Insights into the pathological mechanisms of p85α mutations using a yeast-based phosphatidylinositol 3-kinase model.

In higher eukaryotes, cell proliferation is regulated by class I phosphatidylinositol 3-kinase (PI3K), which transduces stimuli received from neighboring receptors by local generation of PtdIns(3,4,5)P3 in cellular membranes. PI3K is a heterodimeric protein consisting of a regulatory and a catalytic subunit (p85 and p110 respectively). Heterologous expression of p110α in Saccharomyces cerevisiae leads to toxicity by conversion of essential PtdIns(4,5)P2 into futile PtdIns(3,4,5)P3, providing a humanized yeast model for functional studies on this pathway. Here, we report expression and functional characterization in yeast of all regulatory and catalytic human PI3K isoforms, and exploitation of the most suitable setting to functionally assay panels of tumor- and germ line-associated PI3K mutations, with indications to the limits of the system. The activity of p110α in yeast was not compromised by truncation of its N-terminal adaptor-binding domain (ABD) or inactivation of the Ras-binding domain (RBD). In contrast, a cluster of positively charged residues at the C2 domain was essential. Expression of a membrane-driven p65α oncogenic-truncated version of p85α, but not the full-length protein, led to enhanced activity of α, ß, and δ p110 isoforms. Mutations impairing the inhibitory regulation exerted by the p85α iSH2 domain on the C2 domain of p110α yielded the latter non-responsive to negative regulation, thus reproducing this oncogenic mechanism in yeast. However, p85α germ line mutations associated with short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome did not increase PI3K activity in this model, supporting the idea that SHORT syndrome-associated p85α mutations operate through mechanisms different from the canonical disruption of inhibitory p85-p110 interactions typical of cancer.

Cytoplasmic p27Kip1 counteracts the pro-apoptotic function of the open conformation of PTEN by retention and destabilization of PTEN outside of the nucleus.

The tumor suppressor activity of p27Kip1 takes place in the cell nucleus by inhibitory binding to cyclin/CDK complexes. p27Kip1 can also be localized in the cytoplasm, where it has been proposed to have oncogenic properties. Here, we describe a novel role for cytoplasmic p27Kip1 which could account for its activity as an oncoprotein by negative regulation of the PTEN tumor suppressor. p27Kip1 physically interacted with the open conformation of PTEN, which is competent to enter the nucleus. In mammalian cells, cytoplasmic p27Kip1 retained to nuclear-targeted PTEN in the cytoplasm. This retention was exerted by the C-terminal p27Kip1 region, and was independent of cyclin/CDK-binding. The nuclear accumulation of PTEN triggered by pro-apoptotic TNFα treatment was abolished by cytoplasmic p27Kip1. Furthermore, conformationally-open PTEN displayed diminished protein stability and pro-apoptotic activity in the presence of cytoplasmic p27Kip1. Our results support a conformationally-dependent model of cytoplasmic retention and negative regulation of the activity of nuclear PTEN by oncogenic cytoplasmic p27Kip1, and suggest the existence of reciprocal mechanisms to regulate the levels of both p27Kip1 and PTEN.

A comprehensive functional analysis of PTEN mutations: implications in tumor- and autism-related syndromes.

The PTEN (phosphatase and tensin homolog) phosphatase is unique in mammals in terms of its tumor suppressor activity, exerted by dephosphorylation of the lipid second messenger PIP(3) (phosphatidylinositol 3,4,5-trisphosphate), which activates the phosphoinositide 3-kinase/Akt/mTOR (mammalian target of rapamycin) oncogenic pathway. Loss-of-function mutations in the PTEN gene are frequent in human cancer and in the germline of patients with PTEN hamartoma tumor-related syndromes (PHTSs). In addition, PTEN is mutated in patients with autism spectrum disorders (ASDs), although no functional information on these mutations is available. Here, we report a comprehensive in vivo functional analysis of human PTEN using a heterologous yeast reconstitution system. Ala-scanning mutagenesis at the catalytic loops of PTEN outlined the critical role of residues within the P-catalytic loop for PIP(3) phosphatase activity in vivo. PTEN mutations that mimic the P-catalytic loop of mammalian PTEN-like proteins (TPTE, TPIP, tensins and auxilins) affected PTEN function variably, whereas tumor- or PHTS-associated mutations targeting the PTEN P-loop produced complete loss of function. Conversely, Ala-substitutions, as well as tumor-related mutations at the WPD- and TI-catalytic loops, displayed partial activity in many cases. Interestingly, a tumor-related D92N mutation was partially active, supporting the notion that the PTEN Asp92 residue might not function as the catalytic general acid. The analysis of a panel of ASD-associated hereditary PTEN mutations revealed that most of them did not substantially abrogate PTEN activity in vivo, whereas most of PHTS-associated mutations did. Our findings reveal distinctive functional patterns among PTEN mutations found in tumors and in the germline of PHTS and ASD patients, which could be relevant for therapy.