Peroxisomal targeting as a tool for assaying potein-protein interactions in the living cell: cytokine-independent survival kinase (CISK) binds PDK-1 in vivo in a phosphorylation-dependent manner.
Current methods to detect protein-protein interactions are either laborious to implement or not adaptable for mammalian systems or in vitro methods. By adding a peroxisomal targeting signal (PTS) onto one protein, binding partners lacking a targeting signal were co-transported into the peroxisomes in a "piggy-back" fashion, as visualized by confocal and electron microscopy. A fragment of colicin E2 and its tightly interacting immunity protein, ImmE2, were both expressed in the cytosol. When either one contained a PTS tag, both proteins were co-localized in the peroxisomes. The cytokine-independent survival kinase (CISK) containing a PTS tag was not efficiently targeted to the peroxisomes unless the Phox homology (PX) domain, attaching the protein to endosomal membranes, was removed. However, PTS-tagged CISK with deleted PX domain was able to direct 3-phosphoinositide-dependent protein kinase-1 (PDK-1) into the peroxisomes. This demonstrates that the two proteins interact in vivo. Mutating Ser486, which is phosphorylated in activated CISK, to Ala prevented the interaction, indicating that CISK and PDK-1 interact in a phosphorylation-dependent manner. The method therefore allows assessment of protein-protein interactions that depend on post-translational modifications that are cell-specific or dependent on the physiological state of the cell.
Nilsen
Trine
T
Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway.
Slagsvold
Thomas
T
Skjerpen
Camilla Skiple
CS
Brech
Andreas
A
Stenmark
Harald
H
Olsnes
Sjur
S
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D003087:Colicins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D020675:Peroxisomes / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099184:Serum-Glucocorticoid Regulated Kinases
D064413:3-Phosphoinositide-Dependent Protein Kinases; D001426:Bacterial Proteins; D003087:Colicins; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C007104:colicin immunity proteins; C578562:PDPK1 protein, human