Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells.spindle assembly checkpoint | cell cycle | phosphatase | kinase | slippage I n eukaryotic cells, the mitotic state is maintained by the mitotic kinase cyclin B-Cdc2. Historically, mitotic entry and exit was thought to be the direct consequence of cyclin B-Cdc2 activation and inactivation, respectively (1). Recent results have expanded this model to include phosphatases (2). Specifically, recent evidence indicates that protein phosphatase 2A (PP2A) is responsible for dephosphorylation of cyclin B-Cdc2 substrates and that the regulation of this dephosphorylation is required in mitotic entry and exit (3). This finding suggests a previously unexplored model in which the balance between cyclin B-Cdc2 and PP2A controls mitotic entry and exit. Thus, during G2 PP2A activity is high and cyclin B-Cdc2 activity low, thereby preventing phosphorylation of mitotic substrates, whereas at mitotic entry the balance flips, allowing entry into mitosis. The mechanisms controlling cyclin B-Cdc2 activity have been largely described (4). Briefly, cyclin B-Cdc2 is inhibited during G2 by inhibitory phosphorylations on threonine 14 and tyrosine 15 by Wee1 and Myt1 kinases. Upon mitotic entry, these are removed by the Cdc25 phosphatase (4). Finally, at mitotic exit cyclin B-Cdc2 is inhibited by the ubiquitin-dependent degradation of its regulatory subunit cyclin B (5). Unlike cyclin B-Cdc2 regulation, very little is known about the mechanisms controlling PP2A activity during mitosis, and therefore our understanding of G2 and mitosis is incomplete.Recently, Greatwall (Gwl), a unique critical mitotic regulator, has been discovered in Drosophila (6, 7). It is a member of the AGC family of serine/threonine kinases that ph...

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.

The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.