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. Author manuscript; available in PMC: 2010 Sep 1.
Published in final edited form as: Nat Cell Biol. 2010 Feb 14;12(3):286–293. doi: 10.1038/ncb2029

Figure 4.

Figure 4

Phosphorylation of hnRNP E1 at serine-43 by TGFβ-mediated activation of Akt2 disrupts its binding to the BAT element and activates translation of Dab2 and ILEI. (a) IB analysis of immunoprecipitates derived from NMuMG WCLs with α-phospho-serine (p-ser) antibody (top panel) and α-hnRNP E1 antibody (bottom panel) to examine TGFβ-dependent hnRNP E1 phosphorylation. (b) IB analysis of WCLs to examine TGFβ-mediated Akt activation, revealed by phospho-Akt (pS473). (c) IB analysis of α-hnRNP E1 immunoprecipitates derived from NMuMG WCLs were probed with the phospho-Akt substrate antibody that recognizes the RXRXXpS/pT motif. (d) IB analysis of α-hnRNP E1 immunoprecipitates from LY294002 treated and untreated WCLs with α-phospho-serine (p-ser) antibody (top panel) and α-hnRNP E1 antibody (bottom panel) to confirm Akt as the kinase. (e) RNA affinity pull-down and IB analysis of cytosolic extracts from unstimulated and LY294002-treated cells to examine temporal association of hnRNP E1 and the BAT element. (f) Phosphorylated hnRNP E1 does not bind the BAT element. Increasing amounts of phosphorylated-GST-hnRNP E1 protein was subjected to pull-down with Dab2/BAT cRNA. The precipitates and the supernatants post pull-down were analyzed by IB. (j) Akt phosphorylates hnRNP E1 at Ser43. Activated kinases were recovered by anti-p-Akt (pSer473) or PAK1 immunoprecipitation and incubated with 5 µg of GST-hnRNP E1 or serine-43-alanine (S43A) mutant GST-hnRNP E1 in the presence of [γ-32P]-ATP. The kinase reaction products were detected by autoradiography. (g) IB analysis of WCLs derived from NMuMG cells post insulin and TGFβ stimulation to examine insulin and TGFβ-mediated Akt activation (top panel). IB analysis of immunoprecipitates derived from NMuMG WCLs with α-phospho-serine (p-ser) antibody (bottom panel) to examine insulin and TGFβ-dependent hnRNP E1 phosphorylation. (h) IB analysis of Akt1 and Akt2 immunoprecipitates derived from NMuMG WCLs with α-phospho-Akt (pS473) antibody to examine insulin and TGFβ-dependent isoform specific Akt activation. (i) TGFβ activated Akt2 specifically phosphorylates hnRNP E1. Activated Akt1 or Akt2 was recovered by anti-Akt1 or anti-Akt2 immunoprecipitation following TGFβ stimulation and incubated with 5 µg of GST-hnRNP E1 in the presence of [γ-32P]-ATP. Scans of (a), (b), (c), (d), (e), (f), (g), (h) and (i) are shown in SI Fig. S7.