Protein Kinase B/akt and Rab5 Mediate Ras Activation of Endocytosis

• Published in: The Journal of biological chemistry Vol. 273; no. 31; pp. 19367 - 19370
• Main Authors: Barbieri, M. Alejandro, Kohn, Aimee D., Roth, Richard A., Stahl, Philip D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-07-1998; American Society for Biochemistry and Molecular Biology
• Subjects: 3T3 Cells; Animals; Endocytosis - physiology; Gene Expression Regulation; GTP Phosphohydrolases - physiology; GTP-Binding Proteins - genetics; GTP-Binding Proteins - physiology; Horseradish Peroxidase - metabolism; Membrane Fusion - physiology; Mice; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-akt; rab5 GTP-Binding Proteins; ras Proteins - physiology; Signal Transduction - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.31.19367

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.