Figure 9.
MEK1 promotes mitosis at G2/M by phosphorylation of GRASP55 at T222 and T225. (A) The percentage of cells in mitosis (mitotic index) was measured after thymidine release. The MEK1/2 inhibitor U0126 or 0.05% dimethyl sulfoxide carrier were added at 6 h after release. Tandem experiments were carried out in cells transfected with control siRNA (A and B) or siRNA against GRASP55 (B). (C) The degree to which MEK1 inhibition delays mitosis was quantified using an RMSD comparison of control and U0126-treated cells. (D–G) A postmitotic couplet assay was used to estimate the abundance of postmitotic HeLa cells expressing wild-type (D) or nonphosphorylatable GRASP55 (G55 T222,225A; E). Conversely, a functional gene replacement strategy was used (Puthenveedu et al., 2006) to compare wild-type expressors (F) with cells expressing a mutant GRASP55 designed to mimic mitotic ERK phosphorylation (G55 T222,225D; G).