16] Measurement of Rab5 protein kinase B/akt and regulation of Ras-activated endocytosis

Abstract

This chapter describes the methodology to express the small GTPases, Rab5 and H-Ras, and the regulatory kinase, PKB/akt, using the Sindbis virus transient expression system for biochemical and morphological analysis of the endocytic pathway. Functional data are also provided to assess the physiologic relevance of the GTP-binding proteins. Because preparation of recombinant virus stocks and the expression Sindbis virus encoding cDNAs share identical procedures, the method is described only for the Rab5 recombinant Sindbis virus. In this method, cDNAs of Rab5 are subcloned into the unique XbaI site of the Sindbis vector. The plasmid is then linearized by cutting with XhoI. This cut DNA is used as template for the in vitro transcription. The resulting RNA transcripts are used for transfection of confluent BHK-21 cell monolayers using a lipofectin-mediated procedure. It is suggested that cells expressing Rab GTPases (H-Ras or PKB/akt) can be analyzed in a number of ways, including immunofluorescence confocal microscopy, electron microscopy, Western blot analysis, metabolic labeling, and measurements of virus processing during intracellular transport.