title: Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis.
abstract: Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.
authors.affiliation: Department of Cellular and Molecular Pharmacology and the Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
authors.forename: Supriya
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Suzana
authors.initials: S
authors.lastname: Glavas
authors.forename: Alexander
authors.initials: A
authors.lastname: Ott
authors.forename: Fred
authors.initials: F
authors.lastname: Hofmann
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.forename: Daniel
authors.initials: D
authors.lastname: Kalman
authors.forename: Henry R
authors.initials: HR
authors.lastname: Bourne
mesh_terms: D000199:Actins / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology*; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D025461:Feedback, Physiological / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins; D018922:HL-60 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011554:Pseudopodia / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D007295:Inositol Phosphates; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C118500:inositol 3,4,5-trisphosphate; C072292:lethal toxin LT, Clostridium sordellii; D049452:Green Fluorescent Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; C042932:formylmethionyl-leucyl-phenylalanine methyl ester; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D054462:p21-Activated Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins