A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners

Zhiyong Ding, Jiyong Liang, Yiling Lu, Qinghua Yu, Zhou Songyang, Shiaw Yih Lin, Gordon Mills

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

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 a-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.

Original languageEnglish (US)
Pages (from-to)15014-15019
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number41
DOIs
StatePublished - Oct 10 2006
Externally publishedYes

Fingerprint

Retroviridae Proteins
Actinin
Proteins
Protein-Serine-Threonine Kinases
Mutagens
Small Interfering RNA
Exons
Down-Regulation
Fluorescence
Phosphorylation
Cell Proliferation
Membranes

Keywords

  • α-actinin 4
  • AKT1
  • Protein-protein interactions
  • Screen

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. / Ding, Zhiyong; Liang, Jiyong; Lu, Yiling; Yu, Qinghua; Songyang, Zhou; Lin, Shiaw Yih; Mills, Gordon.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 41, 10.10.2006, p. 15014-15019.

Research output: Contribution to journalArticle

Ding, Zhiyong ; Liang, Jiyong ; Lu, Yiling ; Yu, Qinghua ; Songyang, Zhou ; Lin, Shiaw Yih ; Mills, Gordon. / A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners. In: Proceedings of the National Academy of Sciences of the United States of America. 2006 ; Vol. 103, No. 41. pp. 15014-15019.
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