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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 is protected from phosphorylation-mediated degradation upon SCFβTrCP inhibition(A) U2OS cells stably expressing NUAK1 WT or S476A+S480A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with indicated antibodies. (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without MLN-4924 (3 μM) 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) Endogenous NUAK1 was immunoprecipitated from 1 mg of U2OS cell lysates treated with MLN-4924 (3 μM) over the indicated periods of time prior to lysis. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. (D) βTrCP1+/+ (WT) and βTrCP1−/− (knockout) MEFs were lysed and analysed by immunoblotting with the indicated antibodies. Endogenous NUAK1 was immunoprecipitated and its activity was assayed in triplicates with pre-immune IgG as control. Results are means±S.D.
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Figure 3: NUAK1 is protected from phosphorylation-mediated degradation upon SCFβTrCP inhibition(A) U2OS cells stably expressing NUAK1 WT or S476A+S480A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with indicated antibodies. (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without MLN-4924 (3 μM) 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) Endogenous NUAK1 was immunoprecipitated from 1 mg of U2OS cell lysates treated with MLN-4924 (3 μM) over the indicated periods of time prior to lysis. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. (D) βTrCP1+/+ (WT) and βTrCP1−/− (knockout) MEFs were lysed and analysed by immunoblotting with the indicated antibodies. Endogenous NUAK1 was immunoprecipitated and its activity was assayed in triplicates with pre-immune IgG as control. Results are means±S.D.

Mentions: To investigate whether the expression of NUAK1 was controlled by the SCFβTrCP E3 ligase complex, we first treated U2OS cells stably expressing WT NUAK1 with 50 nM calyculin A to induce βTrCP binding. This revealed that following 3 h of calyculin A treatment, the levels of WT NUAK1, but not the non-βTrCP-binding NUAK1[S476A+S480A] mutant, were markedly reduced (Figure 3A).


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 is protected from phosphorylation-mediated degradation upon SCFβTrCP inhibition(A) U2OS cells stably expressing NUAK1 WT or S476A+S480A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with indicated antibodies. (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without MLN-4924 (3 μM) 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) Endogenous NUAK1 was immunoprecipitated from 1 mg of U2OS cell lysates treated with MLN-4924 (3 μM) over the indicated periods of time prior to lysis. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. (D) βTrCP1+/+ (WT) and βTrCP1−/− (knockout) MEFs were lysed and analysed by immunoblotting with the indicated antibodies. Endogenous NUAK1 was immunoprecipitated and its activity was assayed in triplicates with pre-immune IgG as control. Results are means±S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109838&req=5

Figure 3: NUAK1 is protected from phosphorylation-mediated degradation upon SCFβTrCP inhibition(A) U2OS cells stably expressing NUAK1 WT or S476A+S480A mutant were treated with 50 nM calyculin A over the indicated periods of time. The cell lysates were analysed by immunoblotting with indicated antibodies. (B) Endogenous NUAK1 was immunoprecipitated (IP) from 1 mg of U2OS cell lysates treated with calyculin A (50 nM) and with or without MLN-4924 (3 μM) 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) Endogenous NUAK1 was immunoprecipitated from 1 mg of U2OS cell lysates treated with MLN-4924 (3 μM) over the indicated periods of time prior to lysis. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. (D) βTrCP1+/+ (WT) and βTrCP1−/− (knockout) MEFs were lysed and analysed by immunoblotting with the indicated antibodies. Endogenous NUAK1 was immunoprecipitated and its activity was assayed in triplicates with pre-immune IgG as control. Results are means±S.D.
Mentions: To investigate whether the expression of NUAK1 was controlled by the SCFβTrCP E3 ligase complex, we first treated U2OS cells stably expressing WT NUAK1 with 50 nM calyculin A to induce βTrCP binding. This revealed that following 3 h of calyculin A treatment, the levels of WT NUAK1, but not the non-βTrCP-binding NUAK1[S476A+S480A] mutant, were markedly reduced (Figure 3A).

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