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 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.

Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death

“…However, our results indicated that cisplatininduced cell death slightly decreased in NDRG2-silenced SNU-620 cells. Because NDRG2 possesses potential phosphorylation sites (Murray et al, 2004;Chen et al, 2007) and involved signaling cascades (Burchfield et al, 2004), NDRG2 inhibition may disrupt cisplatin-mediated signaling.…”

“…NDRG2 contains several phosphorylation sites at the C-terminus and its phosphorylation is mediated through insulin-stimulated AKT activation (Burchfield et al, 2004). NDRG2 phosphorylation involves Thr330, Ser332 and Thr348 in skeletal muscle cells (Murray et al, 2004), and Thr334 in rat hippocampus tissue (Chen et al, 2007). Most recently, it has been shown that NDRG2 is also down-regulated by Myc via transcriptional repression and interact with MSP58, a 58-kDa microspherule protein (Zhang et al, 2006.…”

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by β-catenin activity

“…Furthermore, we used the selective GSK3β-inhibitor CHIR99021, 38 which stabilizes β-catenin. 39 In organotypic culture, CHIR99021 was tolerated and resulted in a visible expansion of crypt domains after 2 days, probably due to the induction of ectopic β-catenin activity that is normally restricted to the crypt base (Figure 6b).…”

Overexpression of NDRG1 is an indicator of poor prognosis in hepatocellular carcinoma

“…Possible mutations at both the transcriptional and translational levels may account for these discrepancies in its roles in cancer. Specifically, NDRG1 is reported to be a multiphosphorylated protein; 42,43 the role of phosphorylation is unknown but speculated to be related to the multitude of physiological functions of NDRG1.…”