Caratterizzazione nel Saccharomyces cerevisiae di PTEN-L, una variante di trascrizione alternativa dell'oncosoppressore umano PTEN

Mammalian class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimers consisting of a regulatory subunit and a catalytic subunit, which catalyse the conversion of the phosphoinositide PIP2 into PIP3 at the plasma membrane. The PIP3 acts as a second messenger in the PI3K/AKT signalling pathway favouring cell proliferation and inhibiting apoptosis. Mutations of the proteins involved in this signalling pathway lead to the abnormal hyperactivation of its cell signal mediates by PI3P, resulting in tumorigenesis. The tumour suppressor PTEN (Phosphatase and Tensin Homologue deleted from chromosome 10) is a lipid phosphatase that dephosphorylates PIP3 to PIP2, thus counteracting the activity of PI3K and decreasing cell growth. A longer form of PTEN, named PTEN-L, was recently reported as an additional PTEN cell version produced by alternative translation initiation: this PTEN longer form can be secreted and internalized by recipient cells, exerting there its tumour suppressor function. The aim of this study was to detect the PTEN-L domains involved in its cell localisation and to discover a possible correlation between these structural sequences and the PTEN-L tumour suppression function. The cell localisation was showed through the expression of the PTEN-L wild type and the PTEN-L mutational variants in a humanized yeast model, while the inhibition of PI3K-dependent cell growth was analyzed by the co-expression of these PTEN-L variants and the active versions of the catalytic subunit of PI3K under the control of the GAL1 inducible promoter. The mutations in the PTEN-L sequence were involved in the principal structure domains shared with the PTEN wild type, precisely the KRR residues belonging to the Nuclear Localisation Signal (NLS) motif of the N-terminal phosphatase domain and the CBR and Cα2 sequences of the C2 domain. Further mutations were developed in the Membrane-Binding Helix (MBH), a sequence included in the additional N-terminal extension of PTEN-L. Lastly the PTEN-L expression in yeast was analysed.