ARNO but not cytohesin-1 translocation is phosphatidylinositol 3-kinase-dependent in HL-60 cells.
Cytohesin-1 and ARNO are guanine nucleotide-exchange factors (GEFs) for ADP-ribosylation factor (Arf). Here, we show that ARNO is expressed in HL-60 cells and established that granulocytic differentiation induced with Me2SO stimulated cytohesin-1 but not ARNO expression. Cytohesin-1 levels in HL-60 granulocytes were similar to those in human neutrophils. Me2SO-differentiated HL-60 cells expressed ARNO and cytohesin-1 isoforms with a diglycine and a triglycine motif in their PH domains, respectively. In vitro, ARNO diglycine and cytohesin-1 triglycine enhanced phospholipase D1 (PLD1) activation by Arf1 with near-maximal effects at 250 nM. These effects were marked particularly at low Mg2+ concentrations. PLD activation was well-correlated with GTP binding to Arf1, and cytohesin-1 was always more potent than ARNO in the PLD- and GTP-binding assays. Increasing Mg2+ concentrations reduced PLD and Arf1 activation by Arf-GEFs. fMetLeuPhe and phorbol 12-myristate 13-acetate stimulated ARNO and cytohesin-1 as well as Arf1 translocation to HL-60 cell membranes. fMetLeuPhe-mediated ARNO recruitment, but not cytohesin-1 and Arf1 translocation, was blocked by phosphatidylinositol 3-kinase inhibitors. The combined results demonstrate that cytohesin-1 triglycine participates in a major phosphatidylinositol 3-kinase-independent pathway linking cell-surface receptors to Arf1 activation and translocation in human granulocytes.
Bourgoin
Sylvain G
SG
Centre de Recherche en Rhumatologie et Immunologie, Pavillon CHUL et Département d'Anatomie-Physiologie, Faculté de Médecine, Université Laval, Québec, Canada. sylvain.bourgoin@crchul.ulaval.ca
Houle
Martin G
MG
Singh
Indrapal N
IN
Harbour
Danielle
D
Gagnon
Steve
S
Morris
Andrew J
AJ
Brindley
David N
DN
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D001692:Biological Transport; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000032:analysis; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C066755:Sec7 guanine nucleotide exchange factors; C100723:cytohesin-1; C102474:cytohesin-2; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D019869:Phosphatidylinositol 3-Kinases; D010739:Phospholipase D; D020823:ADP-Ribosylation Factor 1; D013755:Tetradecanoylphorbol Acetate