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

ACK是一个重要的致癌驱动因子，但其活动的机制尚不清楚。已知ACK可以激活Akt， Akt是PI3K通路的关键成分。这种激活是通过直接磷酸化，独立于PI3K活性。我们发现ACK在PI3K通路中具有新的调控作用，表明ACK磷酸化了PI3K的所有5个调控亚基，但这并不会导致PI3K活性的直观增加。相反，我们现在假设ACK作用于调控亚基的自由池（p85）。最近发现，p85有两个种群存在。第一种结构与催化亚基结合并执行其经典调节功能；第二种是自由形式，具有绑定PTEN的能力。PTEN是一种重要的肿瘤抑制因子，在约30%的肿瘤中存在功能障碍。它的作用与PI3K相反，使PIP3去磷酸化，从而抑制PIP3信号通路。游离p85 α与PTEN结合，增强其磷酸酶活性。因此，p85alpha以双管齐下的方式控制PIP3水平。我们发现ACK也与PTEN相互作用。我们知道ACK可以磷酸化p85 α，但我们现在要确定哪一组p85 α是ACK底物：自由的p85 α还是与催化亚基络合物的p85 α。我们还将测试PTEN是否是ACK的基板。磷酸化位点的鉴定将表明ACK/PTEN相互作用的功能后果。尽管与p85alpha一样，PTEN被研究得最多的功能是调节PIP3，但现在也知道PTEN具有与PIP3无关的功能，特别是在细胞核中。核PTEN在染色体稳定、DNA修复、细胞周期阻滞和细胞稳定中起作用。已知ACK在细胞核和细胞质之间穿梭，我们已经确定了几个核ACK伙伴。我们将通过细胞分离实验确定细胞间室ACK和PTEN的相互作用（我们发现p85/ACK仅在细胞核中）。PTEN似乎需要核定位来实现其肿瘤抑制功能。我们目前的假设是ACK在细胞质中隔离PTEN，这是ACK的致瘤活性的基础。我们将使用细胞增殖/迁移试验来证明涉及p85alpha的哪些功能对ACK的细胞生长和转移促进特性是重要的。同时，我们将在一组已知对PI3K/Akt通路抑制剂敏感的癌细胞系中检测ACK激活状态。分子突变和表达数据可在AZ面板的300细胞系。我们将鉴定PI3K抑制剂耐药系，评估ACK的突变状态，并确定ACK是否在使用内部和商业抑制剂的背景下激活Akt。在项目结束时，我们将更全面地了解ACK在PI3K信号传导中的作用，并评估抑制ACK的治疗益处。

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.