Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3.

Abstract

We detected a protein in rabbit skeletal muscle extracts that was phosphorylated rapidly by SGK1 (serum- and glucocorticoid- induced kinase 1), but not by protein kinase B ? , and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phos- phorylated NDRG2 at Thr 330 ,Ser 332 and Thr 348 in vitro . All three residues were phosphorylated in skeletal muscle from wild-type mice, but not from mice that do not express SGK1. SGK1 also phosphorylated the related NDRG1 isoform at Thr 328 ,Ser 330 and Thr 346 (equivalent to Thr 330 ,Ser 332 and Thr 348 of NDRG2), as well as Thr 356 and Thr 366 . Residues Thr 346 ,Thr 356 and Thr 366 are lo- cated within identical decapeptide sequences GTRSRSHTSE, re- peated three times in NDRG1. These threonines were phos- phorylated in NDRG1 in the liver, lung, spleen and skeletal muscle of wild-type mice, but not in SGK1 ? / ? mice. Knock-down of SGK1 in HeLa cells using small interfering RNA also sup- pressed phosphorylation of the threonine residues in the repeat region of NDRG1. The phosphorylation of NDRG1 by SGK1 transformed it into an excellent substrate for GSK3 (glycogen synthase kinase 3), which could then phosphorylate Ser 342 ,Ser 352 and Ser 362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electro- phoretic mobility of NDRG1 in HeLa cells, demonstrating that this protein is phosphorylated by GSK3 in cells. Our results identify NDRG1 and NDRG2 as physiological substrates for SGK1, and demonstrate that phosphorylation of NDRG1 by SGK1 primes it for phosphorylation by GSK3.