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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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Related in: MedlinePlus

NUAK1 binds to βTrCP(A) HA–NUAK1 was immunoprecipitated (IP) from U2OS Flp/In cells expressing empty vector (Cntl) or with overexpression of HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and was stained with Coomassic Blue. The gel was divided into the indicated pieces, and proteins in these pieces were identified by mass spectrometry. Previously published interactors ubiquitin and USP9X were identified in band 7, MYPT1, MYPT2 and MBS85 were identified in band 8, PP1β in band 11, and NUAK1 in band 9. βTrCP1 and βTrCP2 was identified in band 10, whereas SKP1 was identified in band 12. Mascot scores of the interactors are detailed in Supplementary Table S1 (http://www.biochemj.org/bj/461/bj4610233add.htm). (B) Overexpressed HA–NUAK1 was immunoprecipitated from U2OS Flp/In cell lysate stably overexpressing HA–NUAK1 and subjected to immunoblotting with the indicated antibodies. U2OS Flp/In cell lysate expressing empty vector was used as a negative control. (C) HEK-293 cells were transfected with expression plasmids for GST-tagged AMPK-related kinases. At 36 h after transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. MARK, MAP/microtubule affinity-regulating kinase.
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Figure 1: NUAK1 binds to βTrCP(A) HA–NUAK1 was immunoprecipitated (IP) from U2OS Flp/In cells expressing empty vector (Cntl) or with overexpression of HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and was stained with Coomassic Blue. The gel was divided into the indicated pieces, and proteins in these pieces were identified by mass spectrometry. Previously published interactors ubiquitin and USP9X were identified in band 7, MYPT1, MYPT2 and MBS85 were identified in band 8, PP1β in band 11, and NUAK1 in band 9. βTrCP1 and βTrCP2 was identified in band 10, whereas SKP1 was identified in band 12. Mascot scores of the interactors are detailed in Supplementary Table S1 (http://www.biochemj.org/bj/461/bj4610233add.htm). (B) Overexpressed HA–NUAK1 was immunoprecipitated from U2OS Flp/In cell lysate stably overexpressing HA–NUAK1 and subjected to immunoblotting with the indicated antibodies. U2OS Flp/In cell lysate expressing empty vector was used as a negative control. (C) HEK-293 cells were transfected with expression plasmids for GST-tagged AMPK-related kinases. At 36 h after transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. MARK, MAP/microtubule affinity-regulating kinase.

Mentions: We employed mass spectrometry to identify proteins that specifically immunoprecipitated with stably overexpressed NUAK1 in osteosarcoma-derived U2OS cells (Fig-ure 1A). NUAK1 was expressed ~20-fold higher than endogenous NUAK1 in these experiments (Supplementary Figure S1 at http://www.biochemj.org/bj/461/bj4610233add.htm). These studies revealed that in addition to previously reported NUAK1 interactors USP9X [17] and PP1βMYPT1 complex components [6], we also observed constituents of the SCFβTrCP E3 ubiquitin ligase complex (Figures 1A and 1B, and Supplementary Table S1 at http://www.biochemj.org/bj/461/bj4610233add.htm). Both closely related βTrCP1 and βTrCP2 isoforms that function as the F-box-containing substrate-recognition components of the SCF E3 ubiquitin ligase complexes [18,19], as well as SKP1 (S-phase kinase-associated protein 1) and Cul1 (cullin 1) subunits, were associated with NUAK1 immunoprecipitates (Figures 1A and 1B). βTrCP co-purified with overexpressed NUAK1 and NUAK2, but not with the eight other AMPK-related protein kinases tested (Figure 1C).


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 binds to βTrCP(A) HA–NUAK1 was immunoprecipitated (IP) from U2OS Flp/In cells expressing empty vector (Cntl) or with overexpression of HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and was stained with Coomassic Blue. The gel was divided into the indicated pieces, and proteins in these pieces were identified by mass spectrometry. Previously published interactors ubiquitin and USP9X were identified in band 7, MYPT1, MYPT2 and MBS85 were identified in band 8, PP1β in band 11, and NUAK1 in band 9. βTrCP1 and βTrCP2 was identified in band 10, whereas SKP1 was identified in band 12. Mascot scores of the interactors are detailed in Supplementary Table S1 (http://www.biochemj.org/bj/461/bj4610233add.htm). (B) Overexpressed HA–NUAK1 was immunoprecipitated from U2OS Flp/In cell lysate stably overexpressing HA–NUAK1 and subjected to immunoblotting with the indicated antibodies. U2OS Flp/In cell lysate expressing empty vector was used as a negative control. (C) HEK-293 cells were transfected with expression plasmids for GST-tagged AMPK-related kinases. At 36 h after transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. MARK, MAP/microtubule affinity-regulating kinase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: NUAK1 binds to βTrCP(A) HA–NUAK1 was immunoprecipitated (IP) from U2OS Flp/In cells expressing empty vector (Cntl) or with overexpression of HA–NUAK1. The immunoprecipitates were resolved on a polyacrylamide gel and was stained with Coomassic Blue. The gel was divided into the indicated pieces, and proteins in these pieces were identified by mass spectrometry. Previously published interactors ubiquitin and USP9X were identified in band 7, MYPT1, MYPT2 and MBS85 were identified in band 8, PP1β in band 11, and NUAK1 in band 9. βTrCP1 and βTrCP2 was identified in band 10, whereas SKP1 was identified in band 12. Mascot scores of the interactors are detailed in Supplementary Table S1 (http://www.biochemj.org/bj/461/bj4610233add.htm). (B) Overexpressed HA–NUAK1 was immunoprecipitated from U2OS Flp/In cell lysate stably overexpressing HA–NUAK1 and subjected to immunoblotting with the indicated antibodies. U2OS Flp/In cell lysate expressing empty vector was used as a negative control. (C) HEK-293 cells were transfected with expression plasmids for GST-tagged AMPK-related kinases. At 36 h after transfection cells were lysed and GST-tagged proteins were immunoprecipitated from 5 mg of cell lysates. Immunoprecipitates were analysed by immunoblotting with indicated antibodies. MARK, MAP/microtubule affinity-regulating kinase.
Mentions: We employed mass spectrometry to identify proteins that specifically immunoprecipitated with stably overexpressed NUAK1 in osteosarcoma-derived U2OS cells (Fig-ure 1A). NUAK1 was expressed ~20-fold higher than endogenous NUAK1 in these experiments (Supplementary Figure S1 at http://www.biochemj.org/bj/461/bj4610233add.htm). These studies revealed that in addition to previously reported NUAK1 interactors USP9X [17] and PP1βMYPT1 complex components [6], we also observed constituents of the SCFβTrCP E3 ubiquitin ligase complex (Figures 1A and 1B, and Supplementary Table S1 at http://www.biochemj.org/bj/461/bj4610233add.htm). Both closely related βTrCP1 and βTrCP2 isoforms that function as the F-box-containing substrate-recognition components of the SCF E3 ubiquitin ligase complexes [18,19], as well as SKP1 (S-phase kinase-associated protein 1) and Cul1 (cullin 1) subunits, were associated with NUAK1 immunoprecipitates (Figures 1A and 1B). βTrCP co-purified with overexpressed NUAK1 and NUAK2, but not with the eight other AMPK-related protein kinases tested (Figure 1C).

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