Involvement of PKCζ and GSK3β in the stability of the metaphase spindle

In the somatic cell, 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 still unclear. Other protein kinases such as, glycogen synthase kinase 3 beta (GSK3 beta) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKC zeta at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3 beta are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3 beta was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKC zeta with phospho(ser9) GSK3 beta. Second, the involvement of inactive GSK3 beta in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKC zeta specific inhibitor to cells can disrupt the mitotic spindle microtubules and some of the proteins associated with it. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKC zeta acting through GSK3 beta. Phospho-PKC zeta is in close molecular proximity to GSK3 beta, whereas the other isoforms of PKC such as pPKC beta II, pPKC gamma, pPKC mu, and pPKC0 are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3 beta may be a substrate of PKC zeta.