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. 2005 Dec;25(24):11019-29.
doi: 10.1128/MCB.25.24.11019-11029.2005.

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

Mario R Stegert  1 , Alexander Hergovich, Rastislav Tamaskovic, Samuel J Bichsel, Brian A Hemmings
Affiliations

Regulation of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3

Mario R Stegert et al. Mol Cell Biol. 2005 Dec.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Activation and hydrophobic motif phosphorylation of NDR2 by MST3. (A) Wild-type recombinant SHP-NDR2 was preincubated for 60 min with or without immunoprecipitated HA-MST3 or HA-MST3KR from untreated and OA-treated (+) HEK293F cells. The activity of SHP-NDR2 was determined using the NDR kinase substrate peptide. The results shown are means ± standard deviations of assays carried out in duplicate and are representative of two independent experiments. Samples from each preincubation were also analyzed for phosphorylation by Western blotting using anti-P-T442 and anti-P-S282 antibodies. HA-MST3wt and HA-MST3KR were quantified by Western blot analysis using the α-HA 12CA5 monoclonal. (B) Phosphorylation of kinase-dead NDR2 by MST3. Kinase-dead NDR2K119A was incubated for 60 min with or without immunoprecipitated HA-MST3 or HA-MST3KR from untreated and OA-treated (+) HEK293F cells. Samples were analyzed by Western blotting as for panel A.
FIG. 2.
FIG. 2.
Analysis of NDR2 activation and hydrophobic motif phosphorylation by MST3 and MOB1A. (A) Activation and phosphorylation of wild-type NDR2. NDR2 SHP-NDR2 was incubated with GST-MOB1A (▪), GST-MST3 from OA-stimulated HEK293F cells (▴), both (•) or alone (○) under our standard NDR kinase assay conditions. (B) Activation and phosphorylation of kinase-dead and MOB-binding-deficient NDR2. SHP-NDR2 (○), SHP-NDR2/GST-MOB1A (▪), SHP-NDR2/MOB1A/MST3+ (•), SHP-NDR2K119A/MOB1A/MST3+ (▴), SHP-NDR2Y32A (□), and SHP-NDR2Y32A/MOB1A/MST3+ (Δ) were incubated under standard NDR kinase assay conditions. (C) Activation and phosphorylation of the NDR2-AIS mutant. SHP-NDR2 (○), SHP-NDR2-AIS (□), SHP-NDR2-AIS/GST-MOB1A (▴), SHP-NDR2-AIS/MST3+ (▪), and SHP-NDR2-AIS/GST-MOB1A/MST3+ (•) were incubated under standard NDR kinase assay conditions. At the time points indicated, NDR kinase activity was assayed with the NDR substrate peptide; results are expressed as specific activity. Results shown are means ± standard deviations of assays carried out in duplicate and are representative of two independent experiments. Error bars are only shown when larger than the size of the symbols. NDR hydrophobic motif phosphorylation was determined at each time point by Western blotting using the anti-P-T442 antibody.
FIG. 3.
FIG. 3.
NDR activation and phosphorylation by MST3 in vivo. (A) Effect of cotransfection of MST3 on NDR2 phosphorylation. COS-7 cells were cotransfected with myc-MST3, kinase-dead myc-MST3KR, and HA-NDR2. OA-treated cells transfected with HA-NDR2 were used as a control for NDR2 phosphorylation. All proteins migrated at the expected molecular mass. Cell lysates were immunoblotted with anti-P-T442 (very long exposure), anti-P-S282, anti-HA, and anti-myc. (B) Inhibition of NDR2 activation and hydrophobic motif phosphorylation by MST3KR. COS-7 cells expressing HA-NDR2, wild-type myc-MST3, and myc-MST3KR in various combinations were treated for 1 h with 1 μM OA or with solvent alone. HA-tagged NDR kinase variants were then immunoprecipitated (100 μg of cell-free protein extracts) with anti-HA 12CA5 monoclonal antibody and assayed for kinase activity using the NDR peptide substrate. Bars represent the means ± standard deviations of triplicate immunoprecipitates and are representative of two independent experiments. All cell lysates were immunoblotted with anti-P-T442 (normal exposure times), anti-P-S282, anti-HA 12CA5, and anti-myc.
FIG. 4.
FIG. 4.
Activity of NDR2-MST3 fusion proteins. HA-tagged variants of NDR2, NDR2-MST3, NDR2KD-MST3, NDR2-MST3KR, and NDR2-PIF in the pcDNA3.1 vector were expressed in HEK293F cells. The NDR kinase variants were then immunoprecipitated (100 μg of cell-free protein extracts) with anti-HA 12CA5 monoclonal antibody, and kinase activity was assayed using the NDR peptide substrate. Bars represent the means ± standard deviations of triplicate immunoprecipitates and are representative of two independent experiments. Additionally, all immunoprecipitates were subjected to SDS-PAGE and Western blotting using anti-HA 12CA5, anti-P-S282, and anti-P-T442 antibodies. All NDR variants migrated at the expected molecular weight.
FIG. 5.
FIG. 5.
Phosphorylation of endogenous NDR by endogenous MST3. (A) Myc-C1-MOB1A was expressed in HEK293F cells transfected with either pcDNA3.1-HA-MST3, pcDNA3.1-HA-MST3KR, or pTER-shMST3. Two days after transfection, the cells were starved for 24 h and then stimulated for 15 min with TPA (100 ng/ml) prior to harvesting. All cell lysates were subjected to SDS-PAGE and immunoblotted with anti-P-T442, anti-NDR-NT, anti-MST3, anti-myc, and an anti-α-tubulin control. (B) Phosphorylation and activation of NDR2 by endogenous MST3. HA-NDR2 was expressed in HEK293F cells and transfected with pcDNA3.1 myc-C1-MOB1A and/or pTER-shMST3. Two days after transfection, the cells were starved for 24 h and subsequently stimulated for 10 min with TPA (100 ng/ml) prior to harvesting. HA-NDR2 was immunoprecipitated with anti-HA 12CA5 monoclonal antibody and assayed for kinase activity using the NDR peptide substrate. Bars represent the means ± standard deviations of duplicate immunoprecipitates and are representative of two independent experiments. All cell lysates were subjected to SDS-PAGE and immunoblotted with anti-P-T442, anti-HA, anti-MST3, anti-myc, and the anti-α-tubulin control.
FIG. 6.
FIG. 6.
Activation and phosphorylation of MOB1-binding-deficient NDR2. HEK293F cells were transfected with HA-tagged NDR2 and NDR2Y32A and variants and stimulated for 1 h with 1 μM OA or solvent prior to harvesting. The NDR kinase variants were then immunoprecipitated (100 μg of cell-free protein) with anti-HA 12CA5 monoclonal antibody and assayed for kinase activity using the NDR peptide substrate. Bars represent the means ± standard deviations of duplicate immunoprecipitates and are representative of two independent experiments. Additionally, all lysates were subjected to SDS-PAGE and immunoblotted using anti-P-T442, anti-P-S282, and anti-HA 12CA5 antibodies.
FIG. 7.
FIG. 7.
Colocalization of NDR2 and MST3 in COS-7 cells. (A) Localization of MST3. COS-7 cells expressing GFP-MST3 (upper panels), HA-MST3wt (middle panel), or HA-MST3KR (lower panels) were processed for immunofluorescence using either no antibody (upper right) or anti-HA Y11 (lower right). Anti-HA antibody was visualized using anti-rabbit antibody-FITC (green) and DNA stained with 1 μM TO-PRO-3 iodide (Molecular Probes Inc.) (blue; left panels). (B) Colocalization of NDR2 and MST3. COS-7 cells expressing HA-MST3 and myc-NDR2 were processed for immunofluorescence using anti-HA Y11 and anti-myc 9E10. Anti-HA Y11 antibody was visualized using anti-rabbit antibody-FITC (green), and anti-myc antibody was visualized using anti-mouse antibody-Texas Red (red). Representative pictures of HA-MST3 and myc-NDR2 localization are shown.
FIG. 8.
FIG. 8.
Fractionation of HA-NDR and MST3 after membrane recruitment of myc-C1-MOB1A. HEK293F cells were transfected with the HA-NDR2 and myc-C1-MOB1A constructs indicated and subjected to S100/P100 fractionation (S, cytoplasm; P, membrane), before immunoblotting with anti-P-T444, anti-HA 12CA5, anti-MST3, and anti-myc antibodies.
FIG. 9.
FIG. 9.
NDR promotes cleavage of MST3 in COS-7 cells. (A) Induction of MST3 cleavage by overexpression of catalytically active NDR2. Lysates of COS-7 cells transfected as indicated with myc-MST3KR, HA-NDR2, HA-NDR2KD, and HA-NDR2-PIF were harvested after 36 h and subjected to Western blotting using anti-HA and anti-myc antibodies. (B) Localization of NDR2-MST3 fusion proteins. COS-7 cells expressing HA-NDR-MST3 (upper panel) or HA-NDR2KD-MST3 (lower panel) were processed for immunofluorescence 24 h after transfection using anti-HA Y11 (right panels). Anti-HA antibody was visualized using anti-rabbit antibody-FITC (green); DNA stained with DAPI (blue; left panels). Representative pictures of HA-NDR2-MST3 and HA-NDR2KD-MST3 localization are shown.
FIG. 10.
FIG. 10.
Model of NDR protein kinase activation by multisite phosphorylation. MOB as well as activated MST is required for full phosphorylation and activation of NDR. MOB binding stimulates auto- and transphosphorylation of NDR by releasing autoinhibition, whereas MST3 phosphorylation at the hydrophobic motif results in an active conformation of the kinase. The kinase complex is negatively regulated by PP2A or PP2A-related phosphatase. Activated NDR kinase in turn promotes cleavage of MST3.
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