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Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1βMYPT1 phosphatase complex and the SCFβTrCP E3 ubiquitin ligase.

Banerjee S, Zagórska A, Deak M, Campbell DG, Prescott AR, Alessi DR - Biochem. J. (2014)

Bottom Line: Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine.We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors.Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.

View Article: PubMed Central - PubMed

Affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.

ABSTRACT
NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFβTrCP (Skp, Cullin and F-boxβTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to βTrCP, the substrate-recognition subunit of the SCFβTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2-M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1βMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1βMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.

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PLK1 interacts with NUAK1 at the CDK priming Ser445 site and phosphorylates NUAK1 at Ser476 and Ser480 and targets it for phosphorylation-mediated degradation(A) U2OS cells stably expressing HA–NUAK1 were treated with the indicated inhibitors for 4 h with or without 50 nM calyculin A treatment. Cell lysates were subjected to immunoblotting with the indicated antibodies. The inhibitors were used at 1 μM final except for MLN-4924 (3 μM), BI605906 (5 μM), WZ4003 (10 μM) and D4476 (30 μM). (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without the indicated concentrations of BI2536 and GSK461364 over the indicated periods of time prior to lysis. Immunoblotting was carried out to detect NUAK1 levels in the immunoprecipitates. Pre-immune IgG was used as a control. (C) U2OS cells stably expressing NUAK1 WT were treated with the indicated inhibitors for 1 h. HA–NUAK1 was immunoprecipitated and analysed by immunoblotting with the indicated antibodies. (D) HA–NUAK1 with or without BI2536 (1 μM) treatment was immunoprecipitated (IP) from U2OS Flp/In cells stably expressing HA–NUAK. XIC analysis of the Ser476- and Ser480-containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was detected in both samples. In the BI253-treated cells, the m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was found to possess a ~3-fold-higher signal intensity than the control immunoprecipitate. (E) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or the indicated mutants. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (F) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or S445A mutant. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (G) HEK-293 cells transiently overexpressing NUAK1 WT or the S445A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with the indicated antibodies.
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Figure 4: PLK1 interacts with NUAK1 at the CDK priming Ser445 site and phosphorylates NUAK1 at Ser476 and Ser480 and targets it for phosphorylation-mediated degradation(A) U2OS cells stably expressing HA–NUAK1 were treated with the indicated inhibitors for 4 h with or without 50 nM calyculin A treatment. Cell lysates were subjected to immunoblotting with the indicated antibodies. The inhibitors were used at 1 μM final except for MLN-4924 (3 μM), BI605906 (5 μM), WZ4003 (10 μM) and D4476 (30 μM). (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without the indicated concentrations of BI2536 and GSK461364 over the indicated periods of time prior to lysis. Immunoblotting was carried out to detect NUAK1 levels in the immunoprecipitates. Pre-immune IgG was used as a control. (C) U2OS cells stably expressing NUAK1 WT were treated with the indicated inhibitors for 1 h. HA–NUAK1 was immunoprecipitated and analysed by immunoblotting with the indicated antibodies. (D) HA–NUAK1 with or without BI2536 (1 μM) treatment was immunoprecipitated (IP) from U2OS Flp/In cells stably expressing HA–NUAK. XIC analysis of the Ser476- and Ser480-containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was detected in both samples. In the BI253-treated cells, the m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was found to possess a ~3-fold-higher signal intensity than the control immunoprecipitate. (E) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or the indicated mutants. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (F) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or S445A mutant. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (G) HEK-293 cells transiently overexpressing NUAK1 WT or the S445A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with the indicated antibodies.

Mentions: In an attempt to pinpoint the kinase that phosphorylates NUAK1 at Ser476 and Ser480, we investigated the effect of a variety of selective protein kinase inhibitors known to target kinases that phosphorylate βTrCP-binding phosphodegron motifs on other targets. These included inhibitors of IKKs (BI605906), GSK3β (CHIR99021), polo-kinases (BI2536 and GSK461364), CK1 (D4476), aurora kinase (VX680) and CDKs (roscovitine). We also tested a number of selective inhibitors of major protein kinase signalling systems namely ATM (ataxia telangiectasia mutated; KU55933), DNA-PK (DNA-dependent protein kinase, catalytic subunit; 401KuDOS), ATR (ataxia telangiectasia and Rad3 related; ETP46464), mTOR (mammalian target of rapamycin; AZD8055), PI3K (phosphoinositide 3-kinase; GDC0941) and Akt (MK2206). This analysis strikingly revealed that two structurally unrelated inhibitors of polo kinases (PLK1, PLK2 and PLK3), namely BI2536 [28] or GSK461364 [29], prevented the calyculin A-mediated decrease in the levels of overexpressed WT NUAK1 (Figure 4A). BI2536 (http://www.kinase-screen.mrc.ac.uk/screening-compounds/341035) and GSK461364 (http://www.kinase-screen.mrc.ac.uk/screening-compounds/224) are highly potent and specific inhibitors of PLK1, PLK2 and PLK3, but not PLK4 [30]. Dose-dependence analysis indicated that the lowest dose of BI2536 and GSK461364 that inhibited the degradation of NUAK1 was 1 μM and 3 μM respectively (Figure 4B), in accordance with the reported cellular IC50 of these compounds [28,29]. Consistent with PLK mediating phosphorylation of Ser476 and Ser480, we found that BI2536 and GSK461364 inhibited binding of βTrCP to overexpressed NUAK1 (Figure 4C). Mass spectrometry analysis also revealed that treatment of U2OS cells with BI2536 inhibitor significantly suppressed phosphorylation of NUAK1 at Ser476 and Ser480 (Figure 4D).


Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1βMYPT1 phosphatase complex and the SCFβTrCP E3 ubiquitin ligase.

Banerjee S, Zagórska A, Deak M, Campbell DG, Prescott AR, Alessi DR - Biochem. J. (2014)

PLK1 interacts with NUAK1 at the CDK priming Ser445 site and phosphorylates NUAK1 at Ser476 and Ser480 and targets it for phosphorylation-mediated degradation(A) U2OS cells stably expressing HA–NUAK1 were treated with the indicated inhibitors for 4 h with or without 50 nM calyculin A treatment. Cell lysates were subjected to immunoblotting with the indicated antibodies. The inhibitors were used at 1 μM final except for MLN-4924 (3 μM), BI605906 (5 μM), WZ4003 (10 μM) and D4476 (30 μM). (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without the indicated concentrations of BI2536 and GSK461364 over the indicated periods of time prior to lysis. Immunoblotting was carried out to detect NUAK1 levels in the immunoprecipitates. Pre-immune IgG was used as a control. (C) U2OS cells stably expressing NUAK1 WT were treated with the indicated inhibitors for 1 h. HA–NUAK1 was immunoprecipitated and analysed by immunoblotting with the indicated antibodies. (D) HA–NUAK1 with or without BI2536 (1 μM) treatment was immunoprecipitated (IP) from U2OS Flp/In cells stably expressing HA–NUAK. XIC analysis of the Ser476- and Ser480-containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was detected in both samples. In the BI253-treated cells, the m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was found to possess a ~3-fold-higher signal intensity than the control immunoprecipitate. (E) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or the indicated mutants. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (F) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or S445A mutant. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (G) HEK-293 cells transiently overexpressing NUAK1 WT or the S445A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with the indicated antibodies.
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Figure 4: PLK1 interacts with NUAK1 at the CDK priming Ser445 site and phosphorylates NUAK1 at Ser476 and Ser480 and targets it for phosphorylation-mediated degradation(A) U2OS cells stably expressing HA–NUAK1 were treated with the indicated inhibitors for 4 h with or without 50 nM calyculin A treatment. Cell lysates were subjected to immunoblotting with the indicated antibodies. The inhibitors were used at 1 μM final except for MLN-4924 (3 μM), BI605906 (5 μM), WZ4003 (10 μM) and D4476 (30 μM). (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without the indicated concentrations of BI2536 and GSK461364 over the indicated periods of time prior to lysis. Immunoblotting was carried out to detect NUAK1 levels in the immunoprecipitates. Pre-immune IgG was used as a control. (C) U2OS cells stably expressing NUAK1 WT were treated with the indicated inhibitors for 1 h. HA–NUAK1 was immunoprecipitated and analysed by immunoblotting with the indicated antibodies. (D) HA–NUAK1 with or without BI2536 (1 μM) treatment was immunoprecipitated (IP) from U2OS Flp/In cells stably expressing HA–NUAK. XIC analysis of the Ser476- and Ser480-containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was detected in both samples. In the BI253-treated cells, the m/z value corresponding to the Ser476- or Ser480-containing phosphopeptide was found to possess a ~3-fold-higher signal intensity than the control immunoprecipitate. (E) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or the indicated mutants. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (F) HEK-293 cells were transfected with expression plasmids for the HA-tagged NUAK1 WT or S445A mutant. At 36 h post-transfection cells were lysed and HA-tagged proteins were immunoprecipitated from 1 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with indicated antibodies. (G) HEK-293 cells transiently overexpressing NUAK1 WT or the S445A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with the indicated antibodies.
Mentions: In an attempt to pinpoint the kinase that phosphorylates NUAK1 at Ser476 and Ser480, we investigated the effect of a variety of selective protein kinase inhibitors known to target kinases that phosphorylate βTrCP-binding phosphodegron motifs on other targets. These included inhibitors of IKKs (BI605906), GSK3β (CHIR99021), polo-kinases (BI2536 and GSK461364), CK1 (D4476), aurora kinase (VX680) and CDKs (roscovitine). We also tested a number of selective inhibitors of major protein kinase signalling systems namely ATM (ataxia telangiectasia mutated; KU55933), DNA-PK (DNA-dependent protein kinase, catalytic subunit; 401KuDOS), ATR (ataxia telangiectasia and Rad3 related; ETP46464), mTOR (mammalian target of rapamycin; AZD8055), PI3K (phosphoinositide 3-kinase; GDC0941) and Akt (MK2206). This analysis strikingly revealed that two structurally unrelated inhibitors of polo kinases (PLK1, PLK2 and PLK3), namely BI2536 [28] or GSK461364 [29], prevented the calyculin A-mediated decrease in the levels of overexpressed WT NUAK1 (Figure 4A). BI2536 (http://www.kinase-screen.mrc.ac.uk/screening-compounds/341035) and GSK461364 (http://www.kinase-screen.mrc.ac.uk/screening-compounds/224) are highly potent and specific inhibitors of PLK1, PLK2 and PLK3, but not PLK4 [30]. Dose-dependence analysis indicated that the lowest dose of BI2536 and GSK461364 that inhibited the degradation of NUAK1 was 1 μM and 3 μM respectively (Figure 4B), in accordance with the reported cellular IC50 of these compounds [28,29]. Consistent with PLK mediating phosphorylation of Ser476 and Ser480, we found that BI2536 and GSK461364 inhibited binding of βTrCP to overexpressed NUAK1 (Figure 4C). Mass spectrometry analysis also revealed that treatment of U2OS cells with BI2536 inhibitor significantly suppressed phosphorylation of NUAK1 at Ser476 and Ser480 (Figure 4D).

Bottom Line: Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine.We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors.Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.

View Article: PubMed Central - PubMed

Affiliation: *MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, U.K.

ABSTRACT
NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFβTrCP (Skp, Cullin and F-boxβTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to βTrCP, the substrate-recognition subunit of the SCFβTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2-M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1βMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1βMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.

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Related in: MedlinePlus