arrest cells respond to stress in the form of dna damage, activated oncogenes, or sub-par growing conditions, and can enter a senescence-like state called "premature senescence". this allows the cell to prevent further replication during periods of damaged dna or general unfavorable conditions. dna damage in a cell can induce prb activation. prb's role in repressing the transcription of cell cycle progression genes leads to the s phase arrest that prevents replication of damaged dna. activation and inactivation when it is time for a cell to enter s phase, complexes of cyclin-dependent kinases (cdk) and cyclins phosphorylate prb, allowing e2f-dp to dissociate from prb and become active. when e2f is free it activates factors like cyclins (e.g. cyclin e and cyclin a), which push the cell through the cell cycle by activating cyclin-dependent kinases, and a molecule called proliferating cell nuclear antigen, or pcna, which speeds dna replication and repair by helping to attach polymerase to dna. inactivation since the 1990s, prb was known to be inactivated via phosphorylation. until, the prevailing model was that cyclin d- cdk 4/6 progressively phosphorylated it from its unphosphorylated to its hyperphosphorylated state (14+ phosphorylations). however, it was recently shown that prb only exists in three states: un-phosphorylated, mono-phosphorylated, and hyper-phosphorylated. each has a unique cellular function. before the development of 2d ief, only hyper-phosphorylated prb was distinguishable from all other forms, i.e. un-phosphorylated prb resembled mono-phosphorylated prb on immunoblots. as prb was either in its active “hypo-phosphorylated” state or inactive “hyperphosphorylated” state. however, with 2d ief, it is now known that prb is un-phosphorylated in g0 cells and mono-phosphorylated in early g1 cells, prior to hyper-phosphorylation after the restriction point in late g1. prb mono phosphorylation when a cell enters g1, cyclin d- cdk4/6 phosphorylates prb at a single phosphorylation site. no progressive phosphorylation occurs because when hff cells were exposed to sustained cyclin d- cdk4/6 activity (and even deregulated activity) in early g1, only mono-phosphorylated prb was detected. furthermore, triple knockout, p16 addition, and cdk 4/6 inhibitor addition experiments confirmed that cyclin d- cdk 4/6 is the sole phosphorylator of prb. throughout early g1, mono-phosphorylated prb exists as 14 different isoforms (the 15th phosphorylation site is not conserved in primates in which the experiments were performed). together, these isoforms represent the “hypo-phosphorylated” active prb state that was thought to exist. each isoform has distinct preferences to associate with different exogenous expressed e2fs. a recent report showed that mono-phosphorylation controls prb's association with other proteins and generates functional distinct forms of prb. all different mono-phosphorylated prb isoforms inhibit e2f transcriptional program and are able to arrest cells in g1-phase. importantly, different mono-phosphorylated forms of prb have distinct transcriptional outputs that are extended beyond e2f regulation. hyper-phosphorylation after a cell passes the restriction point, cyclin e - cdk 2 hyper-phosphorylates all mono-phosphorylated isoforms. while the exact mechanism is unknown, one hypothesis is that binding to the c-terminus tail opens the pocket subunit, allowing access to all phosphorylation sites. this process is hysteretic and irreversible, and it is thought accumulation of mono-phosphorylated prb induces the process. the bistable, switch like behavior of prb can thus be modeled as a bifurcation point: hyper-phosphorylation of mono-phosphorylated prb is an irreversible event that allows entry into s phase. control of prb function by phosphorylation presence of un-phosphorylated prb drives cell cycle exit and maintains senescence. at the end of mitosis, pp1 dephosphorylates hyper-phosphorylated prb directly to its un-phosphorylated state. furthermore, when cycling c2c12 myoblast cells differentiated (by being placed into a differentiation medium), only un-phosphorylated prb was present. additionally, these cells had a markedly decreased growth rate and concentration of dna replication factors (suggesting g0 arrest). this function of un-phosphorylated prb gives rise to a hypothesis for the lack of cell cycle control in cancerous cells: deregulation of cyclin d - cdk 4/6 phosphorylates un-phosphorylated prb in senescent cells to mono-phosphorylated prb, causing them to enter g1. the mechanism of the switch for cyclin e activation is not known, but one hypothesis is that it is a metabolic sensor. mono-phosphorylated prb induces an increase in metabolism, so the accumulation of mono-phosphorylated prb in previously g0 cells then causes hyper-phosphorylation and mitotic entry. since any un-phosphorylated prb is immediately phosphorylated, the cell is then unable to exit the cell cycle, resulting in continuous division. dna damage to g0 cells activates cyclin d - cdk 4/6, resulting in mono-phosphorylation of un-phosphorylated prb. then, active mono-phosphorylated prb causes repression of e2f-targeted genes specifically. therefore, mono-phosphorylated prb is thought to play an active role in dna damage response, so that e2f gene repression occurs until the damage is fixed and the cell can pass the restriction point. as a side note, the discovery that damages causes cyclin d - cdk 4/6 activation even in g0 cells should be kept in mind when patients are treated with both dna damaging chemotherapy and cyclin d - cdk 4/6 inhibitors. activation during the m-to-g1 transition, prb is then progressively dephosphorylated by pp1, returning to its growth-suppressive hypophosphorylated state. prb family proteins are components of the dream complex composed of dp, e2f4/5, rb-like (p130/p107) and muvb (lin9:lin37:lin52:rbabp4:lin54). the dream complex is assembled in go/g1 and maintains quiescence by assembling at the promoters of > 800 cell-cycle genes and mediating transcriptional repression. assembly of dream requires dyrk1a (ser/thr kinase) dependant phosphorylation of the muvb core component, lin52 at serine28. this mechanism is crucial for recruitment of p130/p107 to the muvb core and thus dream assembly. consequences of prb loss consequences of loss of prb function is dependent on cell type and cell cycle status, as prb's tumor suppressive role changes depending on the state and current identity of the cell. in g0 quiescent stem cells, prb is proposed to maintain g0 arrest although the mechanism remains largely unknown. loss of prb leads to exit from quiescence and an increase in the number of cells without loss of cell renewal capacity. in cycling progenitor cells, prb plays a role at the g1, s, and g2 checkpoints and promotes differentiation. in differentiated cells, which make up the majority of cells in the body and are assumed to be in irreversible g0, prb maintains both arrest and differentiation. loss of prb therefore exhibits multiple different responses within different cells that ultimately all could result in cancer phenotypes. for cancer initiation, loss of prb may induce cell cycle re-entry in both quiescent and post-mitotic differentiated cells through dedifferentiation. in cancer progression, loss of prb decreases the differentiating potential of cycling cells, increases chromosomal instability, prevents induction of cellular senescence, promotes angiogenesis, and increases metastatic potential. although most cancers rely on glycolysis for energy production (warburg effect), cancers due to prb loss tend to upregulate oxidative phosphorylation. the increased oxidative phosphorylation can increase stemness, metastasis, and (when enough oxygen is available) cellular energy for anabolism. in vivo, it is still not entirely clear how and which cell types cancer initiation occurs with solely loss of prb, but it is clear that the prb pathway is altered in large number of human cancers. in mice, loss of prb is sufficient to initiate tumors of the pituitary and thyroid glands, and mechanisms of initiation for these hyperplasia are currently being investigated. non-canonical roles the classic view of prb's role as a tumor suppressor and cell cycle regulator developed through research investigating mechanisms of interactions with e2f family member proteins. yet, more data generated from biochemical experiments and clinical trials reveal other functions of prb within the cell unrelated (or indirectly related) to tumor suppression. functional hyperphosphorylated prb in proliferating cells, certain prb conformations (when rxl motif if bound by protein phosphatase 1 or when it is acetylated or methylated) are resistant to cdk phosphorylation and retain other function throughout cell cycle progression, suggesting not all prb in the cell are devoted to guarding the g1/s transition. studies have also demonstrated that hyperphosphorylated prb can specifically bind e2f1 and form stable complexes throughout the cell cycle to carry out unique unexplored functions, a surprising contrast from the classical view of prb releasing e2f factors upon phosphorylation. in summary, many new findings about prb's resistance to cdk phosphorylation are emerging in prb research and shedding light on novel roles of prb beyond cell cycle regulation. genome stability prb is able to be localize to sites of dna breaks during the repair process and assist in non-homologous end joining and homologous recombination through complexing with e2f1. once at the breaks, prb is able to recruit regulators of chromatin structure such as the dna helicase transcription activator brg1. prb has been shown to also be able to recruit protein complexes such as condensin and cohesin to assist in the structural maintenance of chromatin. such findings suggest that in addition to its tumor suppressive role with e2f, prb is also distributed throughout the genome to aid in important processes of genome maintenance such as dna break-repair, dna replication, chromosome condensation, and heterochromatin formation. regulation of metabolism prb has also been implicated in regulating metabolism through interactions with components of cellular metabolic pathways. rb1 mutations can cause alterations in metabolism, including reduced mitochondrial respiration, reduced activity in the electron transport chain, and changes in flux of glucose and/or glutamine. particular forms of prb have been found to localize to the outer mitochondrial membrane and directly interacts with bax to promote apoptosis. as a drug target prb reactivation while the frequency of alterations of the rb gene is substantial for many human cancer types including as lung, esophageal, and liver, alterations in up-steam regulatory components of prb such as cdk4 and cdk6 have been the main targets for potential therapeutics to treat cancers with dysregulation in the rb pathway. this focus has resulted in the recent development and fda clinical approval of three small molecule cdk4/6 inhibitors (palbociclib (ibrance, pfizer inc. 2015), ribociclib (kisquali, novartis. 2017), & abemaciclib (verzenio, eli lilly. 2017)) for the treatment of specific breast cancer subtypes. however, recent clinical studies finding limited efficacy, high toxicity, and acquired resistance of these inhibitors suggests the need to further elucidate mechanisms that influence cdk4/6 activity as well as explore other potential targets downstream in the prb pathway to reactivate prb's tumor suppressive functions. treatment of cancers by cdk4/6 inhibitors depends on the presence of prb within the cell for therapeutic effect, limiting their usage only to cancers where rb is not mutated and prb protein levels are not significantly depleted. direct prb reactivation in humans has not been achieved. however, in murine models, novel genetic methods have allowed for in vivo prb reactivation experiments. prb loss induced in mice with oncogenic kras-driven tumors of lung adenocarcinoma negates the requirement of mapk signal amplification for progression to carcinoma and promotes loss of lineage commitment as well as accelerate the acquisition of metastatic competency. reactivation of prb in these mice rescues the tumors towards a less metastatic state, but does not completely stop tumor growth due to a proposed rewiring of mapk pathway signaling, which suppresses prb through a cdk-dependent mechanism. pro-apoptotic effects of prb loss besides trying to re-activate the tumor suppressive function of prb, one other distinct approach to treat dysregulated prb pathway cancers is to take advantage of certain cellular consequences induced by prb loss. it has been shown that e2f stimulates expression of pro-apoptotic genes in addition to g1/s transition genes, however, cancer cells have developed defensive signaling pathways that protect themselves from death by deregulated e2f activity. development of inhibitors of these protective pathways could thus be a synthetically lethal method to kill cancer cells with overactive e2f. in addition, it has been shown that the pro-apoptotic activity of p53 is restrained by the prb pathway, such that prb deficient tumor cells become sensitive to p53 mediated cell death. this opens the door to research of compounds that could activate p53 activity in these cancer cells and induce apoptosis and reduce cell proliferation. regeneration while the loss of a tumor suppressor such as prb leading to uncontrolled cell proliferation is detrimental in the context of cancer, it may be beneficial to deplete or inhibit suppressive functions of prb in the context of cellular regeneration. harvesting the proliferative abilities of cells induced to a controlled “cancer like” state could aid in repairing damaged tissues and delay aging phenotypes. this idea remains to be thoroughly explored as