A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners.
We developed a retrovirus-based protein-fragment complementation assay (RePCA) screen to identify protein-protein interactions in mammalian cells. In RePCA, bait protein is fused to one fragment of a rationally dissected fluorescent protein, such as GFP, intensely fluorescent protein, or red fluorescent protein. The second, complementary fragment of the fluorescent protein is fused to an endogenous protein by in-frame exon traps in the enhanced retroviral mutagen vector. An interaction between bait and host protein (prey) places the two parts of the fluorescent molecule in proximity, resulting in reconstitution of fluorescence. By using RePCA, we identified a series of 24 potential interaction partners or substrates of the serine/threonine protein kinase AKT1. We confirm that alpha-actinin 4 (ACTN4) interacts physically and functionally with AKT1. siRNA-mediated ACTN4 silencing down-regulates AKT phosphorylation, blocks AKT translocation to the membrane, increases p27(Kip1) levels, and inhibits cell proliferation. Thus, ACTN4 is a critical regulator of AKT1 localization and function.
Ding
Zhiyong
Z
Department of Molecular Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 950, Houston, TX 77030, USA.
Liang
Jiyong
J
Lu
Yiling
Y
Yu
Qinghua
Q
Songyang
Zhou
Z
Lin
Shiaw-Yih
SY
Mills
Gordon B
GB
D000185:Actinin / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics*
C111453:ACTN4 protein, human; D008840:Microfilament Proteins; D000185:Actinin; C494918:AKT1 protein, human; D051057:Proto-Oncogene Proteins c-akt