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

Schematic representation of how NUAK1 expression is regulated by PLK and SCFβTrCPNUAK1 interacts with and phosphorylates PP1βMYPT1 myosin phosphatase complex at Ser445 and Ser472, which promotes 14-3-3 binding. In the S/G2-phase of the cell cycle NUAK1 and MYPT1 are primed by CDKs, which creates a docking site for PLK. PLK1 interacts with MYPT1 at Ser473 which leads to inactivation of PLK1 upon PP1βMYPT1-mediated dephosphorylation of Thr210 at the PLK1 activation loop. PLK docking on to NUAK1 at Ser445 leads to phosphorylation of NUAK1 at Ser476 and Ser480, which promotes interaction of NUAK1 with SCFβTrCP E3 ubiquitin ligase complex that triggers ubiquitylation and degradation of NUAK1 in the G2/M-phase of the cell cycle. PP1βMYPT1 negatively regulates PLK–NUAK1 interaction by possibly dephosphorylating the CDK priming site on NUAK1 and hence protecting NUAK1 from degradation. RBX1, ring-box 1, E3 ubiquitin protein ligase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Schematic representation of how NUAK1 expression is regulated by PLK and SCFβTrCPNUAK1 interacts with and phosphorylates PP1βMYPT1 myosin phosphatase complex at Ser445 and Ser472, which promotes 14-3-3 binding. In the S/G2-phase of the cell cycle NUAK1 and MYPT1 are primed by CDKs, which creates a docking site for PLK. PLK1 interacts with MYPT1 at Ser473 which leads to inactivation of PLK1 upon PP1βMYPT1-mediated dephosphorylation of Thr210 at the PLK1 activation loop. PLK docking on to NUAK1 at Ser445 leads to phosphorylation of NUAK1 at Ser476 and Ser480, which promotes interaction of NUAK1 with SCFβTrCP E3 ubiquitin ligase complex that triggers ubiquitylation and degradation of NUAK1 in the G2/M-phase of the cell cycle. PP1βMYPT1 negatively regulates PLK–NUAK1 interaction by possibly dephosphorylating the CDK priming site on NUAK1 and hence protecting NUAK1 from degradation. RBX1, ring-box 1, E3 ubiquitin protein ligase.

Mentions: NUAK isoforms possess three highly conserved GILK motifs that interact directly with the PP1β subunit of the PP1βMYPT1 complex [6]. Our findings suggest that inhibiting binding of NUAK1 to PP1βMYPT1 by ablating the GILK motifs significantly enhances βTrCP binding to NUAK1 (Figure 2H). This suggests that PP1βMYPT1 acts to dephosphorylate either the PLK1-binding CDK-primed site (Ser445) on NUAK1 and/or the βTrCP-binding residues (Ser476 to Ser480) (Figure 8). PLK1 has also been reported to interact with PP1βMYPT1, which leads to dephosphorylation of the PLK1 T-loop (Thr210) residue and thereby PLK1 inactivation [14,51]. Binding of PLK1 to MYPT1 is triggered following phosphorylation of the MYPT1 subunit by CDK1 at Ser473 [14]. Interestingly, one of the key NUAK1 phosphorylation sites on MYPT1, namely Ser472, lies adjacent to Ser473 that controls PLK1 binding to MYPT1. Phosphorylation of Ser472 triggers 14-3-3 isoforms binding to MYPT1 [6]. Our data indicate that subjecting HEK-293 cells to a condition that promotes endogenous NUAK1 to phosphorylate MYPT1, namely EDTA-induced cell detachment, leads to increased Thr210 phosphorylation of PLK1. This is also accompanied by an electrophoretic band shift, suggesting that stoichiometry of phosphorylation is significant (Figure 7). This phosphorylation of Thr210 as well as the electrophoretic mobility shift is inhibited by treatment with the NUAK1/NUAK2 WZ4003 selective inhibitor. These data support the notion that NUAK1 may indeed play a critical role in modulating PLK1 activity through its ability to phosphorylate MYPT1. In future work it would be interesting to explore whether phosphorylation of MYPT1 by NUAK1 influences association of PLK1 with PP1βMYPT1 and whether 14-3-3 binding is involved. It would also be important to analyse whether the effects that NUAK1 inhibitors have on suppressing mitosis (Figure 6) and cell proliferation [15] are mediated through their ability to induce dephosphorylation and hence inactivation of PLK1.


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)

Schematic representation of how NUAK1 expression is regulated by PLK and SCFβTrCPNUAK1 interacts with and phosphorylates PP1βMYPT1 myosin phosphatase complex at Ser445 and Ser472, which promotes 14-3-3 binding. In the S/G2-phase of the cell cycle NUAK1 and MYPT1 are primed by CDKs, which creates a docking site for PLK. PLK1 interacts with MYPT1 at Ser473 which leads to inactivation of PLK1 upon PP1βMYPT1-mediated dephosphorylation of Thr210 at the PLK1 activation loop. PLK docking on to NUAK1 at Ser445 leads to phosphorylation of NUAK1 at Ser476 and Ser480, which promotes interaction of NUAK1 with SCFβTrCP E3 ubiquitin ligase complex that triggers ubiquitylation and degradation of NUAK1 in the G2/M-phase of the cell cycle. PP1βMYPT1 negatively regulates PLK–NUAK1 interaction by possibly dephosphorylating the CDK priming site on NUAK1 and hence protecting NUAK1 from degradation. RBX1, ring-box 1, E3 ubiquitin protein ligase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Schematic representation of how NUAK1 expression is regulated by PLK and SCFβTrCPNUAK1 interacts with and phosphorylates PP1βMYPT1 myosin phosphatase complex at Ser445 and Ser472, which promotes 14-3-3 binding. In the S/G2-phase of the cell cycle NUAK1 and MYPT1 are primed by CDKs, which creates a docking site for PLK. PLK1 interacts with MYPT1 at Ser473 which leads to inactivation of PLK1 upon PP1βMYPT1-mediated dephosphorylation of Thr210 at the PLK1 activation loop. PLK docking on to NUAK1 at Ser445 leads to phosphorylation of NUAK1 at Ser476 and Ser480, which promotes interaction of NUAK1 with SCFβTrCP E3 ubiquitin ligase complex that triggers ubiquitylation and degradation of NUAK1 in the G2/M-phase of the cell cycle. PP1βMYPT1 negatively regulates PLK–NUAK1 interaction by possibly dephosphorylating the CDK priming site on NUAK1 and hence protecting NUAK1 from degradation. RBX1, ring-box 1, E3 ubiquitin protein ligase.
Mentions: NUAK isoforms possess three highly conserved GILK motifs that interact directly with the PP1β subunit of the PP1βMYPT1 complex [6]. Our findings suggest that inhibiting binding of NUAK1 to PP1βMYPT1 by ablating the GILK motifs significantly enhances βTrCP binding to NUAK1 (Figure 2H). This suggests that PP1βMYPT1 acts to dephosphorylate either the PLK1-binding CDK-primed site (Ser445) on NUAK1 and/or the βTrCP-binding residues (Ser476 to Ser480) (Figure 8). PLK1 has also been reported to interact with PP1βMYPT1, which leads to dephosphorylation of the PLK1 T-loop (Thr210) residue and thereby PLK1 inactivation [14,51]. Binding of PLK1 to MYPT1 is triggered following phosphorylation of the MYPT1 subunit by CDK1 at Ser473 [14]. Interestingly, one of the key NUAK1 phosphorylation sites on MYPT1, namely Ser472, lies adjacent to Ser473 that controls PLK1 binding to MYPT1. Phosphorylation of Ser472 triggers 14-3-3 isoforms binding to MYPT1 [6]. Our data indicate that subjecting HEK-293 cells to a condition that promotes endogenous NUAK1 to phosphorylate MYPT1, namely EDTA-induced cell detachment, leads to increased Thr210 phosphorylation of PLK1. This is also accompanied by an electrophoretic band shift, suggesting that stoichiometry of phosphorylation is significant (Figure 7). This phosphorylation of Thr210 as well as the electrophoretic mobility shift is inhibited by treatment with the NUAK1/NUAK2 WZ4003 selective inhibitor. These data support the notion that NUAK1 may indeed play a critical role in modulating PLK1 activity through its ability to phosphorylate MYPT1. In future work it would be interesting to explore whether phosphorylation of MYPT1 by NUAK1 influences association of PLK1 with PP1βMYPT1 and whether 14-3-3 binding is involved. It would also be important to analyse whether the effects that NUAK1 inhibitors have on suppressing mitosis (Figure 6) and cell proliferation [15] are mediated through their ability to induce dephosphorylation and hence inactivation of PLK1.

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