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 mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α role in multiple cellular processes.
The PPP2R2A gene encodes the B55α regulatory subunit of PP2A. Here, we report that PPP2R2A is hemizygously lost in ~42% of prostate adenocarcinomas, correlating with reduced expression, poorer prognosis, and an increased incidence of hemizygous loss (>75%) in metastatic disease. Of note, PPP2R2A homozygous loss is less common (5%) and not increased at later tumor stages. Reduced expression of B55α is also seen in prostate tumor tissue and cell lines. Consistent with the possibility that complete loss of PPP2R2A is detrimental in prostate tumors, PPP2R2A deletion in cells with reduced but present B55α reduces cell proliferation by slowing progression through the cell cycle. Remarkably, B55α-low cells also appear addicted to lower B55α expression, as even moderate increases in B55α expression are toxic. Reconstitution of B55α expression in prostate cancer (PCa) cell lines with low B55α expression reduces proliferation, inhibits transformation and blocks xenograft tumorigenicity. Mechanistically, we show B55α reconstitution reduces phosphorylation of proteins essential for centrosomal maintenance, and induces centrosome collapse and chromosome segregation failure; a first reported link between B55α/PP2A and the vertebrate centrosome. These effects are dependent on a prolonged metaphase/anaphase checkpoint and are lethal to PCa cells addicted to low levels of B55α. Thus, we propose the reduction in B55α levels associated with hemizygous loss is necessary for centrosomal integrity in PCa cells, leading to selective lethality of B55α reconstitution. Such a vulnerability could be targeted therapeutically in the large pool of patients with hemizygous PPP2R2A deletions, using pharmacologic approaches that enhance PP2A/B55α activity.
Background Immortalization of primary prostate epithelial cells (PrEC) with just hTERT expression is particularly inefficient in the absence of DNA tumor viral proteins or p16 INK4A knockdown. Materials and methods Here, we describe the establishment of immortalized normal prostate epithelial cell line models using CRISPR technology to inactivate the CDKN2A locus concomitantly with ectopic expression of an hTERT transgene. Results Using this approach, we have obtained immortal cell clones that exhibit fundamental characteristics of normal cells, including diploid genomes, near normal karyotypes, normal p53 and pRB cell responses, the ability to form non-invasive spheroids, and a non-transformed phenotype. Based on marker expression, these clones are of basal cell origin. Conclusions Use of this approach resulted in the immortalization of independent clones of PrEC that retained normal characteristics, were stable, and non-transformed. Thus, this approach could be used for the immortalization of normal primary prostate cells. This technique could also be useful for establishing cell lines from prostate tumor tissues of different tumor grades and/or from patients of diverse ethnicities to generate cell line models that facilitate the study of the molecular basis of disease disparity.
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