Regulating Greatwall for mitosis

The mitotic entry is regulated by a series of carefully controlled phosphorylation and dephosphorylation processes. A well-defined regulatory mechanism involves the phosphorylation and activation of protein phosphatase Cdc25, which consequently dephosporylates and activates Cdk1 leading to the entry of mitosis. Recently, a new regulatory network has emerged, which coordinates with Cdk1-cyclin B complex by inhibiting the dephosphorylation of Cdk1 substrates. The principal antagonist of Cdk1-cyclin B activity in mitosis is protein phosphatase 2A (PP2A) with a B55 regulatory subunit.¹ PP2A-B55 activity must be low at mitotic entry to permit the phosphorylation of Cdk1 substrates, and the inhibition of PP2A-B55 at mitotic entry is governed by Greatwall kinase (Gwl), a new cell cycle regulator initially identified in Drosophila in 2004.² In 2 independent studies, the cAMP-regulated phospho protein 19 (Arpp19) and another small protein α-endosulfine (ENSA) were identified as the substrates of Gwl.^3,4 By phosphorylating Arpp19 and ENSA, Gwl promotes the binding of these 2 small proteins to PP2A-B55, and thereby inhibits the activity of this phosphatase. Comparing to what has been discovered for the downstream signaling of Gwl, much less is known about the regulation of Gwl itself. Gwl is classified as a member of the AGC family of kinases, but it contains a unique and poorly conserved ∼500 amino acid central region located between kinase subdomains VII and VIII.² Recent studies have shown that this central region plays an important role in regulating the localization and function of Gwl. In Drosophila, 2 nuclear localization signals (NLSs) have been identified in this region that also appears to be the phosphorylation target of Plk1. Cdk1 has also been reported to phosphorylate the central region and promotes the nuclear exclusion of Gwl in Drosophila and mammalian models.^5,6

The study by Yamamoto et al.⁷ has advanced our further understanding of the mechanism underlying the regulation of Gwl. In search for new regulators of Xenopus Gwl (xGwl), the authors analyzed the co-immunoprecipitation complex of this kinase from Xenopus oocytes, and identified importin α/β and chaperone complex Hsp90/Cdc37 as xGwl binding partners. The study first identified a NLS in xGwl (K456/R457) located in the central region. This is consistent with the finding from mammalian cells, but not in Drosophila cells where 2 NLSs (K518/K520 in NLS1 and K564/R566 in NLS2) are collectively required for Gwl nuclear localization. Then, Yamamoto et al. further showed that the NLS interacts with importin α/β to facilitate the nuclear import of xGwl. It is unclear if the nuclear localization facilitates xGwl to be activated by its upstream kinase(s) or to phosphorylate its downstream substrates. Alternatively, as xGwl negatively regulates the DNA damage checkpoint in Xenopus egg extracts, it is thus possible that the nuclear import of xGwl may suppress a cell cycle inhibitory mechanism such as incomplete DNA replication checkpoint to promote mitotic entry. Yamamoto et al. also revealed a dynamic nucleocytoplasmic localization of xGwl during the cell cycle in Xenopus S3 cells, a result similar to previous findings in Drosophila and mammalian cells. The xGwl is partially relocated to cytoplasm minutes before nuclear envelope breakdown. Though the mechanism is yet to be identified, but in Drosophila, Plk1 phosphorylates Gwl at the central region and promotes the binding of 14–3-3ϵ to the NLS in Gwl, which is thought to mask the NLS, and therefore, prevent the nuclear import of Gwl.⁶ Furthermore, Yamamoto et al. have shown that Hsp90/Cdc37 binds to a conserved glycine-rich loop of xGwl at the N-terminal kinase domain and stabilizes the kinase. Consistent with the previously defined roles of Hsp90/Cdc37 in cell cycle progression, the authors have demonstrated that Gwl is a new client protein of Hsp90/cdc37. Hsp90 inhibition is implicated in cancer treatment, and the ability to target cell cycle proteins through Hsp90 may partially account for this effort. The importance of Gwl in mitotic control is becoming clearer, but more research is needed to fully understand the regulation of this kinase and the significance of its transient nuclear export before mitosis. Nevertheless, the results reported by Yamamoto et al. have provided us with new insights on how the subcellular localization and stability of xGwl are regulated in mitosis.