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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 protein levels are controlled by SCFβTrCP and PLK in the cell cycle(A) U2OS cells were synchronized in the G2 stage of the cell cycle by a DTB and release after 10 μM RO-3306 inhibitor treatment. Cells were lysed after every 2 h over 24 h. Lysates were subjected to immunoblotting with the indicated antibodies. (B) U2OS cells were synchronized in either in the G1/S stage of the cell cycle by a DTB or in the G2 stage by a DTB and release (Rel) after 10 μM RO-3306 inhibitor treatment. Cells were either collected or lysed after indicated times. Collected cells were subjected to quantitative measurement of DNA content (PI staining) by flow cytometry using a FACSCalibur™ (BD Biosciences) and the percentage of G1–S–G2 cells were determined by the Watson (pragmatic) modelling algorithm using FlowJo software (Treestar). Endogenous NUAK1 was immunoprecipitated from 30 mg of cell lysates and immunoblotting was carried out using the indicated antibodies. (C) U2OS Flp/In cells stably expressing HA–NUAK1 WT or S476A+S480A were synchronized as in (B) and the cell lysates were subjected to immunoblotting using the indicated antibodies.
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Figure 5: NUAK1 protein levels are controlled by SCFβTrCP and PLK in the cell cycle(A) U2OS cells were synchronized in the G2 stage of the cell cycle by a DTB and release after 10 μM RO-3306 inhibitor treatment. Cells were lysed after every 2 h over 24 h. Lysates were subjected to immunoblotting with the indicated antibodies. (B) U2OS cells were synchronized in either in the G1/S stage of the cell cycle by a DTB or in the G2 stage by a DTB and release (Rel) after 10 μM RO-3306 inhibitor treatment. Cells were either collected or lysed after indicated times. Collected cells were subjected to quantitative measurement of DNA content (PI staining) by flow cytometry using a FACSCalibur™ (BD Biosciences) and the percentage of G1–S–G2 cells were determined by the Watson (pragmatic) modelling algorithm using FlowJo software (Treestar). Endogenous NUAK1 was immunoprecipitated from 30 mg of cell lysates and immunoblotting was carried out using the indicated antibodies. (C) U2OS Flp/In cells stably expressing HA–NUAK1 WT or S476A+S480A were synchronized as in (B) and the cell lysates were subjected to immunoblotting using the indicated antibodies.

Mentions: PLK1 activity peaks at late S- and G2–M-phase of the cell cycle before declining in the G1- to early S-phase [38]. To investigate whether PLK1 regulates NUAK1 expression in the cell cycle, we synchronized U2OS cells in the G2 stage of the cell cycle using the DTB and CDK1 inhibitor (RO3306) release protocol [39]. Cells were then lysed at intervals over a 21-h period and immunoblotted for NUAK1, PLK1 and other cell cycle control markers (cyclin B1, cyclin A and phosphohistone H3 Ser10). Consistent with a role of PLK1 in targeting NUAK1 for degradation, we observed low levels of NUAK1 during the G2–M-phase (0–1 h time point), when PLK1 as well as cyclin A and B1 were elevated (Figure 5A). Expression levels of NUAK1 remained low in early G1-phase (3–5 h), increased at the 5–7 h time points and were maximal from the 9 h time point onwards (early S-phase to asynchronous) when PLK1 levels are low.


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 protein levels are controlled by SCFβTrCP and PLK in the cell cycle(A) U2OS cells were synchronized in the G2 stage of the cell cycle by a DTB and release after 10 μM RO-3306 inhibitor treatment. Cells were lysed after every 2 h over 24 h. Lysates were subjected to immunoblotting with the indicated antibodies. (B) U2OS cells were synchronized in either in the G1/S stage of the cell cycle by a DTB or in the G2 stage by a DTB and release (Rel) after 10 μM RO-3306 inhibitor treatment. Cells were either collected or lysed after indicated times. Collected cells were subjected to quantitative measurement of DNA content (PI staining) by flow cytometry using a FACSCalibur™ (BD Biosciences) and the percentage of G1–S–G2 cells were determined by the Watson (pragmatic) modelling algorithm using FlowJo software (Treestar). Endogenous NUAK1 was immunoprecipitated from 30 mg of cell lysates and immunoblotting was carried out using the indicated antibodies. (C) U2OS Flp/In cells stably expressing HA–NUAK1 WT or S476A+S480A were synchronized as in (B) and the cell lysates were subjected to immunoblotting using the indicated antibodies.
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Figure 5: NUAK1 protein levels are controlled by SCFβTrCP and PLK in the cell cycle(A) U2OS cells were synchronized in the G2 stage of the cell cycle by a DTB and release after 10 μM RO-3306 inhibitor treatment. Cells were lysed after every 2 h over 24 h. Lysates were subjected to immunoblotting with the indicated antibodies. (B) U2OS cells were synchronized in either in the G1/S stage of the cell cycle by a DTB or in the G2 stage by a DTB and release (Rel) after 10 μM RO-3306 inhibitor treatment. Cells were either collected or lysed after indicated times. Collected cells were subjected to quantitative measurement of DNA content (PI staining) by flow cytometry using a FACSCalibur™ (BD Biosciences) and the percentage of G1–S–G2 cells were determined by the Watson (pragmatic) modelling algorithm using FlowJo software (Treestar). Endogenous NUAK1 was immunoprecipitated from 30 mg of cell lysates and immunoblotting was carried out using the indicated antibodies. (C) U2OS Flp/In cells stably expressing HA–NUAK1 WT or S476A+S480A were synchronized as in (B) and the cell lysates were subjected to immunoblotting using the indicated antibodies.
Mentions: PLK1 activity peaks at late S- and G2–M-phase of the cell cycle before declining in the G1- to early S-phase [38]. To investigate whether PLK1 regulates NUAK1 expression in the cell cycle, we synchronized U2OS cells in the G2 stage of the cell cycle using the DTB and CDK1 inhibitor (RO3306) release protocol [39]. Cells were then lysed at intervals over a 21-h period and immunoblotted for NUAK1, PLK1 and other cell cycle control markers (cyclin B1, cyclin A and phosphohistone H3 Ser10). Consistent with a role of PLK1 in targeting NUAK1 for degradation, we observed low levels of NUAK1 during the G2–M-phase (0–1 h time point), when PLK1 as well as cyclin A and B1 were elevated (Figure 5A). Expression levels of NUAK1 remained low in early G1-phase (3–5 h), increased at the 5–7 h time points and were maximal from the 9 h time point onwards (early S-phase to asynchronous) when PLK1 levels are low.

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