A novel phosphatidylinositol 4,5-bisphosphate-binding domain targeting the Phg2 kinase to the membrane in Dictyostelium cells.
Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P(2)-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P(2)-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P(2) during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P(2) disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P(2) metabolism regulates the dynamics and the function of Phg2.
Blanc
Cédric
C
IFR 128 BioSciences Lyon-Gerland, Institut de Biologie et Chimie des Protéines, UMR5086, CNRS/Université Lyon I, France.
Charette
Steve
S
Cherix
Nathalie
N
Lefkir
Yaya
Y
Cosson
Pierre
P
Letourneur
François
F
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004023:Dictyostelium / Q000166:cytology* / Q000201:enzymology* / Q000502:physiology; D049452:Green Fluorescent Proteins; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010587:Phagocytosis; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010873:Pinocytosis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases