Macropinocytosis: mechanisms and regulation.

Macropinocytosis is defined as an actin-dependent but coat- and dynamin-independent endocytic uptake process, which generates large intracellular vesicles (macropinosomes) containing a non-selective sampling of extracellular fluid. Macropinocytosis provides an important mechanism of immune surveillance by dendritic cells and macrophages, but also serves as an essential nutrient uptake pathway for unicellular organisms and tumor cells. This review examines the cell biological mechanisms that drive macropinocytosis, as well as the complex signaling pathways - GTPases, lipid and protein kinases and phosphatases, and actin regulatory proteins - that regulate macropinosome formation, internalization, and disposition.

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.

Rac1-stimulated macropinocytosis enhances Gßγ activation of PI3Kß.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kß, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gßγ subunits to the C2-helical domain linker of p110ß, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110ß. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gßγ led to an additive PI3Kß-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kß that cannot bind Gßγ, Gßγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kß was blocked. Surprisingly, PI3Kß-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110ß that cannot bind Rac1. Instead of directly binding to PI3Kß, CA-Rac1 acts by enhancing Gßγ coupling to PI3Kß, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gßγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kß by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gßγ coupling to PI3Kß.

Rho A and Rac1: Antagonists moving forward.

Cells detect external stimuli through cell-surface receptors. In cases where the stimulus is a cytokine or a growth factor, the cell responds by inducing modifications in the actin cytoskeleton. These changes are mediated through the Rho family of GTPases. Among these GTPases, RhoA, Rac1 and Cdc42 have been extensively studied. The activity of these proteins is closely monitored and tightly regulated through Guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that turn the "switch" on and off respectively. Crosstalk between Rho GTPases has been long studied; yet many questions are raised regarding the spatiotemporal regulation of these GTPases, particularly RhoA and Rac1. This review sheds a light on the antagonistic relationship between both GTPases and puts emphasis on the importance of cycling of RhoA activation at the focal adhesions for optimal cell migration.

Targeting the MAP kinase pathway in astrocytoma cells using a recombinant anthrax lethal toxin as a way to inhibit cell motility and invasion.

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Understanding and targeting cancer cell invasion is an important therapeutic approach. Cell invasion is a complex process that replies on many signaling pathways including the mitogen-activated protein (MAP) kinase (MAPK). In many cell lines, the use of MAPK-targeted drugs proved to be a potential method to inhibit cancer cell motility. In the present study, we use a recombinant anthrax lethal toxin (LeTx), which selectively inhibits the MAPK pathway, in order to target invasion. LeTx proved ineffective on cell survival in astrocytoma (as well as normal cells). However, astrocytoma cells that were treated with LeTx showed a significant decrease in cell motility as seen by wound healing as well as random 2D motility in serum. The cells also showed a decrease in invasion across a collagen matrix. The effect of LeTx on cell migration was mediated though the deregulation of Rho GTPases, which play a role in cell motility. Finally, the effect of LeTx on cell migration and Rho GTPases was mimicked by the inhibition of the MAPK pathway. In this study, we describe for the first time the effect of the LeTx on cancer cell motility and invasion not cell survival making it a potentially selective brain tumor invasion inhibitor.