Figure 2. The phosphoproteomes of Akt1, Akt2 and Akt3-expressing cells suggest functional differences between Akt isoforms. IWS1 phosphorylation by Akt1 and Akt3.

(A) Heat map showing the probability [-log(p value)] that a given canonical pathway may be regulated by Akt1, Akt2, Akt3 or Akt1/2/3. Analysis based on phosphorylation events with a robust z score ≥2 MAD.

(B) Probability that RNA trafficking and RNA posttranscriptional modifications may be regulated by Akt1, Akt2, or Akt3.

(C) Diagram of the sequential steps involved in RNA processing. Akt targets identified or confirmed by our screen are indicated.

(D) Sequence of the phosphorylated IWS1 peptide. The asterisks show the phosphorylated residues. The numbers show the median absolute deviation of the robust z scores.

(E) Conservation of the phosphorylated peptide and the adjacent sequence.

(F) Upper: Western blots of TKO lung fibroblasts and their Akt isoform-expressing derivatives were probed with anti-myc or anti-Hsp90 (loading control). Lower: The lung fibroblasts in the upper panel were transduced with the indicated constructs and their lysates were used for immunoprecipitation and immunoblotting with the indicated antibodies. Western blots of the same lysates were probed with anti-FLAG or anti-CREB, as indicated (G) HeLa cells transduced with lentiviral constructs of FLAG-IWS1 (wt or mutant) were treated with 5 μM MK2206 or DMSO for 2 hours. Anti-Flag-IWS1 immunoprecipitates were probed with the Akt phosphosubstrate antibody. Western blots of the same cell lysates were probed with the indicated antibodies.

(H) Left: In vitro kinase assays of myc-tagged Akt1, Akt2 or Akt3, using GST-IWS1wt (upper panel) and GSK3α/β (lower panel) recombinant proteins as substrates. Phosphorylation was detected with the Akt phosphosubstrate or the phospho-GSK3α/β (21/9) antibody. Right: In vitro kinase assay of myc-Akt1 using wt or mutant GST-IWS1 as substrates.