The RNA-Binding Protein KSRP Promotes Decay of β-Catenin mRNA and Is Inactivated by PI3K-AKT Signaling (original) (raw)
Figure 6
KSRP Phosphorylation by AKT Promotes Its Interaction with 14-3-3 and Affects Its mRNA-Destabilizing Function
(A) In vitro RNA degradation assays using S100s from either mock αT3-1 (lanes 1–4) or αT3-1–shKSRP cells (lanes 5–28) preincubated with BSA (lanes 1–8), GST (lanes 17–20), KSRP (30 nM, lanes 9–12), AKT2-phosphorylated KSRP (30 nM, lanes 13–16), KH1-4 (S193A) (30 nM, lanes 21–24), or AKT2-phosphorylated KH1-4(S193A) (30 nM, lanes 25–28), respectively. Internally 32P-labeled and capped RNA substrates were added, and their decay was monitored as described above.
(B) Coimmunoprecipitation of FLAG-KSRP and endogenous 14-3-3 in 293T cells transiently transfected with FLAG-KSRP and either pCMV empty vector (mock 293T) or pCMV-myrAKT1 (293T-myrAKT1). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody.
(C) GST pull-down of endogenous 14-3-3 from total extracts of either mock αT3-1 (lanes 1–3) or αT3-1–myrAKT1 (lanes 4–11) cells using either control GST, GST–KH1-4, or the additional KSRP deletion mutants fused with GST (as indicated, see Figure 4B for a schematic representation of KSRP deletion mutants). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody.
(D) Coimmunoprecipitation of either FLAG-KSRP or FLAG-KSRP(S193A) and endogenous 14-3-3 in 293T cells transiently transfected with pCMV-myrAKT1 (293T-myrAKT1) and either FLAG-KSRP or FLAG-KSRP(S193A). Cell lysates were immunoprecipitated as indicated and analyzed by immunoblotting using anti–14-3-3 antibody.
(E) In vitro RNA degradation assays using S100s from αT3-1–shKSRP cells pre-incubated with either GST (lanes 1–4), KH1-4 (30 nM, lanes 5–8), AKT2-phosphorylated KH1-4 (30 nM, lanes 9–12), or AKT2-phosphorylated KH1-4 (30 nM) preincubated with difopein (50 nM) (lanes 13–16), respectively. Internally 32P-labeled and capped RNA substrates were added, and their decay monitored was as described above.
(F) GST pull-down of endogenous 14-3-3 from total extracts of αT3-1-myrAKT1 cells using either control GST, GST–KH1-4, or GST–KH1-4 preincubated with 50 nM difopein (as indicated). Proteins were analyzed by immunoblotting using anti–14-3-3 antibody.