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. 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.
Murray
James T
JT
MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK. j.t.c.murray@dundee.ac.uk
Campbell
David G
DG
Morrice
Nicholas
N
Auld
Gillian C
GC
Shpiro
Natalia
N
Marquez
Rodolpho
R
Peggie
Mark
M
Bain
Jenny
J
Bloomberg
Graham B
GB
Grahammer
Florian
F
Lang
Florian
F
Wulff
Peer
P
Kuhl
Dietmar
D
Cohen
Philip
P
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D018797:Cell Cycle Proteins / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D005810:Multigene Family; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein
D000906:Antibodies; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D017874:Immediate-Early Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D025521:Tumor Suppressor Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; D000099259:N-myc Downstream-Regulated Gene 1 Protein; C466228:NDRG2 protein, human; C069329:RPS6KA1 protein, human; C512964:Rps6ka1 protein, mouse