Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance

Abstract

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...

Genome-wide siRNA Screen Identifies the Radiosensitizing Effect of Downregulation of MASTL and FOXM1 in NSCLC

“…This finding was later confirmed in human cells, where nearly complete knockdown of MASTL led to a severe delay in mitotic entry (31,32). Strikingly, cells with a partial knockdown of MASTL could enter mitosis normally, but exited prematurely, leading to cytokinesis defects (31,32). In our study, in which a partial knockdown of MASTL was achieved, a faster exit of cells from the radiation-induced G 2 -M arrest was observed, suggesting that these cells are exiting mitosis prematurely after encountering DNA damage.…”

“…Depletion of Greatwall kinase, the Xenopus homolog of MASTL, prevented mitotic entry in Xenopus egg extracts (29,30). This finding was later confirmed in human cells, where nearly complete knockdown of MASTL led to a severe delay in mitotic entry (31,32). Strikingly, cells with a partial knockdown of MASTL could enter mitosis normally, but exited prematurely, leading to cytokinesis defects (31,32).…”

“…This is further strengthened by our observation of more chromatin bridges in MASTL knockdown cells after irradiation. The observed cell-cycle arrest in cells exposed to a fractionated dose scheme of irradiation is therefore likely to be caused by a response to the cytokinesis defects that were also observed in earlier studies (31,32).…”

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

“…The quantitative analysis of cellular fluorescence signals following reporter transfection and immunocytochemistry was performed using ImageJ software as described in McCloy et al (2014) and Burgess et al (2010). Multiple sequence alignments were performed using ClustalW, and the secondary structure prediction analysis of the TH 3 ′ UTR was conducted using Mfold (Zuker 2003) and RNAfold (Gruber et al 2008).…”

“…Fluorescence intensity was quantitated using ImageJ software (National Institutes of Health) as described in McCloy et al (2014) and Burgess et al (2010). Briefly, axon bundles of interest were selected and measured.…”