The p14ARF tumor suppressor is a key regulator of cellular proliferation, frequently inactivated in human cancer. The mechanisms that regulate alternative reading frame (ARF) turnover have been obscure for long time, being ARF a relatively stable protein. Recently, it has been described that its degradation depends, at least in part, on the proteasome and that it can be subjected to N-terminal ubiquitination. We have previously reported that ARF protein levels are regulated by TBP-1 (Tat-Binding Protein 1), a multifunctional protein, component of the regulatory subunit of the proteasome, involved in different cellular processes. Here we demonstrate that the stabilization effect exerted by TBP-1 requires an intact N-terminal 39 amino acids in ARF and occurs independently from N-terminal ubiquitination of the protein. Furthermore, we observed that ARF can be degraded in vitro by the 20S proteasome, in the absence of ubiquitination and this effect can be counteracted by TBP-1. These observations seem relevant in the comprehension of the regulation of ARF metabolism as, among the plethora of cellular ARF's interactors already identified, only NPM/B23 and TBP-1 appear to be involved in the control of ARF intracellular levels.

Objectives: Protein tyrosine phosphatases are key regulators of intracellular signaling that contribute to determining cancer cell growth, which thus makes them attractive targets for therapeutic and diagnostic agents. SHP-1 phosphotyrosine phosphatase is rarely expressed in epithelial tumor cells, but expression has been found in several breast cancer cell lines and tumors. To determine the potential significance of SHP-1 as a prognostic marker in the clinical setting, we examined SHP-1 protein expression in breast tumors. Methods: We analyzed SHP-1 expression by immunohistochemistry in a breast tissue microarray composed of 2,081 cores, either alone or in combination with known prognostic markers. Results: Our data showed that SHP-1 expression was confined to a well-defined subset of high-grade tumors characterized by unique biological parameters. SHP-1 expression correlated directly with expression of the tyrosine kinase receptor HER-2 and inversely with expression of the estrogen receptor, while it was weakly associated with Bcl-2 expression. Conclusions: Levels of SHP-1 were correlated with conventional pathologic parameters of tumor aggressiveness and were associated with reduced patient survival, suggesting that elevated expression of SHP-1 is a common molecular abnormality in a defined subset of breast tumors and might be used in routine diagnosis to identify patients with high-risk tumors.

TBP-1 /Tat-Binding Protein 1 (also named Rpt-5, S6a or PSMC3) is a multifunctional protein, originally identified as a regulator of HIV-1-Tat mediated transcription. It is an AAA-ATPase component of the 19S regulative subunit of the proteasome and, as other members of this protein family, fulfils different cellular functions including proteolysis and transcriptional regulation. We and others reported that over expression of TBP-1 diminishes cell proliferation in different cellular contexts with mechanisms yet to be defined. Accordingly, we demonstrated that TBP-1 binds to and stabilizes the p14ARF oncosuppressor increasing its anti-oncogenic functions. However, TBP-1 restrains cell proliferation also in the absence of ARF, raising the question of what are the molecular pathways involved. Herein we demonstrate that stable knock-down of TBP-1 in human immortalized fibroblasts increases cell proliferation, migration and resistance to apoptosis induced by serum deprivation. We observe that TBP-1 silencing causes activation of the Akt/PKB kinase and that in turn TBP-1, itself, is a downstream target of Akt/PKB. Moreover, MDM2, a known Akt target, plays a major role in this regulation. Altogether, our data suggest the existence of a negative feedback loop involving Akt/PKB that might act as a sensor to modulate TBP-1 levels in proliferating cells.