A cDNA library functional screening strategy based on fluorescent protein complementation assays to identify novel components of signaling pathways.
Progress towards a deeper understanding of cellular biochemical networks demands the development of methods to both identify and validate component proteins of these networks. Here, we describe a cDNA library screening strategy that achieves these aims, based on a protein-fragment complementation assay (PCA) using green fluorescent protein (GFP) as a reporter. The strategy combines a simple cell-based cDNA-screening approach (interactions of a "bait" protein of interest with "prey" cDNA products) with specific functional assays that use the same system and provide initial validation of the cDNA products as being biologically relevant. We applied this strategy to identify novel interacting partners of the protein kinase PKB/Akt. This method provides very general means of identifying and validating genes involved in any cellular process and is particularly designed for identifying enzyme substrates or regulatory proteins for which the enzyme specificity can only be defined by their interactions with other proteins in cells in which the proteins are normally expressed.
Remy
Ingrid
I
Département de Biochimie, Université de Montréal, CP 6128, Succursale centre-ville, Montréal, Que, Canada H3C 3J7.
Michnick
Stephen W
SW
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019556:COS Cells; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic / Q000235:genetics; D020798:Two-Hybrid System Techniques; D000077191:Wortmannin
D000730:Androstadienes; D051017:Apoptosis Regulatory Proteins; C109729:Fts protein, mouse; D007328:Insulin; D010446:Peptide Fragments; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011506:Proteins; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin