PI3Kß is selectively required for growth factor-stimulated macropinocytosis.

Macropinocytosis is an actin-dependent but clathrin-independent endocytic process by which cells nonselectively take up large aliquots of extracellular material. Macropinocytosis is used for immune surveillance by dendritic cells, as a route of infection by viruses and protozoa, and as a nutrient uptake pathway in tumor cells. In this study, we explore the role of class I phosphoinositide 3-kinases (PI3Ks) during ligand-stimulated macropinocytosis. We find that macropinocytosis in response to receptor tyrosine kinase activation is strikingly dependent on a single class I PI3K isoform, namely PI3Kß (containing the p110ß catalytic subunit encoded by PIK3CB). Loss of PI3Kß expression or activity blocks macropinocytosis at early steps, before the formation of circular dorsal ruffles, but also plays a role in later steps, downstream from Rac1 activation. PI3Kß is also required for the elevated levels of constitutive macropinocytosis found in tumor cells that are defective for the PTEN tumor suppressor. Our data shed new light on PI3K signaling during macropinocytosis, and suggest new therapeutic uses for pharmacological inhibitors of PI3Kß.

A single discrete Rab5-binding site in phosphoinositide 3-kinase ß is required for tumor cell invasion.

Phosphoinositide 3-kinase ß (PI3Kß) is regulated by receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and small GTPases such as Rac1 and Rab5. Our lab previously identified two residues (Gln596 and Ile597) in the helical domain of the catalytic subunit (p110ß) of PI3Kß whose mutation disrupts binding to Rab5. To better define the Rab5-p110ß interface, we performed alanine-scanning mutagenesis and analyzed Rab5 binding with an in vitro pulldown assay with GST-Rab5GTP Of the 35 p110ß helical domain mutants assayed, 11 disrupted binding to Rab5 without affecting Rac1 binding, basal lipid kinase activity, or Gßγ-stimulated kinase activity. These mutants defined the Rab5-binding interface within p110ß as consisting of two perpendicular α-helices in the helical domain that are adjacent to the initially identified Gln596 and Ile597 residues. Analysis of the Rab5-PI3Kß interaction by hydrogen-deuterium exchange MS identified p110ß peptides that overlap with these helices; no interactions were detected between Rab5 and other regions of p110ß or p85α. Similarly, the binding of Rab5 to isolated p85α could not be detected, and mutations in the Ras-binding domain (RBD) of p110ß had no effect on Rab5 binding. Whereas soluble Rab5 did not affect PI3Kß activity in vitro, the interaction of these two proteins was critical for chemotaxis, invasion, and gelatin degradation by breast cancer cells. Our results define a single, discrete Rab5-binding site in the p110ß helical domain, which may be useful for generating inhibitors to better define the physiological role of Rab5-PI3Kß coupling in vivo.