Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation

Abstract

Budding yeast Spc110, a member of γ-tubulin complex receptor family (γ-TuCR), recruits γ-tubulin complexes to microtubule (MT) organizing centers (MTOCs). Biochemical studies suggest that Spc110 facilitates higher-order γ-tubulin complex assembly (Kollman et al., 2010). Nevertheless the molecular basis for this activity and the regulation are unclear. Here we show that Spc110 phosphorylated by Mps1 and Cdk1 activates γ-TuSC oligomerization and MT nucleation in a cell cycle dependent manner. Interaction between the N-terminus of the γ-TuSC subunit Spc98 and Spc110 is important for this activity. Besides the conserved CM1 motif in γ-TuCRs (Sawin et al., 2004), a second motif that we named Spc110/Pcp1 motif (SPM) is also important for MT nucleation. The activating Mps1 and Cdk1 sites lie between SPM and CM1 motifs. Most organisms have both SPM-CM1 (Spc110/Pcp1/PCNT) and CM1-only (Spc72/Mto1/Cnn/CDK5RAP2/myomegalin) types of γ-TuCRs. The two types of γ-TuCRs contain distinct but conserved C-terminal MTOC targeting domains.DOI: http://dx.doi.org/10.7554/eLife.02208.001

The Centrosome and Its Duplication Cycle

“…The nuclear receptor, Spc110 is a member of the pericentrin family of microtubule-nucleating proteins in which microtubule-nucleating motifs are separated from anchors by extended coiled-coil spacers (Kilmartin et al 1993;Kilmartin and Goh 1996;Sundberg and Davis 1997). These g-tubulin docking motifs are highly conserved from human pericentrin and kendrin through Drosophila centrosomin (CNN) to fission yeast Mto1 and Pcp1 (Flory et al 2002;Zhang and Megraw 2007;Fong et al 2008;Samejima et al 2008;Lin et al 2014). Spc29 links Spc110 to the hexagonal crystalline lattice of Spc42 that comprises the central plaque in a coupling that relies on association of Spc110 with calmodulin (Geiser et al 1993;Stirling et al 1994;Donaldson and Kilmartin 1996;Spang et al 1996;Bullit et al 1997;Sundberg and Davis 1997;Elliott et al 1999).…”

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

“…In yeast, activation might result from γTuSC oligomerization (Kollman et al, 2010;Lin et al, 2014;Lynch et al, 2014). In human cells, however, oligomerization per se might not be sufficient for full nucleation activity: even though oligomeric complexes exist in the form of pre-assembled γTuRCs, the 50-amino-acid CM1 motif of human CDK5RAP2 alone is able to strongly stimulate nucleation activity of these complexes in vitro and in vivo (Choi et al, 2010).…”

“…Phosphorylation of the adapter in the region between the two motifs further enhances the interaction. As a result, γTuSC oligomerizes and becomes a more active nucleation template (Lin et al, 2014). Even in fission yeast, which contains GCP4, GCP5 and GCP6, formation of active higherorder γ-tubulin complexes requires interaction with the CM1-containing protein Mto1 that forms an adapter complex with Mto2 at interphase MTOCs (Lynch et al, 2014).…”

“…Here, interactions with adapter proteins containing CM1 and SPM motifs might trigger formation of γTuSC oligomers that are similar in structure and activity to γTuRC (Kollman et al, 2010;Lin et al, 2015Lin et al, , 2014. The CM1 and SPM motifs of the budding yeast adapter Spc110 have been shown to mediate interaction with γTuSC, through direct interaction with the Nterminus of GCP3 and possibly also GCP2 (Knop and Schiebel, 1997;Lin et al, 2011;Nguyen et al, 1998).…”

“…Pericentrin, AKAP9, CDK5RAP2 and myomegalin have been implicated in γTuRC recruitment to the centrosome (Fong et al, 2008;Takahashi et al, 2002;Zimmerman et al, 2004) and to the Golgi, which functions as a non-centrosomal MTOC (Rivero et al, 2009;Roubin et al, 2013;Wang et al, 2010). All of these proteins contain a CM1 motif (Lin et al, 2014(Lin et al, , 2015Samejima et al, 2008;Sawin et al, 2004), whereas an SPM motif is found only in pericentrin and AKAP9 (Lin et al, 2014(Lin et al, , 2015. Curiously, NEDD1 (also known as GCP-WD), which in human cells is the most crucial factor for centrosomal targeting of γTuRC (Haren et al, 2006;Lüders et al, 2006), does not seem to contain any SPM or CM1 motifs.…”