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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.

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Figure 1: High-throughput, genome-wide analysis of human kinases involved in infectious virus entry (VSV or SV40), cell proliferation and cell death.
Figure 2: Phenotypic profiling.
Figure 3: Hierarchical two-step clustering of RIIs and phenotypic profiles.
Figure 4: Effects of signalling pathways on endocytosis.
Figure 5: Phosphorylation of Cav1 and p38 MAPK.

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Acknowledgements

We thank F. Halley and A. Kroenke for experimental assistance, J. Rose for rVSV, and G. Kochs for hybridomas expressing anti-Tag antibodies. I. Baines (Biopolis Dresden Consultants GmbH), D. Dorris (Ambion Inc.), C. Echeverri (Cenix Bioscience GmbH), R. Günther (Evotec Technologies GmbH) and M. Athelogou (Definiens AG) are acknowledged for making HT RNAi technologies and high-content, automated imaging and analysis technologies available. We thank F. Buchholz, C.-P. Heisenberg, M. Miaczynska, D. Meder, A. Schenck, A. Helenius and K. Simons for discussions and critical reading of the manuscript. L.P. would like to thank A. Helenius for continuous support. This work was supported by grants from the Max Planck Society ‘RNAi interference’ initiative and the Bundesministerium für Bildung und Forschung. L.P. is a Marie Curie fellow.Author Contributions L.P. and M.Z. conceived the experimental idea. L.P. carried out the experiments with help from E.F., H.G. and M.H. Data analysis was carried out by L.P., B.H. and M.Z. L.P., E.K. and M.Z. together with I. Baines conceived and set up the HT-TDS and financed the project. L.P. and M.Z. wrote the manuscript.

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Correspondence to Marino Zerial.

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Supplementary information

Supplementary Figure S1

HT genome-wide analysis of human genes in infectious virus entry. (JPG 351 kb)

Supplementary Figure S2

Hierarchical phenotype clustering of kinases in functional groups. (JPG 596 kb)

Supplementary Table S1

Screening results from all 590 kinases (XLS 167 kb)

Supplementary Table S2

Screening results from 50 random genes (XLS 28 kb)

Supplementary Table S3

Hierarchical clustering of RIIs and phenotypic profiles. (XLS 943 kb)

Supplementary Legends

Legends to accompany the above Supplementary Tables and Supplementary Figures. (DOC 41 kb)

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Pelkmans, L., Fava, E., Grabner, H. et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78–86 (2005). https://doi.org/10.1038/nature03571

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