Skip to main content
. Author manuscript; available in PMC: 2014 May 9.
Published in final edited form as: Mol Cell. 2013 Apr 18;50(3):356–367. doi: 10.1016/j.molcel.2013.03.015

Figure 5. Akt3 Negatively Regulates Ago2-Mediated Cleavage of miRNA-targeted mRNAs.

Figure 5

(A) Akt phosphorylation of Ago2 reduces target mRNA cleavage activity of Ago2 in vitro. Purified Ago2 was incubated with purified Akt proteins in the presence of ATP. The target mRNA cleavage activity of the phosphorylated Ago2 was assayed by incubating the Ago2-Akt mixture with the antisense strand of CXCR4 siRNA and 5′-labeled CXCR4 substrate. Specific cleavage products are indicated with an arrow based on the ladder generated from partial digestion of input substrate by RNase T1 (T1). (B) Knockdown of Akt or MK2 results in increased siRNA-mediated target mRNA cleavage. HeLa-Luc cells with Akt knockdown were transfected with 1nM of an siRNA against the Luc ORF (D1) (NT; represents 50% Luc mRNA cleavage). RNA was analyzed for uncleaved Luc mRNA. (C) Overexpression of Akt results in decreased siRNA-mediated target mRNA cleavage. HeLa-Luc cells over-expressing Akt cDNAs were assayed in as (B). (D) Akt knockdown increases cleavage of endogenous miRNA-targeted mRNAs in HeLa-D8 cells. (E) Cytoplasmic levels of the miR target LYPD3 are subject to tight coordination with cellular Akt levels in A375 melanoma cells. Error bars indicate SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001. See also Figure S5.