Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis

Abstract

Endocytosis is a key cellular process, encompassing different entry routes and endocytic compartments. To what extent endocytosis is subjected to high-order regulation by the cellular signalling machinery remains unclear. Using high-throughput RNA interference and automated image analysis, we explored the function of human kinases in two principal types of endocytosis: clathrin- and caveolae/raft-mediated endocytosis. We monitored this through infection of vesicular stomatitis virus, simian virus 40 and transferrin trafficking, and also through cell proliferation and apoptosis assays. Here we show that a high number of kinases are involved in endocytosis, and that each endocytic route is regulated by a specific kinase subset. Notably, one group of kinases exerted opposite effects on the two endocytic routes, suggesting coordinate regulation. Our analysis demonstrates that signalling functions such as those controlling cell adhesion, growth and proliferation, are built into the machinery of endocytosis to a much higher degree than previously recognized.

p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity

“…However, the mechanisms responsible for the association of caveolin-1 and p38 MAPK and regulation of p38 MAPK activity remain unclear. As several kinases are inextricably linked to the endocytic machinery (18), the interaction of caveolin-1 and p38 MAPK may be important in bringing together components of the endocytic machinery and the signaling modules regulating endothelial cell proliferation and apoptosis.…”

“…This finding suggests that caveolin-1-mediated signaling (22,25) is involved in regulating p38 MAPK activation. Using high-throughput RNA interference and automated image analysis Pelkmans et al (18) showed that Src and MAP kinases regulate caveolae-mediated endocytosis. The findings of Pelkmans et al support our observation that activation of endocytosis regulates p38 MAPK activation and thus may control proliferation and survival of endothelial cells and the integrity of the endothelial monolayer.…”

Genomic RNAi screening in Drosophila S2 cells: what have we learned about host–pathogen interactions?

“…This will be facilitated by the availability of genome-wide libraries of transgenic flies that carry inverted repeat constructs against each gene allowing for directed RNAi in vivo in the animal, and the increasing specificity and robustness of siRNA methodologies. Mammalian RNAi screens are also beginning to contribute to our understanding of host-pathogen interactions [57][58][59], although technical limitations associated with siRNA and shRNA biology still make the Drosophila system ideal for RNAi screening. The comparisons between the host factors involved in pathogenesis in these disparate hosts will also inform the biology.…”

Sésame, ouvre-toi

“…Différents groupes ont ainsi commencé à étudier l'implication de protéines kinases dans le processus d'entrée du VHC. En effet, les protéines kinases sont des protéi-nes-clés de la régulation de nombreux processus cellulaires qui peuvent éga-lement contribuer à l'internalisation de différents virus [8]. La protéine kinase A (PKA) fut la première protéine kinase dont l'implication dans l'entrée du VHC a été identifiée [9].…”

“…Outre le rôle de la PKA dans l'entrée du VHC, il a été montré que l'infection par le VHC conduit à une augmentation de l'activité de cette kinase, ce qui pourrait favoriser la dissémination du virus [9]. Le développement de criblages fonctionnels basés sur l'ARN interférence a repré-senté une avancée considérable dans la découverte de nouvelles interactions entre des virus et leurs cellules hôtes [8]. Au cours de ces dernières années, de tels criblages ont été effectués afin de découvrir des facteurs fonctionnels de l'hôte indispensables pour l'entrée du VHC et pouvant constituer de nouvelles cibles pour de futurs antiviraux [10,11].…”