GSK3β mediates acentromeric spindle stabilization by activated PKCζ

Abstract

Upon fertilization, the mammalian egg undergoes a precise series of signaling events that orchestrate its conversion into a zygote. Mouse eggs contain acentrosomal spindle poles when arrested at meiotic metaphase II. The meiotic spindle is thought to provide a scaffold that mediates spatial and temporal regulation of the signaling pathways orchestrating post-fertilization events. Many kinases have been found to be enriched at the MII meiotic spindle, such as Protein Kinase C (PKC), and are thought to have an important role in regulating signaling events initiated through fertilization. In this study phosphorylated PKC zeta (p-PKC zeta) and Glycogen Synthase Kinase 3beta (GSK3 beta) were found to be enriched at both acentrosomal spindle poles and the kinetochore region. Phosphorylated PKC zeta (p-PKC zeta) was immunopurified from MII eggs and was found to co-localize with known microtubule stabilizing components found in somatic cells, including GSK3 beta and Partition deficit protein 6 (Par6). Both fluorescence resonance energy transfer (FRET) and immunofluorescence confirmed the existence and close association of these proteins with p-PKC zeta at the meiotic spindle. When GSK3 beta is phosphorylated on ser9 its activity is inhibited and the spindle is stabilized. However, when GSK3 beta is dephosphorylated (on ser9) it becomes active and the spindle is destabilized. The mechanism by which p-PKC zeta maintains spindle organization appears to be through GSK3 beta and suggests that p-PKC zeta phosphorylates GSK3 beta on the ser9 position inactivating GSK3 beta and consequently maintaining spindle stability during meiotic metaphase arrest.

Identification and regulation of glycogen synthase kinase-3 during bovine embryo development

“…Lithium also interferes with another second messenger system, the inositol pathway causing selective reductions of PKC (Manji & Lenox 1999), which has been shown to phosphorylate and inactivate GSK3 mediating acentromeric spindle stabilization in mouse oocytes (Baluch & Capco 2008). This reduction in cAMP concentration or PKC by lithium in bovine embryos would lead to a decrease in the phosphorylation of GSK3, as observed here, and may explain the detrimental effect on embryo development as previously shown in mouse, rabbit, and Xenopus embryos (Kao et al 1986, Fahy & Kane 1994, Rogers & Varmuza 1996.…”

PKC Regulation of Gametogenesis and Early Development

“…However, these data leave open the possibility that the knockdown may have several effects on the egg's spindle apparatus (Ma et al, 2008), as the reduction of pericentrin would provide no sites for p-PKC to bind at the poles. This alternative interpretation could explain the disruption of the spindle since p-PKC activity is required for spindle stability as previously described (Baluch et al, 2004(Baluch et al, , 2008. Moreover, both immunopurification and FRET analysis at the spindle poles demonstrates that total and p-PKC are in close molecular proximity with -tubulin, Par6, and ser9-GSK3 (Baluch & Capco, 2008).…”

“…This alternative interpretation could explain the disruption of the spindle since p-PKC activity is required for spindle stability as previously described (Baluch et al, 2004(Baluch et al, , 2008. Moreover, both immunopurification and FRET analysis at the spindle poles demonstrates that total and p-PKC are in close molecular proximity with -tubulin, Par6, and ser9-GSK3 (Baluch & Capco, 2008). The Par6/PKC /GSK3 pathway may have a role in microtubule stability (Baluch & Capco, 2008).…”

“…Total PKC remained behind in the cytoskeleton after the detergent extraction process; the closeness of this binding was confirmed by FRET analysis between -tubulin of the meiotic spindle and the PKC isotypes. FRET analysis demonstrated a close molecular proximity not only between -tubulin and PKC , but also between -tubulin and PKC (Baluch et al, 2004, Baluch & Capco, 2008. Other investigations have shown that PKC exists in the cytoplasm of the fertilization-competent egg (Raz et al, 1998).…”

Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation

“…In mice early embryos treated with lithium or with another GSK3 inhibitor alsterpaullone, chromatin segregation abnormalities were also observed during the first cleavages (Acevedo et al 2007b). Recently, GSK3B has been found to be involved in the stabilisation of meiotic spindle, and it was localised to spindle poles and to kinetochore region in mature mice oocytes (Baluch & Capco 2008). MAPK14 is also considered as a component of the spindle assembly checkpoint in cell cycle.…”