Arf6 and Phosphoinositol-4-Phosphate-5-Kinase Activities Permit Bypass of the Rac1 Requirement for β1 Integrin–mediated Bacterial Uptake

Abstract

Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 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.

Rho and Rho-kinase Mediate Thrombin-induced Phosphatidylinositol 4-Phosphate 5-Kinase Trafficking in Platelets

“…Although PIP5K does relocalize to different subcellular domains following stimulation of the platelet thrombin receptor, our data indicate that platelet PIP5K does not traffic into lipid rafts. (19,26,40). We have previously shown that these GTPases regulate the intracellular localization and catalytic activity of PIP5K␣ in Cos7SH cells (33).…”

The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

“…It is involved in cell migration through membrane ruffle formation and podosome formation (16,17,19). Arf6 also affects uptake of pathogens via endocytosis and recycling of ␤1 integrins (20), membrane delivery during phagocytosis, actin remodeling (21,22), intracellular transport of MHCI molecules (21), and the control of Fc␥ receptor-mediated phagocytosis (23,24). Recently, Arf6 was shown to be essential for two receptors that are related to TLR4.…”

Phosphatidylinositol 4-Phosphate 5-Kinase β Controls Recruitment of Lipid Rafts into the Immunological Synapse

“…Data from other cell types suggest that PIP5Kb is targeted to the plasma membrane and/or activated when overexpressed (24)(25)(26). Overexpression of PIP5Kb induces actin polymerization (27)(28)(29)(30) and actin comets from lipid rafts at the plasma membrane (31).…”