Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Abstract

Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55d subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle-high in interphase and suppressed during mitosis. Depletion of PP2A-B55d (in interphase) from 'cycling' frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A-B55d was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A-B55d in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.

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

“…Similarly, inactivation of the cyclin B-Cdc2 complex by cyclin B degradation was sufficient to trigger mitotic exit. However, recent data from our and other laboratories has demonstrated that PP2A is highly regulated during mitotic entry and exit (3,10,11). Therefore, the old cell-cycle model now must be updated to include a different regulator of mitosis that modulates PP2A during this phase of the cell cycle.…”

“…It has been recently shown that the inhibition of PP2A phosphastase activity is required to allow the phosphorylation of cyclin B-Cdc2 substrates, which promotes entry and maintains mitosis (3,10,11). Therefore, we next analyzed the capacity of these cells to phosphorylate the different cyclin B-Cdc2 substrates at the different doses of siRNA (Fig.…”

“…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.…”

LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

“…24 Furthermore, PP2A silencing has been shown to upregulate downstream Cdk1 transcriptional targets and promote mitosis. 27,28 As such, the Greatwall-PP2A network has been proposed as a key signaling axis that promotes normal Cdk1-driven entry through mitosis. 29 PP2A also acts on other mitotic mediators, including the key mitosis-specific kinase, Polo-like kinase 1 (PLK1), which localizes to centrosomes during mitosis and when inactivated by PP2A is an important hallmark of G2/M arrest and activation of the DNA damage response.…”

The Biochemistry of Mitosis

“…It was a biochemical study using Xenopus extracts that underlined the importance of controlling phosphatases for mitotic entry. Mochida et al showed that activating cyclin BCdk kinase was not sufficient to drive cells into mitosis; a member of the PP2A phosphatase family that antagonized cyclin B-Cdk1 had to be inactivated too (Mochida et al 2009 Williams et al 2014). Phospho-ENSA is then slowly dephosphorylated by PP2A, which allows PP2A to reactivate once cyclin -Cdk and Greatwall kinase activity decline later in mitosis (Williams et al 2014).…”