PROTEIN-KINASE B AKT AND RAB5 MEDIATE RAS ACTIVATION OF ENDOCYTOSIS/

Transient expression of oncogenic Ha-Ras (Ras:V12) stimulates endocyto sis. Using NIH3T3 cells expressing constitutively active protein kinas e B/akt (PKB/akt) or kinase-dead PKB/akt, we show that PKB/akt mediate s the stimulatory effect of Ras on endocytosis. Fluid phase endocytosi s of horseradish peroxidase in cells expressing the constitutively act ive form of PKB/akt was elevated and insensitive to phosphatidylinosit ol 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 a ctivated PKB/akt further increased endocytic rate. However, in cells e xpressing kinase-dead PKB/akt, endocytic rate was not affected by tran sient expression of Rab5:wt, Rab5:L79, on the other hand, increased en docytosis in cells expressing kinase-dead PKB/akt. Similar results wer e obtained using an in vitro endosome fusion reconstitution assay with cytosol prepared from cells expressing the activated PKB/akt or kinas e-dead PKB/akt. Both Rab5:wt and Rab5:L79 stimulated endosome fusion w hen 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.