1= 3). of Merlin with -tubulin and ezrin suggest a potential role for Merlin in cell cycle progression. tumor suppressor gene, inhibits cell proliferation by regulating signaling mediated by Rac1 and Ras GTPases or by mTorc1 and 2. At the plasma membrane, Merlin attenuates growth factor receptor expression and activity in both and mammal (4,C9). In addition, Merlin exerts its growth-suppressive function in the nucleus, where it inhibits the DCAF ubiquitin ligase activity (10). Finally, Merlin is a major regulator of the Hippo signaling pathway by inhibiting Trofosfamide the nuclear accumulation of the co-transcription factors Trofosfamide Yap and Taz in various organisms (11, 12). Although these mechanisms initially appeared distinct, crosstalks were identified between several of them (13,C15). How Merlin modulates mitogenic signaling is extensively studied. In contrast, little information is available on a possible role in regulating cell cycle progression. In glioma and osteosarcoma cell lines, Merlin is nuclear early in G1 and gets exported toward the plasma membrane before S phase entry (16). Also, Bmp15 an interaction between Merlin and HEI10 was reported. HEI10 is involved in the regulation of cyclin B levels (17). However, no specific role of its interaction with Merlin was identified in the control of cell cycle progression. More recently Hebert (18) demonstrated that Merlin regulates polarized cell division by restricting the cortical distribution of ezrin necessary for centrosome positioning and proper orientation of cell division. Indeed, ezrin, radixin, and moesin (ERM)5 have been implicated in several aspects of mitotic progression. In cells, moesin activation by phosphorylation during mitosis increases the Trofosfamide cortical rigidity necessary for proper spindle morphogenesis and chromosome alignment (19, 20). Moesin also regulates spindle length during metaphase and cell shape at a later stage of mitosis (21). In mammalian cells, the phosphorylation of the ERM by the kinase Slk during mitosis is key to the proper orientation of spindle (22). Interestingly, Merlin was shown to directly bind to microtubules and promote their stabilization (23, 24), but the functional consequences were not investigated, notably during mitosis. These observations suggest that Merlin plays a role in cell cycle progression and more specifically during mitosis. In the present study, we show that Merlin is a substrate for Aurora protein kinase A on the main regulatory serine 518, during mitosis. This event facilitates the phosphorylation of a second, newly discovered, site at position 581 that is specific of Merlin isoform 1. When Merlin dual phosphorylation is compromised, it leads to a defect in the stabilization of mitotic spindle orientation prior to metaphase, delaying the onset of anaphase. At the mechanistic level, we show that phosphorylation on Ser-518 controls Merlin interaction with -tubulin whereas Thr-581 phosphorylation modulates Merlin binding to ezrin and subsequently ezrin interactions with both actin and -tubulin. Importantly, specific patient mutations affecting the FERM (Four point one ezrin radixin moesin) domain of Merlin result in abnormal phosphorylation profile and -tubulinCbinding properties, in some case associated with a delay in mitotic progression. Altogether our observations suggest that tight regulation of Merlin by Aurora protein kinase Trofosfamide A is involved in mitotic progression via regulated binding to -tubulin and ezrin and is compromised by mutations found in neurofibromatosis type 2 patients. Results Aurora A binds and phosphorylates Merlin in vitro and in vivo during mitosis The Aurora protein kinase A phosphorylates a variety of substrates during the cell cycle progression. The phosphorylation sites contain a conserved arginine in ?2 position relative to the target serine residue, and leucine is frequently found in ?1 (25). A close examination of the sequence.