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

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 Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 in vitro...

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Published in:	Biochemical journal 2004-12, Vol.384 (Pt 3), p.477-488
Main Authors:	Murray, James T, Campbell, David G, Morrice, Nicholas, Auld, Gillian C, Shpiro, Natalia, Marquez, Rodolpho, Peggie, Mark, Bain, Jenny, Bloomberg, Graham B, Grahammer, Florian, Lang, Florian, Wulff, Peer, Kuhl, Dietmar, Cohen, Philip
Format:	Article
Language:	eng
Subjects:	Amino Acid Sequence Animals Antibodies - immunology Antibody Specificity Cell Cycle Proteins - classification Cell Cycle Proteins - genetics Cell Cycle Proteins - immunology Cell Cycle Proteins - metabolism Cell Line Enzyme Inhibitors - pharmacology Glycogen Synthase Kinase 3 - antagonists & inhibitors Glycogen Synthase Kinase 3 - metabolism HeLa Cells Humans Immediate-Early Proteins Intracellular Signaling Peptides and Proteins Mice Molecular Sequence Data Molecular Weight Multigene Family Nuclear Proteins - genetics Nuclear Proteins - metabolism Phosphatidylinositol 3-Kinases - antagonists & inhibitors Phosphatidylinositol 3-Kinases - metabolism Phosphorylation Phosphoserine - metabolism Protein-Serine-Threonine Kinases - genetics Protein-Serine-Threonine Kinases - metabolism Proteins - classification Proteins - genetics Proteins - immunology Proteins - metabolism Proto-Oncogene Proteins - metabolism Proto-Oncogene Proteins c-akt Rabbits Ribosomal Protein S6 Kinases, 90-kDa - genetics Ribosomal Protein S6 Kinases, 90-kDa - metabolism Substrate Specificity Tumor Suppressor Proteins
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Summary:	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 Ba, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated NDRG2 at Thr330, Ser332 and Thr348 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 Thr328, Ser330 and Thr346 (equivalent to Thr330, Ser332 and Thr348 of NDRG2), as well as Thr356 and Thr366. Residues Thr346, Thr356 and Thr366 are located within identical decapeptide sequences GTRSRSHTSE, repeated three times in NDRG1. These threonines were phosphorylated 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 suppressed 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 Ser342, Ser352 and Ser362 in the repeat region. Incubation of HeLa cells with the specific GSK3 inhibitor CT 99021 increased the electrophoretic 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.
ISSN:	0264-6021 1470-8728