Akt-mediated phosphorylation of argonaute 2 downregulates cleavage and upregulates translational repression of MicroRNA targets

Abstract

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was used to discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

Figure 1. Primary Screening and Reconfirmation of Novel Regulators of siRNA-Mediated Gene Silencing

(A) Schematic diagram of the primary screen to identify novel regulators of RNAi. siRNA-mediated knockdown of only those genes acting as positive regulators of RNAi elicited luciferase de-repression. (B) A histogram showing the results of the primary RNAi genomics screen. (C) Reconfirmation of the main hit, Akt3, relative to known miRNA effector proteins. (D) Confirmation that all three Akt isoforms positively regulate RNAi. HeLa-Luc-D1 cells were transduced with lentiviruses expressing shRNAs to Akt1, Akt2, Akt3, MAPKAPK2 (MK2) or a non-targeting control (NT) and assayed for FLuc de-repression. Error bars indicate SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001. See also Figure S1.

Figure 2. Akt3 Interacts With and Phosphorylates Ago2 at S387

(A) Akt and Ago2 physically interact in cells. His-Akt-expressing lysates and IP extracts were immunoblotted for Ago2, His or β-actin. Relative amounts of Ago2 protein are indicated using His-Vector as a reference for input and His-Akt1 as a reference for IP. (B) Kinase reaction showing Akt3 phosphorylating Ago2 at S387. Purified His-tagged Akt1, Akt2, Akt3 or a kinase-impaired Akt3 mutant (T305A) were incubated with purified WT or S387A Ago2 in the presence of ³²P-γ-ATP, and resolved on a denaturing gel. Histone H2B was used as a positive control for Akt phosphorylation. (C) The MS/MS spectrum of the Ago2 SApSFNTDPYVR phosphopeptide from the kinase reaction in (B). Prominent y- and b- ions are shown and [M+2H-H₃PO₄]²⁺ = 620. (D) Akt3 binds Ago2 at the S387 site. HeLa cells were transiently transfected with FLAG-tagged Ago2 constructs and lysates and IP extracts were immunoblotted for Akt3, FLAG or β-actin. Relative amounts of Akt3 protein are indicated using FLAG vector as a reference for input and FLAG-Ago2 (WT) as a reference for IP. (E) Knockdown of Akt3 reduces phosphorylation of Ago2 S387. Akt knockdown HeLa cell lysates and IP fractions were immunoblotted for Ago2-pS387, FLAG or β-actin. Relative amounts of pS387 protein are indicated using siNT as a reference for input and IP. The experiment was repeated four times and the average pS387 levels along with standard deviations as error bars are shown in the inset graph. See also Figure S2.

Figure 3. Knockdown of Akt Reduces Ago2-mediated mRNA Repression

(A) Ago2 S387 phosphorylation positively affects CXCR4 siRNA-mediated repression. HeLa cells harboring endogenous Ago2 knockdown and expressing ectopic Ago2 mutants were assayed for CXCR4 siRNA targeting to the FL6X mRNA repression reporter. (B) Knockdown of Akt3 significantly disrupts miR-21-mediated repression in HeLa-D8 cells. (C) Phospho-dominant Ago2-S387E mutant partially rescues the Akt knockdown phenotype. HeLa-D8 cells were co-transfected with siRNAs targeting Akt3 and Ago2 and Ago2 constructs. (D) Knockdown of Akt3 increases PTEN protein but not pten mRNA levels in HeLa cells. Bars indicate pten mRNA levels (normalized to GAPDH) and triangles represent pten protein levels relative to the NT control sample. (E) Knockdown of Akt3 up-regulates translation of miR-21 targets PTEN and PDCD4 in HEK293T cells measured by ³⁵S methionine incorporation. Relative amounts of PTEN and PDCD4 proteins are indicated using siNT as a reference for input and IP. Error bars indicate SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001. See also Figure S3.

Figure 4. Akt-mediated Phosphorylation of Ago2 S387 Promotes Interactions of Ago2 with GW182 and P-bodies

(A) Akt positively regulates Ago2-GW182 interactions. FLAG-Ago2 and GW812 were co-immunoprecipitated in Akt knockdown HeLa cells. Relative amounts of GW182 protein are indicated using siNT as a reference for input and IP. (B) S387 phosphorylation status guides Ago2-GW182 interactions. Lysates and IP fractions from HeLa cells expressing FLAG-Ago2 constructs were analyzed for GW182 interaction and pS387 levels. Relative amounts of GW182 protein are indicated using FLAG-Ago2 (WT) as a reference. (C) HeLa-EGFP-Ago2 cells show disrupted Ago2/GW182 co-localization upon knockdown of Akt3. Representative HeLa-EGFP-Ago2 cells stained with immunofluorescent antibodies to GW182, Ago2-pS387 and Hoechst; EGFP represents endogenous Ago2. White arrows indicate Ago2 not co-localized with GW182 bodies. (D) Co-localization quantifications from (C). (E) Knockdown of Akt3 decreases total P-body formation in cells. Quantitation of phospho-Ago2 and GW182 foci in cells from (C); foci were enumerated on a confocal microscope with n = 10 cells. Error bars indicate SD. *P ≤ .05, **P ≤ .01, ***P ≤ .001. See also Figure S4.

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

(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.

Figure 6. Akt3-Mediated Phosphorylation of Ago2 is a Molecular Switch Between Target mRNA Cleavage and Translational Repression

Ago2 is capable of cleaving miRNA-targeted mRNAs or directing them for translational repression, which is dependent on its S387 phosphorylation status. This is dictated by Akt3, MK2 or to a lesser extent Akt1 or Akt2. Ago2 unphosphorylated at S387 maintains high endonucleolytic cleavage activity which results in suppressed expression of developmental genes. Ago2 phosphorylated at S387 switches to high translational repression which results in suppressed expression of tumor suppressor genes.