Retinoblastoma-Like Protein p107
Retinoblastoma-Like Protein p130
Permanent link to this recordhttp://hdl.handle.net/20.500.12613/5089
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AbstractThe destiny of a cell - whether it undergoes division, differentiation or death - results from an intricate balance of many regulators, including oncoproteins, tumor-suppressor proteins and cell-cycle-associated proteins. One of the better-studied tumor suppressors is the retinoblastoma protein, known as pRb or p105. Two recently identified proteins, pRb2/p130 and p107, show structural and functional similarities to pRb, and these proteins and their orthologs make up the retinoblastoma (Rb) family. Members of the family have been found in animals and plants, and a related protein is known in the alga Chlamydomonas. Members of the Rb family are bound and inactivated by viral proteins and, in turn, bind cellular transcription factors and repress their function, and can also form complexes with cyclins and cyclin-dependent kinases and with histone deacetylases. The are found in the nucleus and their subnuclear localization depends on binding to the nuclear matrix. Members of the family form part of a signal-transduction pathway called the Rb pathway, which is important in cell-cycle regulation and have roles in growth suppression, differentiation and apoptosis in different organisms and cell types.
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PP2A/B55α Substrate Recruitment As Defined By The Retinoblastoma-Related Protein p107Graña-Amat, Xavier; Shore, Scott K.; Rothberg, Brad S.; Dunbrack, Roland L. (Temple University. Libraries, 2021)Protein phosphorylation is a reversible post-translation modification that is essential in cell signaling. It is estimated that a third of all cellular proteins are phosphorylated (reviewed in Ficarro et al., 2002), with more than 98% of those phosphorylation events occurring on serine and threonine residues (Olsen et al., 2006). Kinases are the necessary enzymes for phosphorylation and protein phosphatases dynamically reverse this action. While the mechanisms of substrate recognition for kinases have been well-characterized to date, the same is not true for phosphatases that play an equally important role in opposing kinase function and determining global phosphorylation levels in cells. This dichotomy has also translated into the clinic, where there has been a persistently narrow research focus on the development of small-molecule kinase inhibitors for use as chemotherapeutic agents, without an equal effort being placed into the generation of the analogous phosphatase activators (reviewed in Westermarck, 2018). Members of the phosphoprotein phosphatase (PPP) family of serine/threonine phosphatases are responsible for the majority of dephosphorylation in eukaryotic cells, with protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) accounting for more than 90% of the total phosphatase activity (Moorhead et al., 2007; Virshup and Shenolikar, 2009). Structurally, PP2A is a trimeric holoenzyme consisting of a scaffold (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. B55α is a ubiquitous regulatory subunit that is reported to target many substrates with critical functions in processes including cell division. A long-standing question that has persisted in the field of cellular signaling is as to how the most abundant serine/threonine PP2A holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site for subsequent dephosphorylation. Such critical data have only recently become well understood for the B56 family of ‘B’ regulatory subunits, where an LxxIxE short linear motif (or SLiM) has been identified in a subset of protein targets and shown via crystal structure analysis to dock into a 100% conserved binding pocket on the B56 surface (Hertz et al., 2016; Wang et al., 2016a; Wang et al., 2016b; Wu et al., 2017). Here, we show how 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” SLiM in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous additional PP2A/B55α substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, allowing us to propose a consensus SLiM sequence, “p[ST]-P-x(5-10)-[RK]-V-x-x-[VI]-R”. In support of this, mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. Moreover, a data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation to the PP2A/C active site. 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 the role of PP2A/B55α in many key cellular processes. As a parallel continuation of our efforts to identify novel B55α substrates/regulators, we generated mutant B55α constructs that occlude PP2A/A-C dimer engagement but retain substrate binding to the β-propeller structure (allowing us to interrogate direct interactors). Our preliminary AP-MS data led to the identification of several proteins that bound better to our “monomeric B55α” mutant compared to wild-type B55α in the context of the PP2A/B55α heterotrimer, including the centrosomal proteins HAUS6 and CEP170 (two substrates previously validated in a phosphoproteomic screen by our lab), suggesting that these mutants trap substrates as they cannot be dephosphorylated by PP2A/C. These analyses also identified an enrichment of T-complex protein 1 subunits in the “monomeric B55α” mutant elutions, further supporting the notion that these mutants may function as dominant negatives. Several additional proteins of interest were identified in the two independent rounds of mass spectrometry, including subunits of the DNA-directed RNA polymerases I, II, and IV, as well as the double-strand break repair protein MRE11, which can be followed up as potential novel B55α substrates. These studies can contribute to significant advances in our understanding of the network of proteins that B55α interacts with, and thus the signaling pathways that can be modulated by PP2A/B55α complexes in cells. Moreover, these advances can also provide translational benefits as has been demonstrated through the study of PP2A activators termed SMAPs, which demonstrate selective stabilization of PP2A/B56α complexes in cells that result in selective dephosphorylation of substrates including the oncogenic target c-MYC.
Misidentified Human Gene Functions with Mouse Models: The Case of the Retinoblastoma Gene Family in SenescenceAlessio, N; Capasso, S; Ferone, A; Di Bernardo, G; Cipollaro, M; Casale, F; Peluso, G; Giordano, A; Galderisi, U; Giordano, Antonio|0000-0002-5959-016X (2017-10-01)© 2017 The Authors Although mice models rank among the most widely used tools for understanding human genetics, biology, and diseases, differences between orthologous genes among species as close as mammals are possible, particularly in orthologous gene pairs in which one or more paralogous (i.e., duplicated) genes appear in the genomes of the species. Duplicated genes can possess overlapping functions and compensate for each other. The retinoblastoma gene family demonstrates typical composite functionality in its three member genes (i.e., RB1, RB2/P130, and P107), all of which participate in controlling the cell cycle and associated phenomena, including proliferation, quiescence, apoptosis, senescence, and cell differentiation. We analyzed the role of the retinoblastoma gene family in regulating senescence in mice and humans. Silencing experiments with each member of the gene family in mesenchymal stromal cells (MSCs) and fibroblasts from mouse and human tissues demonstrated that RB1 may be indispensable for senescence in mouse cells, but not in human ones, as an example of species specificity. Furthermore, although RB2/P130 seems to be implicated in maintaining human cell senescence, the function of RB1 within any given species might differ by cell type, as an example of cell specificity. For instance, silencing RB1 in mouse fibroblasts induced a reduced senescence not observed in mouse MSCs. Our findings could be useful as a general paradigm of cautions to take when inferring the role of human genes analyzed in animal studies and when examining the role of the retinoblastoma gene family in detail.
Role of Retinoblastoma Protein Family (Rb/p105 and Rb2/p130) Expression in the Histopathological Classification of Borderline Ovarian TumorsMasciullo, V; Valdivieso, P; Amadio, G; Santoro, A; Angelico, G; Sgambato, A; Boffo, S; Giordano, A; Scambia, G; Zannoni, GF; Giordano, Antonio|0000-0002-5959-016X; Boffo, Silvia|0000-0002-6352-160X (2020-11-11)© Copyright © 2020 Masciullo, Valdivieso, Amadio, Santoro, Angelico, Sgambato, Boffo, Giordano, Scambia and Zannoni. Borderline ovarian tumors (BOT) are uncommon but not rare epithelial ovarian neoplasms, intermediate between benign and malignant categories. Emerging knowledge supports the notion that subtypes of borderline ovarian tumors comprise distinct biologic, pathogenetic, and molecular entities, precluding a single unifying concept for BOT. The identification of valuable markers for the diagnosis and classification of these tumors is in need. Among the molecular candidates, the Retinoblastoma (Rb) family members Rb/p105 and Rb2/p130 seem to play a pivotal role in ovarian cancer. In particular, Rb/p105, when in the unphosphorylated form, acts as a growth suppressor controlling cell cycle and tumor progression; whereas, the phosphorylated form activates gene transcription and cellular proliferation. While Rb/p105 is ubiquitously confined to the nuclei of cycling and quiescent cells, Rb2/p130 activity is also regulated by intracellular localization. According to this, Rb family members could represent a novel marker in diagnosis and classification risk for patients with BOT. In this study, we evaluated the expression and subcellular localization of proteins of the retinoblastoma (Rb) gene family in 65 ovarian borderline tumors. Statistically significant differences were found in nuclear and cytoplasmic expressions of Rb/p105 and Rb2/p130 according to different examined histotypes. In detail, the nuclear expression of Rb/p105 and Rb2/p130 was more frequently detected in serous (84.6%) than sero-mucinous (42.1%) and mucinous (50%) types. Conversely, the cytoplasmic expression of Rb2/p130 was not detected in serous tumors and frequently observed in mucinous subtypes (80%). Our findings suggest that Rb proteins do not play a key role in the tumor progression of serous borderline tumors since any cases showed cytoplasmic localization. By contrast, the observed higher cytoplasmic expression of Rb2/p130 in intestinal mucinous BOTs is indicative of Rb protein family involvement in the cancerogenesis pathway of mucinous ovarian tumors. Also, mucinous BOTs of intestinal-type, exhibiting low nuclear and high cytoplasmic levels of Rb2/p130 might potentially be considered a high-risk category of malignant evolution. Further studies on larger series are needed to clarify how BOTs could be stratified in different prognostic groups according to their Rb proteins immunohistochemical profile.