FIGURE 4.

Phosphorylation of Ser-825 is required for insulin to induce Sort1 in AML12 cells. A, sequence illustrates phosphorylated serine residues Ser-793 and Ser-825 (S) identified in human Sort1 protein by LC-MS/MS and the conserved DXXLL motifs among species. B, AML12 cells were transfected with wild type and mutant human FLAG-Sort1 plasmids, as indicated. Cells were serum-starved for 16 h and treated with 100 nm insulin for 8 h. Ectopically expressed Sort1 was detected with anti-FLAG antibody in immunoblot. C, serum-starved (16 h) AML12 cells were pretreated with 15 μm TBCA for 1 h followed by 100 nm insulin treatment as indicated. Sort1 protein was measured by immunoblot. D, AML12 cells transfected with wild type and mutant human FLAG-Sort1 plasmids were treated with 100 μg/ml cycloheximide (CHX). FLAG-Sort1 protein was measured by immunoblot. P-AKT, phospho-AKT.