title: Arf6 and phosphoinositol-4-phosphate-5-kinase activities permit bypass of the Rac1 requirement for beta1 integrin-mediated bacterial uptake.
abstract: Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to beta1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP-bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.
authors.affiliation: Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA. ralph.isberg@tufts.edu
authors.forename: Ka-Wing
authors.initials: KW
authors.lastname: Wong
authors.forename: Ralph R
authors.initials: RR
authors.lastname: Isberg
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D018829:Adhesins, Bacterial / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D054803:Phospholipase C delta; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism; D015011:Yersinia pseudotuberculosis / Q000166:cytology / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D018829:Adhesins, Bacterial; D019012:Integrin beta1; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; C053476:invasin, Yersinia; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D020830:rac1 GTP-Binding Protein