An intriguing association between the p14ARF oncosuppressor and TBP-1, a multifunctional protein with a potential role in the control of cell growth.
Sepe, Maria (2008) An intriguing association between the p14ARF oncosuppressor and TBP-1, a multifunctional protein with a potential role in the control of cell growth. [Tesi di dottorato] (Inedito)
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The p14ARF oncosuppressor is among the most relevant oncogenic stress sensor in mammalian cells. It is activated as part of a checkpoint response that counters oncogenic signals by promoting cell cycle arrest or apoptosis through both p53-dependent and independent mechanisms. ARF response is exerted through the activation of a complex signalling networks accomplished by the interaction with a multitude of different cellular partners that explain, at least in part, its function. The discovery of multiple ARF interactors and the observation that, aside oncogenic stimuli, also viral, genotoxic, hypoxic and oxidative stresses activate an ARF dependent response, suggest that ARF could exert a wider role to protect the cell. Although ARF intracellular levels increase very rapidly following oncogenic stimuli, the mechanisms regulating its turnover are not completely clarified. ARF is a relatively stable protein, although prevalently unstructured and largely disordered in solution. For long time it has been thought that it could not be degraded by the proteasome since its sequence lacks lysine residues (the mouse protein presenting only one lysine residue) that can allow ubiquitination in a canonical way. Recently, I demonstrated that ARF is degraded, at least in part, by the proteasome, Furthermore, it has been reported that ARF can be subjected to N-terminal ubiquitination, a process independent from p53 and MDM2 whose physiological role is still elusive. I demonstrated that ARF can be degraded by the 20S proteasome in vitro in the absence of ubiquitination; moreover, it has been described a very interesting role of the REG regulatory particle of the proteasome in the regulation of the ARF turnover. We previously identified as ARF binding partner, TBP1, a multifunctional protein component of the 19S regulatory subunit of the proteasome with chaperone-like activity involved in different cellular processes independent from the proteolytic activity of the proteasome. TBP-1 interacts with and regulates ARF protein levels delaying its turnover. Thus I investigated more precisely on the mechanisms through which the stabilization effect is exerted by TBP-1 on p14ARF and I demonstrated that silencing of TBP-1 expression causes a concomitant reduction of ARF intracellular protein levels, strongly suggesting that basal TBP-1 levels controls basal ARF levels. Interestingly, a point mutation in the ATPase domain of TBP-1 that destroys its chaperone-like activity, impairs TBP-1’s capacity to stabilize ARF, leading me to postulate the hypothesis that, upon binding, TBP-1 could cause ARF to fold, rendering it a poor substrate for proteasome destruction. The effect on ARF indicates a potential oncosuppressive role of TBP-1 also supported by other evidences. Thus, I focused my attention on the role of TBP-1 in the control of cell growth. My data indicate that stable cell clones in which TBP-1 expression has been silenced show higher proliferation rate respect to the control, also in conditions of serum deprivation. Furthermore, silenced clones display an increase of the S-phase and are more resistant to serum starvation induced apoptosis. TBP-1 silenced clones exhibit higher levels of activated pAkt, a protein kinase controlling the balance between cell survival and apoptosis, leading to the hypothesis that TBP-1 could exert a down-modulation of activated Akt levels and activity. Consistently, transient overexpression of TBP-1 causes a reduced activation of pAkt following insulin stimulation. Moreover, my data demonstrate that TBP-1 is likely a downstream target of Akt activation. Acute insulin stimulation of U2OS osteosarcoma cell line causes a rapid drop in TBP-1 intracellular levels. Conversely, the inhibition of the PI3K/Akt pathway by making use of specific drugs (Wortmannin and LY) determine a reproducible increase of TBP-1 intracellular levels. These data demonstrate that TBP-1 intracellular levels are critical to control cell duplication and are tightly regulated by a double-negative feedback loop that is mediated by the activation of the PKB/Akt kinase that thus seems to act as a sensor that modulate the TBP-1 levels in actively duplicating cells.
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