Involvement of PKC? and GSK3? 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? (GSK3?) have also been shown to be associated with the mitotic spindle apparatus. In this study, we show the enrichment of active (phosphorylated) PKC? 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? are associated with the mitotic spindle, first, the co-localization of phosphorylated PKC isoforms with GSK3? was studied at the poles in metaphase cells. Fluorescence resonance energy transfer (FRET) analysis was used to demonstrate close molecular proximity of phospho-PKC? with phospho(ser9)GSK3?. Second, the involvement of inactive GSK3? in maintaining an intact mitotic spindle in 3T3 cells was shown. Third, this study also showed that addition of a phospho-PKC? 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? acting through GSK3?. Phospho-PKC? is in close molecular proximity to GSK3?, whereas the other isoforms of PKC such as pPKC?II, pPKC?, pPKC?, and pPKC? are not close enough to have significant FRET readings. The close molecular proximity supports the idea that GSK3? may be a substrate of PKC?.