Figure 4
Residues L191 and V263 in the p85α BH domain are important for Rab5 binding. (a) The BH domain of human p85α with the proposed G protein binding (i.e. Rab5) residues indicated. Residues with little or no effect on Rab5 binding are shown in grey (K187, I267 [L267 in bovine p85α], M271 [L271 in bovine p85α]), with catalytically important R151 in orange. Residues important for both Rab5 binding and catalytic activity are shown in red (L191, V263, R274). Residues that help mediate BH–BH domain dimerization within the crystal structure are shown in M176 (purple) from one BH domain fitting into a hydrophobic pocket containing L161, I177 and V181 (pink) on the other BH domain29. (b) Pull-down assay with GST and GST-Rab5 mutants immobilized on glutathione Sepharose beads and loaded with the indicated nucleotide. Binding of purified p85α wild type or mutant protein was detected using an immunoblot analysis. The input lanes contain 0.4% of the purified p85α protein used in the pull-down assay. Full-length images of the cropped blots are in Supplementary Fig. S6. (c) The ability of each p85α mutant protein to regulate Rab5 GTPase activity was determined using a Rab5 GAP assay. Mean ± SEM from three independent experiments. ***P < 0.001 as compared to wild type p85α. (d) Immobilized GST and wild type GST-PTEN were allowed to bind purified p85α wild type (wt) or mutant proteins as indicated. The input lanes contain 0.4% of the purified p85α protein used in the pull-down assay. Full-length images of the cropped blots are in Supplementary Fig. S6. (e) PTEN lipid phosphatase activity was measured either alone (no p85α) or with the added p85α WT or mutant protein. Mean ± SEM from five independent assays. No significant differences were measured for the mutants as compared to wild type p85α.
