Tufano, Martina (2022) The role of FKBP51 in the activation of Akt oncogenic pathway. [Tesi di dottorato]

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FKBP51 is an immunophilin with a relevant role in sustaining cancer cell growth and aggressiveness of different human tumors and, particularly, melanoma. The protein contains two N-terminal FKBP-like domains FK1 and FK2, with FK1 exerting an isomerase activity, and a C-terminal region with a tetratricopeptide repeat (TPR) domain for protein/protein interaction. Based on its scaffold and isomerase activities, it participates in several signaling pathways, including NF-κB and Akt signaling. The role of FKBP51 in the Akt pathway is controversial. In 2009 Pei et al. proposed a role for FKBP51 as a scaffold promoting interaction between Akt and PHLPP phosphatase, thus negatively regulating Akt activation, in a pancreatic cancer context. In 2010, Romano et al. showed an association between FKBP51 upregulation and high pAkt levels in different tumor types. In 2013, Fabian et al. reported an increase in pAkt S473 upon FKBP51 overexpression. Deregulation of the Akt pathway is one of the major mechanisms that sustain cancer survival and progression. Especially in an incurable tumor as melanoma, in depth knowledge of the mechanisms underlying Akt activation is needed. In this thesis work, the interactions of FKBP51 with Akt and PHLPP are investigated along with the mechanism of Akt activation by the immunophilin. The study benefited from the recent identification of the FKBP51 spliced isoform (lacking the TPR domain), that reconciled the diverging results, generating the hypothesis that a unique gene regulated phosphorylation and de-phosphorylation of Akt. Thanks to the use of TPR-mutants, I demonstrated that the TPR domain of the canonical isoform mediates Akt phosphorylation through the activation of Akt-K63-ubiquitination. Conversely, upregulation of the spliced FKBP51s decreased K63-Ub residues binding to Akt. I also show that Hsp90, a known interactor of FKBP51-TPR domain, is a further important element in Akt activation. The pharmacological inhibition of Hsp90, indeed, impaired Akt K63-ubiquitination. Lack of TPR in FKBP51s explains the defective K63-ubiquitination capability. Unexpectedly, PHLPP silencing did not foster phosphorylation of Akt, whereas its overexpression even promoted K63-ubiquitination and phosphorylation of Akt. As TRAF6 is a E3 ubiquitin ligase and a known FKBP51 interactor that undergoes degradation upon phosphorylation, we hypothesized a role for PHLPP in stabilizing TRAF6. The finding that PHLPP silencing decreased TRAF6 level, supported such a hypothesis. Collectively, these results suggest that FKBP51 promotes Akt activation by serving as a scaffold to build the macrocomplex deputed to Akt ubiquitination and phosphorylation. Moreover, the present study introduces an unknown oncogenic role for the phosphatase PHLPP. A preliminary mass spectrometry-based proteome profile of melanoma cells overexpressing PHLPP, highlighted a relevant contribution of PHLPP especially in improvement of melanoma cell proliferation. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51 and FKBP51s that may affect Akt activity in cancer.

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