Fig. 1.

Aly is an Akt substrate in vitro and in vivo. (A) Schematic diagram of Aly. Aly is a 257-aa protein containing an RNA recognition motif (RRM). Aly has several Akt phosphorylation motifs. (B) Aly N terminus and C terminus are phosphorylated by Akt. Except GST-ALY 107–181 (RRM domain), both C and N termini of Aly were phosphorylated by Akt (Upper). Coomassie brilliant blue staining of used GST recombinant proteins is shown in Lower. (C) S34 and T219 residues in Aly are Akt phosphorylation sites. S34A and T219A mutation disrupted Aly phosphorylation by Akt in vitro. (D) ALY phosphorylation by EGF in vivo. The transfected HEK293 cells were serum-starved and fed with ³²P-orthophosphate for 4 h. The cells were treated with 100 ng/ml EGF for 20 min after wortmannin (100 nM) or LY294002 (10 μM) treatment. GST-Aly was pulled down and separated on SDS/PAGE and analyzed by autoradiography or Western blotting. (E) T219 is the major phosphorylation site by Akt in vivo. GST-Aly wild type, S34A, and T219A construct transfected HEK293 cells were treated with NLS-Akt adenovirus. After 36 h of infection, cells were metabolically labeled. GST-Aly were pulled down and analyzed by autoradiography and immunoblotting. S34A mutation weakly decreased Aly phosphorylation, and T219A mutation substantially abolished Aly phosphorylation (first panel). The T219A mutant did not bind to Akt (second panel). (F) Ablation of Akt abolishes Aly phosphorylation. Knocking down of Akt eliminated Aly T219 phosphorylation, whereas overexpression of active myristoylated Akt enhanced Aly phosphorylation (top panel in Left). Aly T219 phosphorylation was selectively diminished in Akt1 but not Akt2 knockout MEF cells (top panel in Right).