Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3.
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.
Stegert
Mario R
MR
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Hergovich
Alexander
A
Tamaskovic
Rastislav
R
Bichsel
Samuel J
SJ
Hemmings
Brian A
BA
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry; D004789:Enzyme Activation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047908:Intracellular Signaling Peptides and Proteins; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D000099317:Hippo Kinases
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D019319:Okadaic Acid; D017346:Protein Serine-Threonine Kinases; D013912:Threonine; C496255:MOB1B protein, human; C109163:STK24 protein, human; D000099317:Hippo Kinases; C117090:MAP4K4 protein, human; C496014:STK38L protein, human