Fig. 3.

Effect of p101 mutations on cellular activity and transformation. (A) Schematic representation of the two states compared by HDX-MS to identify changes in p101 induced by prenylated Gβγ on membranes. In this experiment, p101 was always in complex with p110γ. (B) Linear plot of changes in HDX rates in p101 upon binding prenylated Gβγ as a function of central residue number. White indicates regions for which no peptides were detected in the HDX-MS experiment. (C) Schematic representation of p101 tetra-alanine mutants that were generated for biochemical characterization. Above the blocks illustrating positions of mutations is a bar colored according to the HDX results (blue represents a decrease in HDX, gray no change, and white regions not covered by peptides in the HDX-MS experiment). (D) Representative images of HEK293T cells transfected with the described PI3Kγ constructs. PIP₃ production is indicated by translocation of the GFP-Grp1_PH domain construct to the plasma membrane (as for Fig. 2B). (E) A quantitation of Grp1_PH translocation for all of the p101 tetra-alanine mutants. (F) In vitro lipid kinase activity as a function of Gβγ concentration for two selected p101 mutants in complexes with wild-type or ⁵⁵²DD-mutated p110γ. Error bars show SD for at least three replicates. (G) Transformation of NIH 3T3 cells measured by colony formation in soft agar resulting from transfection with the indicated constructs.