Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3
Exploitation of KESTREL to identify NDRG family members as physiological substrates for SGK1 and GSK3
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DOI：
10.1042/bj20041057
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发表时间：
2004-12-15
影响因子：
4.1
通讯作者：
Cohen, P
中科院分区：
文献类型：
--
作者：
Murray, JT;Campbell, DG;Cohen, P
我们在兔骨骼肌提取物中检测到一种蛋白，它可以被SGK1（血清和糖皮质激素诱导的激酶1）快速磷酸化，但不能被蛋白激酶Balpha磷酸化，并将其鉴定为NDRG2 （N-myc下游调节基因2）。SGK1在体外磷酸化NDRG2的Thr（330）、Ser（332）和Thr（348）位点。所有三个残基在野生型小鼠的骨骼肌中都被磷酸化，但在不表达SGK1的小鼠中没有磷酸化。SGK1还磷酸化了相关的NDRG I亚型Thr（328）、Ser（330）和Thr(346)（相当于NDRG2的Thr（330）、Ser（332）和Thr(348)），以及Thr（356）和Thr（366）。残基Thr（346）、Thr（356）和Thr（366）位于相同的十肽序列GTRSRSHTSE中，在NDRG1中重复三次。这些苏氨酸在野生型小鼠的肝脏、肺、脾脏和骨骼肌的NDRG1中被磷酸化，但在SGK1（-/-）小鼠中没有磷酸化。使用小干扰RNA敲除HeLa细胞中的SGK1也抑制了NDRG1重复区苏氨酸残基的磷酸化。NDRG1被SGK1磷酸化后，转化为GSK3（糖原合成酶激酶3）的优良底物，可以磷酸化重复区域的Ser（342）、Ser（352）和Ser（362）。特异性GSK3抑制剂CT 99021在HeLa细胞中孵育，提高了NDRG1在HeLa细胞中的电泳迁移率，表明该蛋白在细胞中被GSK3磷酸化。我们的研究结果确定NDRG1和NDRG2是SGK1的生理底物，并证明SGK1磷酸化NDRG1会引发GSK3的磷酸化。
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 Balpha, and identified it as NDRG2 (N-myc downstream-regulated gene 2). SGK1 phosphorylated 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 NDRG I 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 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-clown 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 Ser(342), Ser(352) and Ser(362) 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.