mitosis but not DNA replication (S phase). NF-κB stimulates the expression of several genes, the products of which are important for mitosis. Quantitative polymerase chain reaction experiments confirmed that transcripts for cyclin B1 and B2, polo-like kinase-1, and the protein phosphatase Cdc25B were increased at the time of G2 and that the increase in their transcription was blocked if NF-κB activation was inhibited. Thus, ERK5, acting through RSK2, appears to activate NF-κB to drive the expression of genes essential for the completion of mitosis.
K. Cude, Y. Wang, H.-J. Choi, S.-L. Hsuan, H. Zhang, C.-Y. Wang, Z. Xia, Regulation of the G2-M cell cycle progression by the ERK5-NFκB signaling pathway. J. Cell Biol. 177, 253-264 (2007). [Abstract] [Full Text]