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The retinoblastoma family of phosphoproteins consisting of the retinoblastoma protein (pRB) and the two structurally related proteins p130 and p107 play an important role in the negative regulation of cell cycle progression. Hypophosphorylated pocket proteins interact with the different members of the E2F family and repress the transcription of E2F-dependent genes and consequently suppress cell cycle progression through the G0/G1 transition and the restriction point in G1. Mitogenic stimulation results in sequential activation of cyclin/CDK complexes in mid to late G1, leading to subsequent hyperphosphorylation at multiple Ser/Thr sites of pocket proteins triggering dissociation of pocket protein/E2F complexes. This disruption leads to de-repression of many E2F dependent genes whose products are essential for cell cycle progression. The traditional view has been that pocket proteins continue to be hyperphosphorylated through the S and G2 phases and following cyclin/CDK inactivation during mitotic exit become dephosphorylated by action of PP1. However, our lab observed that upon treatment of asynchronously growing cells with the CDK inhibitor Flavopiridol or CHX, pocket proteins, are rapidly dephosphorylated correlating with the inactivation of G1/CDKs and down regulation of D-type cyclins, respectively. Pocket protein dephosphorylation was prevented by pre-treating these cells with phosphtase inhibitors at a concentration selective for PP2A, implicating PP2A or PP2A-like serine/threonine phosphatase in this iii process. The involvement of PP2A on pocket protein dephosphorylation was further strengthened by the observation that SV40 small t antigen (ST) delays/prevents p107 dephosphorylation. Moreover, a physical association between PP2A/C and p130/p107 was observed throughout the cell cycle that was not affected by CHX treatment, strongly suggesting that CHX-induced dephosphorylation is not the result of increased pocket protein targeting by PP2A, but rather that a dynamic equilibrium between CDKs and PP2A is shifted to dephosphorylation when CDK activity is compromised. This dynamic equilibrium operates throughout the cell cycle. PP2A is a trimeric enzyme complex consisting of a catalytic C, a structural A and substrate specific B subunit. There are four families of regulatory B subunits designated B, B’, B’’ and B’’’, each with several members encoded by genes with multiple splice variants that mediate substrate specificity and subcellular localization. It has been reported recently that in excess of 200 functional distinct PP2A holoenzymes can assemble with distinct specificities. Therefore, to gain insight into the mechanisms that regulate the steady state phosphorylation of pocket proteins throughout the cell cycle, it was essential to identify the specific holoenzyme complexes involved. To this end, it was identified that a PP2A trimeric holoenzyme containing B55α specifically targets and dephosphorylates p107/p130 both in vitro and in mammalian cells. B55α associates directly with the spacer of p107 and this interaction seems to be indirectly enhanced by the C-terminus of p107. The decreased association of p107 with PP2A/C of the B55α/PP2A holoenzyme complex upon treatment with ST further confirmed the role of B55α in mediating p107-PP2A/C interaction. Our data also revealed an interaction between B55α and p130, but not pRb, which appears to prefer a PR70, suggesting selectivity in the interaction of pocket proteins with distinct PP2A holoenzymes. In accordance with this, recombinant purified B55α dephosphorylates p107 in vitro. Limited ectopic expression of B55α but not other subunits, result in ST sensitive dephosphorylation of p107 and p130 in cells. Further shRNA mediated knockdown of B55α results in hyperphosphorylation of p107 and p130. This suggests that the cellular levels of B55α are critical in modulating the phosphorylation status of p107/p130 rather than just catalyzing the dephosphorylation of these proteins when the activity of CDKs is compromised. Since ST disrupts the B55α/PP2A holoenzyme complex by binding to the PP2A-A-C dimer and leads to hyperphosphorylation of pocket proteins it is conceivable that ST mediates its effects on cell proliferation at least in part, via inactivation of the PP2A holoenzymes that activates pocket proteins. Given the sensitivity of p107 phosphorylation to the cellular levels of B55α, future analyses should ascertain if deregulation of B55α leads to hyperphosphorylation of pocket proteins and abnormal cell cycle progression. / Molecular Biology and Genetics

The cell cycle is negatively regulated by members of the pocket protein family, which consists of the tumor suppressor pRB and two closely related paralogs, p107 and p130. In their hypophosphorylated state, they are associated with E2F transcription factors which result in the repression of transcription of E2F-dependent genes that are required for cell cycle progression. The phosphorylation state of pocket proteins during the cell cycle is determined at least in part by an equilibrium between inducible CDKs and the serine/threonine protein phosphatase PP2A. Protein Phosphatase 2A (PP2A), is a serine/threonine phosphatase that functions as as a collection of trimeric holoenzymes. The trimeric PP2A holoenzyme is composed of the "A" scaffolding subunit, the "C" catalytic subunit, and a "B" regulatory subunit. The B subunit is the major determinant in substrate specificity and subcellular localization. Two holoenzymes consisting of the core PP2A dimer and either the B55α or PR70 regulatory subunits have been implicated in the activation of p107/p130 and pRB, respectively. While the phosphorylation state of p107 is very sensitive to forced changes of B55α levels in human cell lines, regulation of p107 in response to physiological modulation of PP2A/B55α has not been previously elucidated. In this thesis, I show that FGF1, which induces maturation and cell cycle exit in chondrocytes, triggers rapid accumulation of p107/PP2A/B55α complexes coinciding with p107 dephosphorylation without an increase in B55α protein expression in RCS cells. Reciprocal solution-based mass-spectrometry analysis identified the PP2A/B55α complex as a major component of a subset of p107 complexes, which also contain E2F/DPs, DREAM subunits and cyclin/CDK complexes. p107 is one of the major partners of B55α, which also associates with pRB in RCS cells. FGF1 induces dephosphorylation of p107, its translocation to the nucleus, remodeling of p107 complexes, and enhances its interaction with E2F4 and other p107 partners. Consistent with an essential role of B55α in the rapid activation of p107 in chondrocytes, limited ectopic expression of B55α results in marked dephosphorylation of p107, while B55α knockdown results in hyperphosphorylation. More importantly, limited knockdown of B55α dramatically delays FGF1 induced dephosphorylation of p107. Moreover, dephosphorylation of p107 in response to FGF1 treatment results in selective recruitment of p107 to regulated genes including CMYC. Our results suggest a model where FGF1 mediates rapid dephosphorylation and activation of p107 independently of the CDK activities that maintain p130 and pRB hyperphosphorylated for several hours post p107 dephosphorylation in maturing chondrocytes. Additionally, we provide preliminary evidence that PPP2R2A may act as a haploinsufficient tumor suppressor in prostate cancer cell lines. PPP2R2A is hemizygously deleted in various prostate cancer cell lines and tumor samples. We identified three cell lines that express less B55α the gene product of PPP2R2A, than cell lines that are reported to have both alleles intact. Furthermore, ectopic expression of B55α in PC3 cells results in a phenotype reminiscent of senescence, ultimately leading to cell death. These cells are unable to form colonies in soft agar and have increased DNA content and euploidy. Combined with their larger cell and nuclear size, this suggests that ectopic expression of B55α in PC3 cells results in endoreplication. Altogether these suggest that reduced B55α expression in these cells confers a growth advantage in PCa cell lines, which is extinguished when B55α is reintroduced, supporting the notion that hemizygous deletion of PPP2R2A in prostate tumors may help promote tumorigenesis. / Molecular Biology and Genetics

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

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. / Biomedical Sciences

The philosophy of linguistics is a rich philosophical domain which encompasses various disciplines. One of the aims of this thesis is to unite theoretical linguistics, the philosophy of language, the philosophy of science (particularly mathematics and modelling) and the ontology of language. Each part of the research presented here targets separate but related goals with the unified aim of bringing greater clarity to the foundations of linguistics from a philosophical perspective. Part I is devoted to the methodology of linguistics in terms of scientific modelling. I argue against both the Conceptualist and Platonist (as well as Pluralist) interpretations of linguistic theory by means of three grades of mathematical involvement for linguistic grammars. Part II explores the specific models of syntactic and semantics by an analogy with the harder sciences. In Part III, I develop a novel account of linguistic ontology and in the process comment on the type-token distinction, the role and connection with mathematics and the nature of linguistic objects. In this research, I offer a structural realist interpretation of linguistic methodology with a nuanced structuralist picture for its ontology. This proposal is informed by historical and current work in theoretical linguistics as well as philosophical views on ontology, scientific modelling and mathematics.