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

ACK是一个重要的致癌因素，但其活性的机制尚不清楚。已知ACK激活Akt，Akt是PI3K途径的关键组成部分。这种激活是通过直接磷酸化实现的，与PI3K活性无关。我们发现了ACK在PI3K途径中的一个新的调节作用，表明ACK磷酸化了PI3K的所有5个调节亚基，但这并不会导致PI3K活性的直观增加。相反，我们现在假设ACK作用于调节亚基的自由池(P85)。最近变得明显的是，P85作为两个种群存在。第一种是结构性地结合到催化亚单位上，并执行其经典的调节功能；第二种是自由形式，能够与PTEN结合。PTEN是一种重要的肿瘤抑制因子，在约30%的癌症中功能失调。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p85α与PTEN结合，增强其磷酸酶活性。因此，p85pha以双管齐下的方式控制PIP3水平。我们已经发现ACK也与PTEN相互作用。我们知道ACK可以磷酸化p85α，但我们现在将确定哪个p85α池是ACK底物：游离的p85α还是与催化亚基形成复合体的底物。我们还将测试PTEN是否为ACK的底物。磷酸化位点的确定将提示ACK/PTEN相互作用的功能后果。虽然像p85pha一样，PTEN的功能是在PIP3调节中被研究得最好的，但现在也知道PTEN具有PIP3不依赖的功能，特别是在细胞核中。核PTEN在染色体稳定、DNA修复、细胞周期停滞和细胞稳定等方面发挥重要作用。ACK在细胞核和细胞质之间穿梭是已知的，我们已经确定了几个核ACK伙伴。我们将在细胞分裂实验中确定ACK和PTEN之间的相互作用(我们只在细胞核中发现P85/ACK)。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将PTEN隔离在胞浆中，这是ACK致瘤活性的基础。我们将使用细胞增殖/迁移分析来证明哪些涉及p85α的功能对ACK的细胞生长和转移促进特性是重要的。同时，我们将检测一组已知对PI3K/Akt通路抑制剂敏感的癌细胞株的ACK激活状态。分子突变和表达数据可在AZ获得一个由300个细胞系组成的小组。我们将鉴定PI3K抑制剂抗性株系，评估ACK的突变状态，并确定ACK是否在这一背景下使用内部和商业抑制剂激活Akt。在项目结束时，我们将更彻底地了解ACK在PI3K信号转导中的作用，以及对抑制ACK的治疗益处进行评估。

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.