Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3
Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3
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DOI：
10.1128/mcb.25.24.11019-11029.2005
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发表时间：
2005-12-01
影响因子：
5.3
通讯作者：
Hemmings, BA
中科院分区：
文献类型：
--
作者：
Stegert, MR;Hergovich, A;Hemmings, BA
NDR蛋白激酶参与细胞周期进程和形态的调控。NDR1/NDR2蛋白激酶通过激活环磷酸化位点Ser281/Ser282和疏水基序磷酸化位点Thr444/Thr442磷酸化而激活。NDR的自磷酸化负责Ser281/Ser282的磷酸化，而Thr444/Thr442则由上游激酶靶向。我们发现哺乳动物ste20样蛋白激酶MST3能够磷酸化NDR蛋白激酶Thr444/Thr442位点。在体外，MST3选择性磷酸化NDR2的Thr442，导致NDR活性的10倍刺激。MOB1A （Mps one binder 1A）蛋白的活性进一步提高，形成了一个完全活跃的激酶。在体内，冈田酸刺激后Thr442的磷酸化被MST3KR （MST3的激酶死亡突变体）有效抑制。在HEK293F细胞中，使用短发夹结构敲除MST3可消除NDR的Thr442疏水基序磷酸化。我们得出结论，NDR的激活是一个多步骤的过程，包括MST3对疏水基序位点Thr444/2的磷酸化、Ser281/2的自磷酸化和MOB1A的结合。
NDR protein kinases are involved in the regulation of cell cycle progression and morphology. NDR1/NDR2 protein kinase is activated by phosphorylation on the activation loop phosphorylation site Ser281/Ser282 and the hydrophobic motif phosphorylation site Thr444/Thr442. Autophosphorylation of NDR is responsible for phosphorylation on Ser281/Ser282, whereas Thr444/Thr442 is targeted by an upstream kinase. Here we show that MST3, a mammalian Ste20-like protein kinase, is able to phosphorylate NDR protein kinase at Thr444/Thr442. In vitro, MST3 selectively phosphorylated Thr442 of NDR2, resulting in a 10-fold stimulation of NDR activity. MOB1A (Mps one binder 1A) protein further increased the activity, leading to a fully active kinase. In vivo, Thr442 phosphorylation after okadaic acid stimulation was potently inhibited by MST3KR, a kinase-dead mutant of MST3. Knockdown of MST3 using short hairpin constructs abolished Thr442 hydrophobic motif phosphorylation of NDR in HEK293F cells. We conclude that activation of NDR is a multistep process involving phosphorylation of the hydrophobic motif site Thr444/2 by MST3, autophosphorylation of Ser281/2, and binding of MOB1A.