Involvement of PKCzeta, GSK3beta, and MAPK in maintenance of the mitotic spindl

In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3beta (GSK3beta) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCzeta at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3beta are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3beta was studied in metaphase cells. Second, the involvement of inactive GSK3beta in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCzeta specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCzeta acting through GSK3beta. 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. GSK3beta and PKCzeta also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3beta, PKCzeta, and a-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3beta and PKCzeta 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 also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3beta and PKCzeta enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3beta and PKCzeta may not function as a part of the MAPK pathway to regulate the mitotic spindle.