Limits...
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.

Show MeSH

Related in: MedlinePlus

NUAK1 degradation is required for controlled mitotic progression(A) U2OS cells treated with or without 10 μM of WZ4003 or 3 μM HTH-01015 and U2OS Flp/In cells stably expressing NUAK1 WT and NUAK1 S476A+S480A were fixed in ice-cold 70% ethanol, stained with PI and analysed for cell cycle distribution by flow cytometry (as described in the Materials and methods section). The percentage of S-phase cells were calculated between control and inhibitor treated using GraphPad Prism. *P<0.05. (B) U2OS cells were treated with or without DMSO (control) or 10 μM of WZ4003 or 3 μM HTH-01015 or 10 μM of RO-3306 (control of mitotic defect). Mitotic cells were manually counted and plotted using GraphPad Prism software as elaborated in the Materials and methods section. (C) NUAK1 levels in U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) were compared using immunoblotting of the cell lysates. Proliferation rate between U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) was compared over 5 days using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. Data were represented as percentage of growth times the Day 0 counts. (D) U2OS Flp/In NUAK1 WT (with or without 10 μM of WZ4003 treatment) and S476A+S480A (SS/AA) cells were harvested by centrifugation, fixed in 1% paraformaldehyde, permeabilized with 90% ice-cold methanol and stained with anti-phosphohistone H3–Alexa Fluor® 488 antibody to quantify the mitotic population. The data are represented as the percentage of mitotic population and graphs were developed using GraphPad Prism. Results are means±S.D. *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4109838&req=5

Figure 6: NUAK1 degradation is required for controlled mitotic progression(A) U2OS cells treated with or without 10 μM of WZ4003 or 3 μM HTH-01015 and U2OS Flp/In cells stably expressing NUAK1 WT and NUAK1 S476A+S480A were fixed in ice-cold 70% ethanol, stained with PI and analysed for cell cycle distribution by flow cytometry (as described in the Materials and methods section). The percentage of S-phase cells were calculated between control and inhibitor treated using GraphPad Prism. *P<0.05. (B) U2OS cells were treated with or without DMSO (control) or 10 μM of WZ4003 or 3 μM HTH-01015 or 10 μM of RO-3306 (control of mitotic defect). Mitotic cells were manually counted and plotted using GraphPad Prism software as elaborated in the Materials and methods section. (C) NUAK1 levels in U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) were compared using immunoblotting of the cell lysates. Proliferation rate between U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) was compared over 5 days using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. Data were represented as percentage of growth times the Day 0 counts. (D) U2OS Flp/In NUAK1 WT (with or without 10 μM of WZ4003 treatment) and S476A+S480A (SS/AA) cells were harvested by centrifugation, fixed in 1% paraformaldehyde, permeabilized with 90% ice-cold methanol and stained with anti-phosphohistone H3–Alexa Fluor® 488 antibody to quantify the mitotic population. The data are represented as the percentage of mitotic population and graphs were developed using GraphPad Prism. Results are means±S.D. *P<0.05.

Mentions: We next investigated the effect that inhibiting NUAK1 catalytic activity had on the cell cycle by treating asynchronous U2OS cells for 8 h with two structurally distinct and highly selective NUAK1 inhibitors termed WZ4003 and HTH-01-015 [15]. This revealed that both WZ4003 and HTH-01-015, under conditions which they inhibit phosphorylation of the NUAK1 substrate MYPT1, induced a ~50% reduction in the population of cells in S-phase (Figure 6A).


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 degradation is required for controlled mitotic progression(A) U2OS cells treated with or without 10 μM of WZ4003 or 3 μM HTH-01015 and U2OS Flp/In cells stably expressing NUAK1 WT and NUAK1 S476A+S480A were fixed in ice-cold 70% ethanol, stained with PI and analysed for cell cycle distribution by flow cytometry (as described in the Materials and methods section). The percentage of S-phase cells were calculated between control and inhibitor treated using GraphPad Prism. *P<0.05. (B) U2OS cells were treated with or without DMSO (control) or 10 μM of WZ4003 or 3 μM HTH-01015 or 10 μM of RO-3306 (control of mitotic defect). Mitotic cells were manually counted and plotted using GraphPad Prism software as elaborated in the Materials and methods section. (C) NUAK1 levels in U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) were compared using immunoblotting of the cell lysates. Proliferation rate between U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) was compared over 5 days using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. Data were represented as percentage of growth times the Day 0 counts. (D) U2OS Flp/In NUAK1 WT (with or without 10 μM of WZ4003 treatment) and S476A+S480A (SS/AA) cells were harvested by centrifugation, fixed in 1% paraformaldehyde, permeabilized with 90% ice-cold methanol and stained with anti-phosphohistone H3–Alexa Fluor® 488 antibody to quantify the mitotic population. The data are represented as the percentage of mitotic population and graphs were developed using GraphPad Prism. Results are means±S.D. *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: NUAK1 degradation is required for controlled mitotic progression(A) U2OS cells treated with or without 10 μM of WZ4003 or 3 μM HTH-01015 and U2OS Flp/In cells stably expressing NUAK1 WT and NUAK1 S476A+S480A were fixed in ice-cold 70% ethanol, stained with PI and analysed for cell cycle distribution by flow cytometry (as described in the Materials and methods section). The percentage of S-phase cells were calculated between control and inhibitor treated using GraphPad Prism. *P<0.05. (B) U2OS cells were treated with or without DMSO (control) or 10 μM of WZ4003 or 3 μM HTH-01015 or 10 μM of RO-3306 (control of mitotic defect). Mitotic cells were manually counted and plotted using GraphPad Prism software as elaborated in the Materials and methods section. (C) NUAK1 levels in U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) were compared using immunoblotting of the cell lysates. Proliferation rate between U2OS Flp/In NUAK1 WT and S476A+S480A (SS/AA) was compared over 5 days using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. Data were represented as percentage of growth times the Day 0 counts. (D) U2OS Flp/In NUAK1 WT (with or without 10 μM of WZ4003 treatment) and S476A+S480A (SS/AA) cells were harvested by centrifugation, fixed in 1% paraformaldehyde, permeabilized with 90% ice-cold methanol and stained with anti-phosphohistone H3–Alexa Fluor® 488 antibody to quantify the mitotic population. The data are represented as the percentage of mitotic population and graphs were developed using GraphPad Prism. Results are means±S.D. *P<0.05.
Mentions: We next investigated the effect that inhibiting NUAK1 catalytic activity had on the cell cycle by treating asynchronous U2OS cells for 8 h with two structurally distinct and highly selective NUAK1 inhibitors termed WZ4003 and HTH-01-015 [15]. This revealed that both WZ4003 and HTH-01-015, under conditions which they inhibit phosphorylation of the NUAK1 substrate MYPT1, induced a ~50% reduction in the population of cells in S-phase (Figure 6A).

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