ATM-mediated phosphorylation activates the tumor-suppressive function of B56γ–PP2A

Abstract

Protein phosphatase 2A (PP2A) is a family of heterotrimeric protein phosphatases that has a multitude of functions inside the cell, acting through various substrate targets in cell-signaling pathways. Recent evidence suggests that a subset of PP2A holoenzymes function as tumor suppressors and one particular family of B subunits, B56, are implicated in this function. However, the regulatory mechanisms that govern activation of B56-PP2A tumor-suppressive function have not been elucidated. In the present study, we demonstrate that ataxiatelangiectasia mutated (ATM) directly phosphorylates and specifically regulates B56c3, B56c2 and B56d, after DNA damage. We further show that phosphorylation of B56c3 at Ser510 leads to an increase in B56c3-PP2A complexes, and direction of PP2A phosphatase activity toward the substrate p53, activating its tumor-suppressive functions. In addition, we found that under cell growth conditions B56c3 is kept at low levels through the actions of the E3 ubiquitin ligase MDM2, and, importantly, phosphorylation of B56c3 by ATM leads to upregulation of the protein by blocking MDM2-mediated B56c3 ubiquitination. Finally, we show that Ser510 phosphorylation significantly enhances the ability of B56c3 to inhibit cell proliferation and anchorage-independent growth. These results provide mechanistic insight into the regulation of PP2A tumor-suppressive function, and suggest a model for parallel regulation of p53 and B56c3.

Checkpoint control and cancer

“…So far the only well-characterized phosphatase that has been described to actively participate to establish the cellcycle arrest is PP2A in complex with its regulatory subunit, B56g. Thus, B56g is stabilized in an ATMdependent manner and activates p53 through dephosphorylation of pThr-55 (Li et al, 2007;Shouse et al, 2011). On the other hand, the major cellular phosphatase PP2A has also been shown to limit basal phosphorylation of ATM-pSer-1981, Chk2-pThr-68, Chk1-pSer-317 andgH2AX (Goodarzi et al, 2004;Chowdhury et al, 2005;Leung-Pineda et al, 2006;Carlessi et al, 2010;.…”

The same, only different – DNA damage checkpoints and their reversal throughout the cell cycle

“…Indeed, PP2A phosphatase activity towards cH2AX-containing nucleosomes is repressed in the first hour, but increases by twofold 5 hours after DSB induction by camptothecin (Chen et al, 2014). PP1, PP2A, Wip1 and PP6 are regulated through distinct mechanisms by DNA damage, and both PP2A and Wip1 have been shown to accumulate at DSBs (Chen et al, 2014;Chowdhury et al, 2005;Dozier et al, 2004;Guo et al, 2002;Macůrek et al, 2010;Naito et al, 2012;Shouse et al, 2011), whereas PP4 appears to be selective for chromatin-dissociated cH2AX in yeast (Keogh et al, 2006), revealing that these phosphatases modulate specific pools of cH2AX at different times. Although less thoroughly assessed for other checkpoint phosphorylation events, redundancy and spatiotemporal regulation are a recurrent theme.…”

“…Inhibition of PP2A-B56c in G2 results in increased levels of cyclin E1 in mitotic cells and accelerates Cdk2-dependent S-phase entry in daughter cells. As ATM contributes to the activation of PP2A-B56c (Shouse et al, 2011), ATM might thus synergise with p21 to inhibit Cdk2 activity in checkpoint-adapting cells, which are then programmed to enter a state of quiescence after cell division to safeguard genomic stability.…”

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

“…In response to DNA damage, ataxia telangiectasia-mutated (ATM) signaling directly activates and stabilizes PP2A via phosphorylating a site targeted by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), for inducing proteasomal degradation. 16,17 Subsequently, PP2A suppresses protein kinase B (AKT1) signaling which diminishes levels of activated phosphorylated MDM2 (pMDM2) and prevents MDM2-mediated proteasomal degradation of p53. 17,18 PP2A also directly dephosphorylates sites on p53, promoting p53 stability, cell cycle arrest, expression of Bax, and apoptosis in the context of irreparable DNA damage.…”

“…16,17 Subsequently, PP2A suppresses protein kinase B (AKT1) signaling which diminishes levels of activated phosphorylated MDM2 (pMDM2) and prevents MDM2-mediated proteasomal degradation of p53. 17,18 PP2A also directly dephosphorylates sites on p53, promoting p53 stability, cell cycle arrest, expression of Bax, and apoptosis in the context of irreparable DNA damage. 19,20 Mitotic Cell Cycle Progression PP2A activity plays a key role in the regulation of normal mitotic progression.…”