PP2A­-B55γ counteracts Cdk1 and regulates proper spindle orientation through the cortical dynein adaptor NuMA

Abstract

Proper orientation of the mitotic spindle is critical for accurate development and morphogenesis. In human cells, spindle orientation is regulated by the evolutionarily conserved protein NuMA, which interacts with dynein and enriches it at the cell cortex. Pulling forces generated by cortical dynein orient the mitotic spindle. Cdk1-mediated phosphorylation of NuMA at threonine 2055 (T2055) negatively regulates its cortical localization. Thus, only NuMA not phosphorylated at T2055 localizes at the cell cortex. However, the identity and the mechanism of action of the phosphatase complex involved in T2055 dephosphorylation remains elusive. Here, we characterized the PPP2CA-B55γ (PPP2R2C)–PPP2R1B complex that counteracts Cdk1 to orchestrate cortical NuMA for proper spindle orientation. In vitro reconstitution experiments revealed that this complex is sufficient for T2055 dephosphorylation. Importantly, we identified polybasic residues in NuMA that are critical for T2055 dephosphorylation, and for maintaining appropriate cortical NuMA levels for accurate spindle elongation. Furthermore, we found that Cdk1-mediated phosphorylation and PP2A-B55γ-mediated dephosphorylation at T2055 are reversible events. Altogether, this study uncovers a novel mechanism by which Cdk1 and its counteracting PP2A-B55γ complex orchestrate spatiotemporal levels of cortical force generators for flawless mitosis.

NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

“…To investigate the mobility of NuMA in the interphase nucleus, we sought to conduct a fluorescence recovery after photobleaching (FRAP) analysis in the HeLa cell line that stably expresses AcGFP (Aequorea coerulescens GFP), and a mono-FLAG tagged NuMA (AcGFP-NuMA; Figure 1A ; Keshri et al. , 2020 ).…”

“…The protocol for generating stable HeLa Kyoto cells stably expressing AcGFP-NuMA was described recently ( Keshri et al. , 2020 ).…”

Phospho‐regulation of mitotic spindle assembly

“…Cdk1 phosphorylates Thr2055 of NuMA; this phosphorylation event inhibits NuMa localization at the cortex, such that only NuMA not phosphorylated at this residue localizes to the cortex (Kotak et al, 2013). The proper extent of NuMA localization at the cortex is regulated by PP2A-B55γ (Keshri et al, 2020) and by PP1-Repo-Man (Lee et al, 2018). Expression of a construct of NuMA that could not be phosphorylated on Thr2055 resulted in spindle oscillations (i.e., the mitotic spindle wobbled unsteadily) (Kotak et al, 2013), and inhibition of either phosphatase complex resulted in improper metaphase spindle positioning (Keshri et al, 2020;Kotak et al, 2013).…”

“…The proper extent of NuMA localization at the cortex is regulated by PP2A-B55γ (Keshri et al, 2020) and by PP1-Repo-Man (Lee et al, 2018). Expression of a construct of NuMA that could not be phosphorylated on Thr2055 resulted in spindle oscillations (i.e., the mitotic spindle wobbled unsteadily) (Kotak et al, 2013), and inhibition of either phosphatase complex resulted in improper metaphase spindle positioning (Keshri et al, 2020;Kotak et al, 2013). During anaphase, when Cdk1 activity is low, these phosphatases promote increased NuMA localization to the cortex, generating dynein-dependent forces that result in spindle elongation and progression through anaphase (Keshri et al, 2020;Kotak et al, 2013).…”

“…Expression of a construct of NuMA that could not be phosphorylated on Thr2055 resulted in spindle oscillations (i.e., the mitotic spindle wobbled unsteadily) (Kotak et al, 2013), and inhibition of either phosphatase complex resulted in improper metaphase spindle positioning (Keshri et al, 2020;Kotak et al, 2013). During anaphase, when Cdk1 activity is low, these phosphatases promote increased NuMA localization to the cortex, generating dynein-dependent forces that result in spindle elongation and progression through anaphase (Keshri et al, 2020;Kotak et al, 2013). At the spindle poles, NuMA also forms the END (Emi1, NuMA, dynein-dynactin) network, a complex that localizes the APC/C at the spindle poles and inhibits its activity, allowing for proper Cdk1-mediated spindle assembly in early mitosis (Ban et al, 2007).…”