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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 regulated PLK1 T-loop Thr210 phosphorylationHEK-293 cells were treated in the absence (DMSO) or presence of 10 μM of WZ4003 over 8 h. Cell medium was then replaced with either normal DMEM (−) or EDTA/PBS-based cell dissociation buffer (+) containing the same concentration of WZ4003 or DMSO that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were rapidly lysed either by scraping for (−) cells or by resuspending in lysis buffer for (+) cells and the lysates were subjected to immunoblotting with the indicated antibodies. Results shown in the histogram are means±S.D.
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Figure 7: NUAK1 regulated PLK1 T-loop Thr210 phosphorylationHEK-293 cells were treated in the absence (DMSO) or presence of 10 μM of WZ4003 over 8 h. Cell medium was then replaced with either normal DMEM (−) or EDTA/PBS-based cell dissociation buffer (+) containing the same concentration of WZ4003 or DMSO that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were rapidly lysed either by scraping for (−) cells or by resuspending in lysis buffer for (+) cells and the lysates were subjected to immunoblotting with the indicated antibodies. Results shown in the histogram are means±S.D.

Mentions: As discussed in the Introduction section, PP1βMYPT1 binds to and inhibits PLK1 by dephosphorylating the T-loop residue (Thr210) [14]. The ability PP1βMYPT1 to bind to and dephosphorylate and hence inactivate PLK1 is dependent upon phosphorylation of MYPT1 at Ser473 by the CDK1, which creates a binding site for the PLK1 Polo-box domains [14]. As Ser473 lies adjacent to the NUAK1 phosphorylation site (Ser472) that controls 14-3-3 binding [6], we decided to explore whether NUAK1 could indeed influence PLK1 T-loop phosphorylation. We treated HEK-293 cells with EDTA to induce cell dissociation, a condition that has previously been shown to promote phosphorylation of MYPT1 by NUAK1 [6]. Immunoblotting with phospho-specific antibodies confirmed that EDTA treatment induced phosphorylation of MYPT1 at Ser445 and Ser472 (Figure 7). Strikingly, we observed that EDTA also induced a significant stimulation of PLK1 phosphorylation at Thr210 that was accompanied by marked electrophoretic band shift in PLK1 protein. Treatment of cells with the WZ4003 dual NUAK1/NUAK2 inhibitor prior to stimulation with EDTA, inhibited phosphorylation of Thr210 as well as the electrophoretic mobility shift of PLK1 protein (Figure 7).


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 regulated PLK1 T-loop Thr210 phosphorylationHEK-293 cells were treated in the absence (DMSO) or presence of 10 μM of WZ4003 over 8 h. Cell medium was then replaced with either normal DMEM (−) or EDTA/PBS-based cell dissociation buffer (+) containing the same concentration of WZ4003 or DMSO that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were rapidly lysed either by scraping for (−) cells or by resuspending in lysis buffer for (+) cells and the lysates were subjected to immunoblotting with the indicated antibodies. Results shown in the histogram are means±S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: NUAK1 regulated PLK1 T-loop Thr210 phosphorylationHEK-293 cells were treated in the absence (DMSO) or presence of 10 μM of WZ4003 over 8 h. Cell medium was then replaced with either normal DMEM (−) or EDTA/PBS-based cell dissociation buffer (+) containing the same concentration of WZ4003 or DMSO that the cells were previously incubated in. Cell detachment was induced with gentle tapping of the plates followed by gentle centrifugation at 70 g for 3 min. Cells were rapidly lysed either by scraping for (−) cells or by resuspending in lysis buffer for (+) cells and the lysates were subjected to immunoblotting with the indicated antibodies. Results shown in the histogram are means±S.D.
Mentions: As discussed in the Introduction section, PP1βMYPT1 binds to and inhibits PLK1 by dephosphorylating the T-loop residue (Thr210) [14]. The ability PP1βMYPT1 to bind to and dephosphorylate and hence inactivate PLK1 is dependent upon phosphorylation of MYPT1 at Ser473 by the CDK1, which creates a binding site for the PLK1 Polo-box domains [14]. As Ser473 lies adjacent to the NUAK1 phosphorylation site (Ser472) that controls 14-3-3 binding [6], we decided to explore whether NUAK1 could indeed influence PLK1 T-loop phosphorylation. We treated HEK-293 cells with EDTA to induce cell dissociation, a condition that has previously been shown to promote phosphorylation of MYPT1 by NUAK1 [6]. Immunoblotting with phospho-specific antibodies confirmed that EDTA treatment induced phosphorylation of MYPT1 at Ser445 and Ser472 (Figure 7). Strikingly, we observed that EDTA also induced a significant stimulation of PLK1 phosphorylation at Thr210 that was accompanied by marked electrophoretic band shift in PLK1 protein. Treatment of cells with the WZ4003 dual NUAK1/NUAK2 inhibitor prior to stimulation with EDTA, inhibited phosphorylation of Thr210 as well as the electrophoretic mobility shift of PLK1 protein (Figure 7).

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