Control of the PI3K regulatory subunits and PTEN underpins the oncogenic driver activity of the small G protein regulated tyrosine kinase, ACK.

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ACK is an important oncogenic driver but the mechanisms underpinning its activity are unclear. ACK is known to activate Akt, a key component of the PI3K pathway. This activation is via direct phosphorylation, independent of PI3K activity. We have discovered a new regulatory role for ACK in the PI3K pathway by showing that ACK phosphorylates all 5 regulatory subunits of PI3K, however this does not lead to the intuitive increase in PI3K activity. Instead we now hypothesize that ACK is acting on the free pool of regulatory subunit (p85).It has recently become apparent that the p85 exist as two populations. The first is constitutively bound to the catalytic subunit and performs its classic regulatory functions; the second is a free form, with ability to bind to PTEN. PTEN is an important tumour suppressor which is dysfunctional in ~30% of cancers. It acts in opposition to PI3K, dephosphorylating PIP3, so inhibiting PIP3-signalling pathways. Free p85alpha binds to PTEN enhancing its phosphatase activity. Thus p85alpha controls PIP3 levels in a two-pronged approach. We have uncovered that ACK also interacts with PTEN.We know that ACK can phosphorylate p85alpha but we will now identify which pool of p85alpha is the ACK substrate: free p85alpha or that in complex with the catalytic subunit. We will also test whether PTEN is a substrate for ACK. Identification of the phosphorylation site will indicate the functional consequence of the ACK/PTEN interaction.Although like p85alpha, the best-studied function of PTEN is in PIP3 regulation, it is also now known that PTEN has PIP3-independent functions, especially in the nucleus. Nuclear PTEN plays roles in chromosomal stability, DNA repair, cell cycle arrest and cellular stability. ACK is known to shuttle between the nucleus and the cytoplasm and we have identified several nuclear ACK partners. We will determine the cellular compartment ACK and PTEN interact in using cell fractionation experiments (we find p85/ACK only in the nucleus). PTEN appears to require nuclear localization to fulfil its tumour suppressor functions. Our current hypothesis is that ACK sequesters PTEN in the cytosol and this underlies the tumourigenic activity of ACK.We will use cell proliferation/migration assays to demonstrate which functions involving p85alpha are important to ACK's cell growth and metastatic promoting properties.In parallel we will examine ACK activation status in a panel of cancer cell lines with known sensitivity to inhibitors of the PI3K/Akt pathway. Molecular mutation and expression data is available at AZ for a panel of 300 cell lines. We will identify PI3K inhibitor resistant lines and assess the mutational status of ACK and determine whether ACK is activating Akt in this background using both in-house and commercial inhibitors.At the end of the project we will have in hand a more thorough understanding of the role of ACK in PI3K signalling, together with an assessment of the therapeutic benefit of inhibiting ACK.