NuMA Phosphorylation by Aurora-A Orchestrates Spindle Orientation

Abstract

Spindle positioning is essential for tissue morphogenesis and homeostasis. The signaling network synchronizing spindle placement with mitotic progression relies on timely recruitment at the cell cortex of NuMA:LGN:Gαi complexes, in which NuMA acts as a receptor for the microtubule motor Dynein. To study the implication of Aurora-A in spindle orientation, we developed protocols for the partial inhibition of its activity. Under these conditions, in metaphase NuMA and Dynein accumulate abnormally at the spindle poles and do not reach the cortex, while the cortical distribution of LGN remains unperturbed. FRAP experiments revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle pole-localized pools of NuMA. We show that Aurora-A phosphorylates directly the C terminus of NuMA on three Ser residues, of which Ser1969 determines the dynamic behavior and the spindle orientation functions of NuMA. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif. Our study demonstrates that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in transformed and non-transformed cells in culture. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules or for LGN but rather determines the mobility of the protein at the spindle poles. The finding that NuMA can associate concomitantly with LGN and microtubules suggests that its microtubule-binding activity contributes to anchor Dynein-loaded microtubule +TIPs at cortical sites with LGN.

NuMA Recruits Dynein Activity to Microtubule Minus-Ends at Mitosis

“…The data also indicate that NuMA's minus-end localization at mitosis is mediated by the tail1+2A region of its C-terminus, while previously identified microtubule-binding domains tail2A or tail2B (Du et al, 2002;Gallini et al, 2016;Haren and Merdes, 2002) are not sufficient. We propose three hypotheses for tail1+2A-mediated minus-end recognition.…”

“…The second half of the C-terminus ('C-tail2', a.a. 1882-2115), which contains residues previously implicated in NuMA-microtubule interactions (Chang et al, 2017;Du et al, 2002;Gallini et al, 2016;Haren and Merdes, 2002), localized all along spindle microtubules with no minus-end preference ( Figure 6B). Similarly, C-tail2A (a.a. bound along the lattice, while C-tail1 (a.a. 1701-1881) did not bind microtubules ( Figure 6figure supplement 1).…”

“…Second, NuMA may recognize both plus-and minus-ends via microtubule curvature sensing, as previously proposed (Seldin et al, 2016), and yet be actively excluded from plus-ends within the spindle by other proteins. Third, NuMA tail1+2A may bind minus-ends directly in cells, perhaps using posttranslational modifications (Compton and Luo, 1995;Gallini et al, 2016;Magescas et al, 2017;Yan et al, 2015) not previously recapitulated in vitro. Canonical microtubule binding domains in tail2 localize all along spindle microtubules, while the addition of tail1 to tail2A confers minus-end localization.…”

“…See also Figure 6 and Localizes to preferentially to minus-ends (Kotak et al, 2012). 'C-tail2' was previously implicated in microtubule (MT) binding (Chang et al, 2017;Du et al, 2002;Gallini et al, 2016; Figure 6 continued on next page domain ('N-Tau'). N-Tau localized along the length of spindle microtubules and recruited dynactin there ( Figure 6E).…”

Epithelial spindle orientation diversities and uncertainties: recent developments and lingering questions

“…Additionally, both ABL1 and Aurora A have been shown to phosphorylate the C-terminus of NuMA to promote its release from spindle poles and recruitment to the cortex in metaphase, where its interactions with the membrane-associated factor 4.1 help stabilize its residence at the cortex 24– 26 . Cdk1, conversely, phosphorylates the C-terminus of NuMA to maintain its localization to spindle poles in metaphase, which can be counterbalanced by PPP2CA phosphatase activity, while anaphase-driven Cdk1 inactivation promotes extensive NuMA and dynein cortical accumulation 25, 27 .…”