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Regulation of PP2A-subfamily holoenzyme function by carboxyl-terminal methylation

ABSTRACT: The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes with catalytic, scaffolding, and regulatory subunits. Although there are only ten catalytic PPP subunits encoded in the human genome, the formation of holoenzymes creates hundreds of unique enzymes that oppose kinases and carry out specific dephosphorylation reactions. Thus, to achieve the correct cellular phosphorylation state, the assembly of PPP holoenzymes needs to be tightly controlled. Indeed, changes in the cellular repertoire of PPPs are frequently linked to human disease, including cancer and neurodegeneration. The Protein Phosphatase 2A (PP2A) subfamily of PPPs consists of PP2A, PP4, PP6, and holoenzyme formation is at least in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as methylation) of the catalytic subunits. This reversible modification is catalyzed by a Leucine Carboxyl Methyltransferase-1 (LCMT1) that utilizes S-adenosyl-methionine (SAM) as the methyl donor and removed by Protein Phosphatase Methylesterase 1 (PME1). For PP2A, C-terminal methylation controls regulatory subunit selection. Notably, different types of regulatory subunits display differential methylation sensitivity. For PP4 and PP6, the role of C-terminal methylation is less well defined. Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to comprehensively elucidate the role of C-terminal methylation in PP2A, PP4, and PP6 function in multiple cell lines. Using these approaches, we quantitatively determine the effects of reduced C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly in an unbiased, isoform-specific manner.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion, Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Arminja Kettenbach

PROVIDER: MSV000086183 | MassIVE |

SECONDARY ACCESSION(S): PXD021598

REPOSITORIES: MassIVE

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Project description: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 B55a 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 B55a/PP2A. This conserved SLiM is necessary for FAM122A binding to B55a in vitro and in cells. Computational structure prediction with AlphaFold-Multimer 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 a-helix (helix 1) to dock to the B55a top groove, thereby blocking substrate binding to the same site. In this model, FAM122A also spatially constrains substrate access by occluding the catalytic subunit with a second a-helix immediately adjacent to helix 1. Consistently, FAM122A functions as a competitive inhibitor as it prevents the binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55a/PP2A in cell lysates. The 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 short helical motif (SHeM)-dependent, substrate-competitive inhibitor of B55a/PP2A that suppresses multiple functions of B55a in the DNA damage response and in timely progression through the cell cycle interphase.

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Regulation of PP2A-subfamily holoenzyme function by carboxyl-terminal methylation

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