Beschreibung:
Abstract Dysregulation of phosphoinositide levels is associated with many cancer phenotypes. A variety of mechanisms activate phosphoinositide 3-kinases (PI3Ks), including association with G-proteins. Increased activity of PI 4-kinases (PI4Ks) also has been associated with cancer phenotypes. Although PI4Ks associate with G proteins, the relationship with enzyme activity is more complex. To understand regulation of these enzymes, we have used hydrogen/deuterium exchange mass spectrometry (HDX-MS) in conjunction with X-ray crystallography. The method enables us to explore not only three-dimensional structures but also the dynamics of the activation process and to map key regulatory interactions with G-proteins. PI3Ks and PI4Ks, along with PI3K-related protein kinases such as TOR are members of a closely related family that all have a common core structure, consisting of a helical domain closely associated with a kinase domain. The helical domains have important roles both in stabilizing the structures and forming regulatory interactions. For example, PI3Kγ is activated by associating with Gβγ heterodimers released upon activation of GPCRs, and HDX-MS shows how the helical domain of the enzyme interacts with the p101 regulatory subunit and with Gβγ heterodimers. When the p110γ/p101 complex interacts with lipid membranes a conformational change takes place that prepares the helical domain for association with prenylated Gβγ heterodimers on membranes. Type IIIβ PI4K (PI4KB) synthesizes PI4P on Golgi membranes. The enzyme is important for processes that require rapid expansion and remodeling of PI4P-containing membranes, including replication of a range of viruses. Its activity has also been associated with decreased cell-cell adhesion in invasive cancer cell lines. Using both HDX-MS and X-ray crystallography we have shown how the helical domain of PI4KB interacts with the Ras superfamily G protein Rab11. In this unique interaction, PI4KB grasps the Rab11 but leaves the switch regions of the G protein unencumbered and free to interact with Rab11 effectors. Consistent with this type of interaction, PI4K can interact with complexes of Rab11 bound to both GDP and GTP. Furthermore, the structure of the complex of the Rab11 effector FIP3 bound to the Rab11-PI4KB complex shows that PI4KB does not perturb Rab11 effector binding and suggests that PI4KB may be able to recruit both Rab11 and its downstream effectors to Golgi membranes. Citation Format: John E. Burke, Alison J. Inglis, Oscar Vadas, Olga Perisic, Glenn R. Masson, Stephen H. McLaughlin, Roger L. Williams. G-proteins regulating PI3Ks and PI4KIIIβ regulating a G-protein. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA02.