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STIL binding to Polo-box 3 of PLK4 regulates centriole duplication.

Arquint C, Gabryjonczyk AM, Imseng S, Böhm R, Sauer E, Hiller S, Nigg EA, Maier T - Elife (2015)

Bottom Line: STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region.In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding.We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box-peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.

No MeSH data available.


Related in: MedlinePlus

PB3 of PLK4 directly interacts with STIL-CC.(A) Schematic illustration of PLK4 fragments used to map the STIL-CC binding site. Kinase domain (KD), grey; PB1, yellow; PB2, orange; PB3, blue. The relative strengths of the interactions are indicated (+, strong; -, not detected). (B–D) Western blots of co-immunoprecipitation experiments using HEK293T cells co-transfected with plasmids expressing PLK4 fragments and FLAG-STIL (B) or EGFP-STIL-CC (C and D). Antibodies used for Western blot detection are indicated. (E) Isothermal titration calorimetry of STIL-CC into a solution of PLK4-PB3. Left panel: Direct measurement of the Gibbs energy associated with STIL-CC binding to PLK4-PB3. Right panel: integrated and fitted raw data using a one-site binding model.DOI:http://dx.doi.org/10.7554/eLife.07888.009
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fig4: PB3 of PLK4 directly interacts with STIL-CC.(A) Schematic illustration of PLK4 fragments used to map the STIL-CC binding site. Kinase domain (KD), grey; PB1, yellow; PB2, orange; PB3, blue. The relative strengths of the interactions are indicated (+, strong; -, not detected). (B–D) Western blots of co-immunoprecipitation experiments using HEK293T cells co-transfected with plasmids expressing PLK4 fragments and FLAG-STIL (B) or EGFP-STIL-CC (C and D). Antibodies used for Western blot detection are indicated. (E) Isothermal titration calorimetry of STIL-CC into a solution of PLK4-PB3. Left panel: Direct measurement of the Gibbs energy associated with STIL-CC binding to PLK4-PB3. Right panel: integrated and fitted raw data using a one-site binding model.DOI:http://dx.doi.org/10.7554/eLife.07888.009

Mentions: To determine which regions of PLK4 are involved in STIL binding, we generated a series of EGFP- and FLAG-tagged PLK4 fragments comprising either the N-terminal PLK4 part (residues 1–570), including the kinase domain (1–271) and the linker region L1 (265–570), or the C-terminal region containing the Polo-boxes (PB1/2 and PB3, residues 570–970; PB1/2, residues 570–820; L2-PB3, residues 814–970; PB3, residues 880–970) (Figure 4A). Co-expression of the EGFP-tagged PLK4 fragments with FLAG-tagged full-length STIL, followed by anti-EGFP co-immunoprecipitation, revealed that both, the N-terminal PLK4 fragment spanning 1–570 or all C-terminal fragments harboring PB3 were sufficient for the interaction with full-length STIL (Figure 4B). Moreover, the STIL-CC motif alone was sufficient to bind both the N-terminus (residues 1–570) and PB3 (residues 880–970) of PLK4 (Figure 4C). Within the N-terminal PLK4 part, the linker region (265–570) participated in the interaction, whereas the kinase domain itself (1–271) did not (Figure 4D). Attempts to further narrow down the interaction region in L1 were unsuccessful, indicating a non-linear folded binding region. To quantify the PLK4-PB3/STIL-CC interaction we determined the binding affinity of PLK4-PB3 and STIL-CC using isothermal titration calorimetry. For this purpose, PB3 (residues 884–970) was recombinantly expressed in Escherichia coli and purified, and a synthetic peptide corresponding to STIL-CC was subsequently titrated into a solution of PLK4-PB3. The integrated raw data are well fitted by a one-site binding model demonstrating a direct interaction between PB3 and STIL-CC with a dissociation constant Kd of 280 ± 60 nM and an equimolar stoichiometry (Figure 4E).10.7554/eLife.07888.009Figure 4.PB3 of PLK4 directly interacts with STIL-CC.


STIL binding to Polo-box 3 of PLK4 regulates centriole duplication.

Arquint C, Gabryjonczyk AM, Imseng S, Böhm R, Sauer E, Hiller S, Nigg EA, Maier T - Elife (2015)

PB3 of PLK4 directly interacts with STIL-CC.(A) Schematic illustration of PLK4 fragments used to map the STIL-CC binding site. Kinase domain (KD), grey; PB1, yellow; PB2, orange; PB3, blue. The relative strengths of the interactions are indicated (+, strong; -, not detected). (B–D) Western blots of co-immunoprecipitation experiments using HEK293T cells co-transfected with plasmids expressing PLK4 fragments and FLAG-STIL (B) or EGFP-STIL-CC (C and D). Antibodies used for Western blot detection are indicated. (E) Isothermal titration calorimetry of STIL-CC into a solution of PLK4-PB3. Left panel: Direct measurement of the Gibbs energy associated with STIL-CC binding to PLK4-PB3. Right panel: integrated and fitted raw data using a one-site binding model.DOI:http://dx.doi.org/10.7554/eLife.07888.009
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Related In: Results  -  Collection

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fig4: PB3 of PLK4 directly interacts with STIL-CC.(A) Schematic illustration of PLK4 fragments used to map the STIL-CC binding site. Kinase domain (KD), grey; PB1, yellow; PB2, orange; PB3, blue. The relative strengths of the interactions are indicated (+, strong; -, not detected). (B–D) Western blots of co-immunoprecipitation experiments using HEK293T cells co-transfected with plasmids expressing PLK4 fragments and FLAG-STIL (B) or EGFP-STIL-CC (C and D). Antibodies used for Western blot detection are indicated. (E) Isothermal titration calorimetry of STIL-CC into a solution of PLK4-PB3. Left panel: Direct measurement of the Gibbs energy associated with STIL-CC binding to PLK4-PB3. Right panel: integrated and fitted raw data using a one-site binding model.DOI:http://dx.doi.org/10.7554/eLife.07888.009
Mentions: To determine which regions of PLK4 are involved in STIL binding, we generated a series of EGFP- and FLAG-tagged PLK4 fragments comprising either the N-terminal PLK4 part (residues 1–570), including the kinase domain (1–271) and the linker region L1 (265–570), or the C-terminal region containing the Polo-boxes (PB1/2 and PB3, residues 570–970; PB1/2, residues 570–820; L2-PB3, residues 814–970; PB3, residues 880–970) (Figure 4A). Co-expression of the EGFP-tagged PLK4 fragments with FLAG-tagged full-length STIL, followed by anti-EGFP co-immunoprecipitation, revealed that both, the N-terminal PLK4 fragment spanning 1–570 or all C-terminal fragments harboring PB3 were sufficient for the interaction with full-length STIL (Figure 4B). Moreover, the STIL-CC motif alone was sufficient to bind both the N-terminus (residues 1–570) and PB3 (residues 880–970) of PLK4 (Figure 4C). Within the N-terminal PLK4 part, the linker region (265–570) participated in the interaction, whereas the kinase domain itself (1–271) did not (Figure 4D). Attempts to further narrow down the interaction region in L1 were unsuccessful, indicating a non-linear folded binding region. To quantify the PLK4-PB3/STIL-CC interaction we determined the binding affinity of PLK4-PB3 and STIL-CC using isothermal titration calorimetry. For this purpose, PB3 (residues 884–970) was recombinantly expressed in Escherichia coli and purified, and a synthetic peptide corresponding to STIL-CC was subsequently titrated into a solution of PLK4-PB3. The integrated raw data are well fitted by a one-site binding model demonstrating a direct interaction between PB3 and STIL-CC with a dissociation constant Kd of 280 ± 60 nM and an equimolar stoichiometry (Figure 4E).10.7554/eLife.07888.009Figure 4.PB3 of PLK4 directly interacts with STIL-CC.

Bottom Line: STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region.In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding.We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box-peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.

No MeSH data available.


Related in: MedlinePlus