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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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NUAK1 degron requires phosphorylation to interact with βTrCP(A) U2OS cells were treated with or without 50 nM calyculin A for 10 min and the cells were lysed and endogenous NUAK1 was immunoprecipitated (IP) from 15 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. Pre-immune IgG was used as a control. (B) Endogenous NUAK1 was immunoprecipitated from U2OS cells treated with 50 nM calyculin A for 30 min. The immunoprecipitates were subjected to lambda-phosphatase assay with or without prior inactivation of lambda-phosphatase with EDTA. The immunoprecipitates were washed thoroughly and were analysed by immunoblotting with indicated antibodies. (C) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or NUAK1 3IL/KK mutant. At 36 h post-transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with the indicated antibodies. (D) Alignment showing the conservation of the ESGYYS degron of NUAK1 and NUAK2 within vertebrate orthologues. The sequences of Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalisi, Anolis carolinensis and Danio rerio NUAK1 and NUAK2 are from the NCBI. There are two copies of NUAK1 in D. rerio: one on chr. 4 and another on chr. 25. Alignment was performed using T-Coffee software and visualized using Jalview. (E) Alignment of the candidate phosphodegron sequence DSGxxS between NUAK1 and the canonical βTrCP-interacting target proteins IkB, β-catenin, Wee1, Emi1, claspin and Bora. (F) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblottings were carried out as in (C). (G) HA–NUAK1 with or without calyculin A treatment and HA–NUAK1 S476A+S480A were immunoprecipitated (IP) from U2OS Flp/In cells expressing either the WT or the mutant HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie Blue. After trypsin digestion and precursor ion scanning (−79 Da) on the QTrap 4000 an XIC analysis of the Ser476 and Ser480 containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to this phosphopeptide was detected in NUAK1 WT (both DMSO- and calyculin A-treated), but not in the S476A+Ser480A mutant. In the calyculin A-treated cells, the m/z value corresponding to the Ser476- or Ser480 -containing phosphopeptide was found to possess a >6-fold higher signal intensity than DMSO treated (control) immunoprecipitate. (H) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblotting was carried out as in (C).
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Figure 2: NUAK1 degron requires phosphorylation to interact with βTrCP(A) U2OS cells were treated with or without 50 nM calyculin A for 10 min and the cells were lysed and endogenous NUAK1 was immunoprecipitated (IP) from 15 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. Pre-immune IgG was used as a control. (B) Endogenous NUAK1 was immunoprecipitated from U2OS cells treated with 50 nM calyculin A for 30 min. The immunoprecipitates were subjected to lambda-phosphatase assay with or without prior inactivation of lambda-phosphatase with EDTA. The immunoprecipitates were washed thoroughly and were analysed by immunoblotting with indicated antibodies. (C) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or NUAK1 3IL/KK mutant. At 36 h post-transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with the indicated antibodies. (D) Alignment showing the conservation of the ESGYYS degron of NUAK1 and NUAK2 within vertebrate orthologues. The sequences of Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalisi, Anolis carolinensis and Danio rerio NUAK1 and NUAK2 are from the NCBI. There are two copies of NUAK1 in D. rerio: one on chr. 4 and another on chr. 25. Alignment was performed using T-Coffee software and visualized using Jalview. (E) Alignment of the candidate phosphodegron sequence DSGxxS between NUAK1 and the canonical βTrCP-interacting target proteins IkB, β-catenin, Wee1, Emi1, claspin and Bora. (F) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblottings were carried out as in (C). (G) HA–NUAK1 with or without calyculin A treatment and HA–NUAK1 S476A+S480A were immunoprecipitated (IP) from U2OS Flp/In cells expressing either the WT or the mutant HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie Blue. After trypsin digestion and precursor ion scanning (−79 Da) on the QTrap 4000 an XIC analysis of the Ser476 and Ser480 containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to this phosphopeptide was detected in NUAK1 WT (both DMSO- and calyculin A-treated), but not in the S476A+Ser480A mutant. In the calyculin A-treated cells, the m/z value corresponding to the Ser476- or Ser480 -containing phosphopeptide was found to possess a >6-fold higher signal intensity than DMSO treated (control) immunoprecipitate. (H) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblotting was carried out as in (C).

Mentions: To investigate whether interaction between βTrCP isoforms and NUAK1 is controlled by phosphorylation, we first treated U2OS cells in the presence or absence of the protein phosphatase inhibitor calyculin A [24] to see whether this enhances phosphorylation of NUAK1 and hence binding to βTrCP. This revealed that calyculin A within 30 min markedly enhanced association of βTrCP isoforms to endogenous NUAK1 (Figure 2A). Consistent with the association of βTrCP with NUAK1 being mediated through phosphorylation, treatment of endogenous NUAK1 immunoprecipitates with lambda phosphatase-induced dissociation of βTrCP isoforms (Figure 2B).


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)

NUAK1 degron requires phosphorylation to interact with βTrCP(A) U2OS cells were treated with or without 50 nM calyculin A for 10 min and the cells were lysed and endogenous NUAK1 was immunoprecipitated (IP) from 15 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. Pre-immune IgG was used as a control. (B) Endogenous NUAK1 was immunoprecipitated from U2OS cells treated with 50 nM calyculin A for 30 min. The immunoprecipitates were subjected to lambda-phosphatase assay with or without prior inactivation of lambda-phosphatase with EDTA. The immunoprecipitates were washed thoroughly and were analysed by immunoblotting with indicated antibodies. (C) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or NUAK1 3IL/KK mutant. At 36 h post-transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with the indicated antibodies. (D) Alignment showing the conservation of the ESGYYS degron of NUAK1 and NUAK2 within vertebrate orthologues. The sequences of Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalisi, Anolis carolinensis and Danio rerio NUAK1 and NUAK2 are from the NCBI. There are two copies of NUAK1 in D. rerio: one on chr. 4 and another on chr. 25. Alignment was performed using T-Coffee software and visualized using Jalview. (E) Alignment of the candidate phosphodegron sequence DSGxxS between NUAK1 and the canonical βTrCP-interacting target proteins IkB, β-catenin, Wee1, Emi1, claspin and Bora. (F) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblottings were carried out as in (C). (G) HA–NUAK1 with or without calyculin A treatment and HA–NUAK1 S476A+S480A were immunoprecipitated (IP) from U2OS Flp/In cells expressing either the WT or the mutant HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie Blue. After trypsin digestion and precursor ion scanning (−79 Da) on the QTrap 4000 an XIC analysis of the Ser476 and Ser480 containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to this phosphopeptide was detected in NUAK1 WT (both DMSO- and calyculin A-treated), but not in the S476A+Ser480A mutant. In the calyculin A-treated cells, the m/z value corresponding to the Ser476- or Ser480 -containing phosphopeptide was found to possess a >6-fold higher signal intensity than DMSO treated (control) immunoprecipitate. (H) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblotting was carried out as in (C).
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Figure 2: NUAK1 degron requires phosphorylation to interact with βTrCP(A) U2OS cells were treated with or without 50 nM calyculin A for 10 min and the cells were lysed and endogenous NUAK1 was immunoprecipitated (IP) from 15 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. Pre-immune IgG was used as a control. (B) Endogenous NUAK1 was immunoprecipitated from U2OS cells treated with 50 nM calyculin A for 30 min. The immunoprecipitates were subjected to lambda-phosphatase assay with or without prior inactivation of lambda-phosphatase with EDTA. The immunoprecipitates were washed thoroughly and were analysed by immunoblotting with indicated antibodies. (C) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or NUAK1 3IL/KK mutant. At 36 h post-transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates (IP) were analysed by immunoblotting with the indicated antibodies. (D) Alignment showing the conservation of the ESGYYS degron of NUAK1 and NUAK2 within vertebrate orthologues. The sequences of Homo sapiens, Mus musculus, Gallus gallus, Xenopus tropicalisi, Anolis carolinensis and Danio rerio NUAK1 and NUAK2 are from the NCBI. There are two copies of NUAK1 in D. rerio: one on chr. 4 and another on chr. 25. Alignment was performed using T-Coffee software and visualized using Jalview. (E) Alignment of the candidate phosphodegron sequence DSGxxS between NUAK1 and the canonical βTrCP-interacting target proteins IkB, β-catenin, Wee1, Emi1, claspin and Bora. (F) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblottings were carried out as in (C). (G) HA–NUAK1 with or without calyculin A treatment and HA–NUAK1 S476A+S480A were immunoprecipitated (IP) from U2OS Flp/In cells expressing either the WT or the mutant HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and stained with Coomassie Blue. After trypsin digestion and precursor ion scanning (−79 Da) on the QTrap 4000 an XIC analysis of the Ser476 and Ser480 containing phosphopeptide (R.ESGYYSSPER.S+1P) was carried out. The m/z value corresponding to this phosphopeptide was detected in NUAK1 WT (both DMSO- and calyculin A-treated), but not in the S476A+Ser480A mutant. In the calyculin A-treated cells, the m/z value corresponding to the Ser476- or Ser480 -containing phosphopeptide was found to possess a >6-fold higher signal intensity than DMSO treated (control) immunoprecipitate. (H) HEK-293 cells were transfected with expression plasmids for the GST-tagged NUAK1 WT or indicated mutants and immunoprecipitations and immunoblotting was carried out as in (C).
Mentions: To investigate whether interaction between βTrCP isoforms and NUAK1 is controlled by phosphorylation, we first treated U2OS cells in the presence or absence of the protein phosphatase inhibitor calyculin A [24] to see whether this enhances phosphorylation of NUAK1 and hence binding to βTrCP. This revealed that calyculin A within 30 min markedly enhanced association of βTrCP isoforms to endogenous NUAK1 (Figure 2A). Consistent with the association of βTrCP with NUAK1 being mediated through phosphorylation, treatment of endogenous NUAK1 immunoprecipitates with lambda phosphatase-induced dissociation of βTrCP isoforms (Figure 2B).

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.

Show MeSH
Related in: MedlinePlus