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 3β (GSK3β) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKCζ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases PKC and GSK3β are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3β was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKCζ with phospho(ser9)GSK3β. Second, the involvement of inactive GSK3β in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKCζ 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 PKCζ acting through GSK3β. Phospho-PKCζ is in close molecular proximity to GSK3β, whereas the other isoforms of PKC such as pPKCβII, pPKCγ, pPKCμ, and pPKCθ are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3β may be a substrate of PKCζ.