Exploring the PI3Kβ-Rab5 Interface
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Class I Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that regulate cell motility, growth, and survival. Class IA PI3Ks are obligate heterodimers that contain a regulatory subunit (p85α/β, p55α/γ, or p50α) and a catalytic (p110α, β, or δ) subunit. The full-length regulatory p85 subunit consists of a N-terminal SH3 domain, two proline-rich domains (nPRD and cPRD) that flank a BCR homology domain (BCR), and two SH2 domains (nSH2 and cSH2) linked by the inter-SH2 (iSH2) domain. The p110 catalytic subunit contains an N-terminal adaptor binding domain (ABD), a Ras-binding domain (RBD), a C2 domain, a helical domain, and a C-terminal kinase domain. The Class IA PI3Kβ can be activated by both receptor tyrosine kinase (RTK) binding to the p85 SH2 domains and by G-protein coupled receptors (GPCRs), which stimulate G3γ binding to the C2-helical linker region of p110β (the G3y binding loop). In contrast to the other Class IA PI3Ks, which bind Ras, p110β binds the small GTPases Rac1 and Cdc42 via the RBD domain. RabS was first identified as a p110β binding partner in a screen for RabS effectors. RabS localizes to early endosomes and other vesicular structures, and P13Kβ has been implicated in endocytic trafficking of cell surface receptors. The Rab5-PI3Kβ interaction was found to be critical for macroautophagy induced by growth factor limitation. Our lab initially identified a region of p110β that mediates binding to Rab5 using a conservation-based mutagenesis approach that evaluated the C-terminal end of p110β (C2, helical, and kinase domains), which is sufficient to bind Rab5. From this screen, two residues in the helical domain were identified (Q596C and I597S) whose mutation disrupted binding to Rab5. Beyond these two residues, the extent of Rab5 binding to p110β is not known. The aim of my project was to define the Rab5 binding interface within PI3Kβ. To better define the Rab5-P13Kβ interface, we performed scanning alanine mutagenesis on the catalytic subunit p110β and analyzed binding using an in vitro pull down assay with active GST-Rab5. While it has been previously reported that the regulatory subunit p85 can bind to Rab5 and act as a GTPase activating protein (GAP), we failed to see any direct binding of p85 to GST-Rab5 using an in vitro pull down assay. Focusing on the catalytic subunit, we assessed binding of 35 helical domain mutants in p110β to Rab5. Of the helical domain mutants tested, only 12 were found to reduce binding to GST-Rab5 by more than 33% as compared to wildtype p110β. Based on research from another lab, we also tested p110β mutants from the RBD domain that were reported to affect Rab5 binding. In our hands, all the RBD domain mutants were able to bind to Rab5. All of the p110β mutants that were found to disrupt Rab5 binding were assessed for protein functionally. We used a GST-Rac1 in vitro pull down assay to confirm that the Rab5-uncoupled mutants could bind a known interactor of p110β. Using in vitro kinase assays, we found that mutations affecting Rab5 binding did not impact the basal or Gβγ-stimulated p110β kinase activity. Compiling the data, we found the Rab5 binding interface within p110β is restricted to two perpendicular α-helices in the helical domain that are adjacent to the initially identified Q596 and 1597 residues. Analysis of the Rab5-P13Kβ interactions by deuterium exchange-mass spectrometry identified p110β peptides that overlapped with these helices; no interactions were detected between Rab5 and other regions of p110β or p85α. Overall, we identified a discrete binding site for Rab5 in the helical domain of p110β. Using the Rab5-p110β interface data gathered, a collaborator selected a list of 31 small molecule compounds from a library of thousands, based on in silico modeling, that were predicted to disrupt the Rab5 binding interface. We adapted the GST-Rab5 in vitro pull down assay to assess the small molecule compounds for inhibitory action. Currently, we have not found any compounds to affect the interaction between p110β and Rab5. Despite this, we believe our data, which defines a single discrete Rab5 binding site in the p110β helical domain, will be useful for generating mutants and inhibitors to better define the physiological role of Rab5-PI3Kβ coupling in vivo.