GSK-3β Regulates Proper Mitotic Spindle Formation in Cooperation with a Component of the γ-Tubulin Ring Complex, GCP5

Abstract

Glycogen synthase kinase-3␤ (GSK-3␤) is known to play a role in the regulation of the dynamics of microtubule networks in cells. Here we show the role of GSK-3␤ in the proper formation of the mitotic spindles through an interaction with GCP5, a component of the ␥-tubulin ring complex (␥TuRC). GCP5 bound directly to GSK-3␤ in vitro, and their interaction was also observed in intact cells at endogenous levels. Depletion of GCP5 dramatically reduced the GCP2 and ␥-tubulin in the ␥TuRC fraction of sucrose density gradients and disrupted ␥-tubulin localization to the spindle poles in mitotic cells. GCP5 appears to be required for the formation or stability of ␥TuRC and the recruitment of ␥-tubulin to the spindle poles. A GSK-3 inhibitor not only led to the accumulation of ␥-tubulin and GCP5 at the spindle poles but also enhanced microtubule nucleation activity at the spindle poles. Depletion of GCP5 rescued this disrupted organization of spindle poles observed in cells treated with the GSK-3 inhibitor. Furthermore, the inhibition of GSK-3 enhanced the binding of ␥TuRC to the centrosome isolated from mitotic cells in vitro. Our findings suggest that GSK-3␤ regulates the localization of ␥TuRC, including GCP5, to the spindle poles, thereby controlling the formation of proper mitotic spindles.

The where, when and how of microtubule nucleation – one ring to rule them all

“…In addition to GCP-WD, centrosomal targeting of c-tubulin in humans requires an intact cTuRC (N. T.-T., J. R. and J. L., unpublished observations; Izumi et al, 2008). By contrast, depletion of GCP-WD, GCP4, GCP5 and GCP6 in Drosophila does not abolish centrosomal recruitment of c-tubulin and microtubule nucleation (Vérollet et al, 2006).…”

“…In epithelial cells, CDK1 phosphorylates GCP6 to disrupt the interaction of cTuRCs with keratin and to remove the complexes from the apical domain, which might be important for the remodeling of the microtubule array on mitotic entry (Oriolo et al, 2007). In vitro, human GCP5 is a substrate for glycogen synthase kinase 3 beta (GSK3b), which negatively regulates the amount of c-tubulin at mitotic centrosomes (Izumi et al, 2008). It is unclear, however, whether this regulation occurs at the level of GCP5 or involves other PCM components.…”

“…Depletion of c-tubulin or any of the Grip-GCPs destabilizes cTuRC in sucrose gradients, suggesting that they all have important structural roles (Izumi et al, 2008;Vérollet et al, 2006;Vogt et al, 2006;Xiong and Oakley, 2009;Zhang et al, 2000). cTuRCs are formed by the helical arrangement of smaller Y-shaped subcomplexes, the so-called c-tubulin small complexes (cTuSCs), which are composed of two molecules of c-tubulin and one molecule each of GCP2 and GCP3 ( Fig.…”

MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation

“…However, in human cells γTuRC pre-assembly, targeting and activation are linked in the sense that all of these steps seem to involve the γTuRC-specific GCP4, GCP5 and GCP6 (Bahtz et al, 2012;Choi et al, 2010;Izumi et al, 2008;Scheidecker et al, 2015), but the underlying mechanism has not been revealed. In fact, a systematic comparative analysis of the roles of all human GCPs in γTuRC assembly and function has never been conducted.…”

“…Thus, the activity of γTuRC is tightly controlled by targeting and activation factors that spatially restrict nucleation to microtubule-organizing centers (MTOCs) such as the centrosome. Surprisingly, whereas assembly and targeting of human γTuRC seem to involve all GCPs (Bahtz et al, 2012;Choi et al, 2010;Izumi et al, 2008;Scheidecker et al, 2015), only γ-tubulin, GCP2 and GCP3, which form the smaller subcomplex γTuSC, are essential in flies and fungi, and are sufficient for microtubule nucleation and targeting to MTOCs (Anders et al, 2006;Fujita et al, 2002;Lin et al, 2015;Venkatram et al, 2004;Vérollet et al, 2006;Xiong and Oakley, 2009). These observations suggest that there are important organism-specific differences in the manner by which nucleation template assembly is linked to targeting and activation.…”