Involvement of MEK, ERK, PKC and GSK3B in Maintaining the Mitotic Spindle

The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. Glycogen synthase kinase 3B (GSK3B) and protein kinase C (PKC) also have been implicated in cell cycle regulation due to their association with the centrosomes along with MEK and ERK. In the current study, we tested the effects of inhibiting MEK on the activities of ERK, GSK3B, PKC, and ?-tubulin. Two types of MEK inhibitors were used, a siRNA inhibitor and U0126. The effects of MEK inhibition were tested by immunocytochemistry and confocal analysis, western blotting, RT-PCR analysis and study of cell numbers in M-phase stages. Results from this study indicate that inhibition of MEKdid not inhibit GSK3B and PKC enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was used next. Our results indicated a further decrease in mitotic spindle microtubules than what was seen with the MEK inhibitors and an absence of cells in most of the M-phase stages. GSK3B and PKC enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3B and PKC may not function as a part of the MAPK pathway to regulate the mitotic spindle.