Sequential activities of phosphoinositide 3-kinase, PKB/Akt, and Rab7 during macropinosome formation in Dictyostelium

Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostel ium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3- kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/A kt) are important in regulating completion of macropinocytosis. Although ac tin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent prot ein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) i ndicates that D3 phosphoinositides are enriched in the forming macropinocyt ic cup and remain associated with newly formed macropinosomes for <1 minute . A fusion protein, consisting of GFP fused to an F-actin binding domain, o verlaps with GFP-PHPKB in the timing of association with forming macropinos omes. Although macropinocytosis is reduced in cells expressing dominant neg ative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates onl y with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F- actin modulating proteins and vesicle trafficking proteins coordinately reg ulate the formation and maturation of macropinosomes.