FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase.

The Ser/Thr protein phosphatase 2A (PP2A) is a highly conserved collection of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). PP2A/B55 substrate recruitment was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM [ RK ]- V -x-x-[ VI ]- R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A. This conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational and biochemical data and supports a mechanism whereby FAM122A uses the 'SLiM' in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, progression through cell cycle transitions and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a 'SLiM'-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase.

Chemogenetic profiling reveals PP2A-independent cytotoxicity of proposed PP2A activators iHAP1 and DT-061.

Protein phosphatase 2A (PP2A) is an abundant phosphoprotein phosphatase that acts as a tumor suppressor. For this reason, compounds able to activate PP2A are attractive anticancer agents. The compounds iHAP1 and DT-061 have recently been reported to selectively stabilize specific PP2A-B56 complexes to mediate cell killing. We were unable to detect direct effects of iHAP1 and DT-061 on PP2A-B56 activity in biochemical assays and composition of holoenzymes. Therefore, we undertook genome-wide CRISPR-Cas9 synthetic lethality screens to uncover biological pathways affected by these compounds. We found that knockout of mitotic regulators is synthetic lethal with iHAP1 while knockout of endoplasmic reticulum (ER) and Golgi components is synthetic lethal with DT-061. Indeed we showed that iHAP1 directly blocks microtubule assembly both in vitro and in vivo and thus acts as a microtubule poison. In contrast, DT-061 disrupts both the Golgi apparatus and the ER and lipid synthesis associated with these structures. Our work provides insight into the biological pathways perturbed by iHAP1 and DT-061 causing cellular toxicity and argues that these compounds cannot be used for dissecting PP2A-B56 biology.

Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division.

The formin INF2 polymerizes a calcium-activated cytoplasmic network of actin filaments, which we refer to as calcium-induced actin polymerization (CIA). CIA plays important roles in multiple cellular processes, including mitochondrial dynamics and vesicle transport. Here, we show that nonmuscle myosin II (NMII) is activated within 60 s of calcium stimulation and rapidly recruited to the CIA network. Knockout of any individual NMII in U2OS cells affects the organization of the CIA network, as well as three downstream effects: endoplasmic-reticulum-to-mitochondrial calcium transfer, mitochondrial Drp1 recruitment, and mitochondrial division. Interestingly, while NMIIC is the least abundant NMII in U2OS cells (>200-fold less than NMIIA and >10-fold less than NMIIB), its knockout is equally deleterious to CIA. On the basis of these results, we propose that myosin II filaments containing all three NMII heavy chains exert organizational and contractile roles in the CIA network. In addition, NMIIA knockout causes a significant decrease in myosin regulatory light chain levels, which might have additional effects.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107.

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.

Selective dephosphorylation by PP2A-B55 directs the meiosis I-meiosis II transition in oocytes.

Meiosis is a specialized cell cycle that requires sequential changes to the cell division machinery to facilitate changing functions. To define the mechanisms that enable the oocyte-to-embryo transition, we performed time-course proteomics in synchronized sea star oocytes from prophase I through the first embryonic cleavage. Although we found that protein levels were broadly stable, our analysis reveals that dynamic waves of phosphorylation underlie each meiotic stage. We found that the phosphatase PP2A-B55 is reactivated at the meiosis I/meiosis II (MI/MII) transition, resulting in the preferential dephosphorylation of threonine residues. Selective dephosphorylation is critical for directing the MI/MII transition as altering PP2A-B55 substrate preferences disrupts key cell cycle events after MI. In addition, threonine to serine substitution of a conserved phosphorylation site in the substrate INCENP prevents its relocalization at anaphase I. Thus, through its inherent phospho-threonine preference, PP2A-B55 imposes specific phosphoregulated behaviors that distinguish the two meiotic divisions.