Protein kinase B/akt and rab5 mediate Ras activation of endocytosis.
Transient expression of oncogenic Ha-Ras (Ras:V12) stimulates endocytosis. Using NIH3T3 cells expressing constitutively active protein kinase B/akt (PKB/akt) or kinase-dead PKB/akt, we show that PKB/akt mediates the stimulatory effect of Ras on endocytosis. Fluid phase endocytosis of horseradish peroxidase in cells expressing the constitutively active form of PKB/akt was elevated and insensitive to phosphatidylinositol 3-kinase inhibitors. However, expression of dominant negative Rab5:N34 blocked endocytosis in cells expressing the constitutively active form of PKB/akt. Transient expression of either Rab5:wt or Rab5:L79, a GTPase deficient mutant of Rab5, in cells expressing constitutively activated PKB/akt further increased endocytic rate. However, in cells expressing kinase-dead PKB/akt, endocytic rate was not affected by transient expression of Rab5:wt. Rab5:L79, on the other hand, increased endocytosis in cells expressing kinase-dead PKB/akt. Similar results were obtained using an in vitro endosome fusion reconstitution assay with cytosol prepared from cells expressing the activated PKB/akt or kinase-dead PKB/akt. Both Rab5:wt and Rab5:L79 stimulated endosome fusion when assayed in cytosol containing the activated PKB/akt, whereas only Rab5:L79 activated fusion when the assay utilized cytosol from kinase-dead expressing cells. We conclude that Ras activation of endocytosis requires both PKB/akt and Rab5 and that active kinase is required for activation Rab5.
Barbieri
M A
MA
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Kohn
A D
AD
Roth
R A
RA
Stahl
P D
PD
D016475:3T3 Cells; D000818:Animals; D004705:Endocytosis / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000502:physiology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005786:Gene Expression Regulation; D006735:Horseradish Peroxidase / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology; D051379:Mice; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000502:physiology; D020696:rab5 GTP-Binding Proteins; D018631:ras Proteins / Q000502:physiology*
D011518:Proto-Oncogene Proteins; D006735:Horseradish Peroxidase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D018631:ras Proteins