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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

STIL is a phosphorylation target of PLK4 and PLK4-ND.(A) FLAG-STIL is phosphorylated by GST-PLK41–430 in vitro. HEK293T cells were transfected with FLAG-STIL for 48 hr. After cell lysis, FLAG-STIL was purified using anti-FLAG antibodies and subjected to an in vitro kinase assay with recombinant GST-PLK41–430 or, as a control, with a kinase-inactive version of GST-PLK41–430 (D154A). On the left, the Western blot probed with anti-FLAG antibody shows the amounts of FLAG-STIL used for the reactions. The autoradiograph (right side) shows the phosphorylation of FLAG-STIL by GST-PLK41–430 (upper band) as well as the autophosphorylation of GST-PLK41–430 (lower band). (B) HEK293T cells were transfected with the indicated plasmids and cell extracts were subjected to anti-myc co-immunoprecipitations followed by Western blot analysis. PLK4-ND—the non-degradable PLK4 mutant used throughout this study (S285A and T289A [Guderian et al., 2010])—exhibits enhanced stabilization and thus facilitates visualization of STIL binding (higher amounts of FLAG-STIL were detected in the precipitate of myc-PLK4-ND compared to wild-type myc-PLK4). Note the upshift of the STIL band upon co-expression with PLK4-ND, indicating that FLAG-STIL is phosphorylated by myc-PLK4-ND.DOI:http://dx.doi.org/10.7554/eLife.07888.005
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fig1s2: STIL is a phosphorylation target of PLK4 and PLK4-ND.(A) FLAG-STIL is phosphorylated by GST-PLK41–430 in vitro. HEK293T cells were transfected with FLAG-STIL for 48 hr. After cell lysis, FLAG-STIL was purified using anti-FLAG antibodies and subjected to an in vitro kinase assay with recombinant GST-PLK41–430 or, as a control, with a kinase-inactive version of GST-PLK41–430 (D154A). On the left, the Western blot probed with anti-FLAG antibody shows the amounts of FLAG-STIL used for the reactions. The autoradiograph (right side) shows the phosphorylation of FLAG-STIL by GST-PLK41–430 (upper band) as well as the autophosphorylation of GST-PLK41–430 (lower band). (B) HEK293T cells were transfected with the indicated plasmids and cell extracts were subjected to anti-myc co-immunoprecipitations followed by Western blot analysis. PLK4-ND—the non-degradable PLK4 mutant used throughout this study (S285A and T289A [Guderian et al., 2010])—exhibits enhanced stabilization and thus facilitates visualization of STIL binding (higher amounts of FLAG-STIL were detected in the precipitate of myc-PLK4-ND compared to wild-type myc-PLK4). Note the upshift of the STIL band upon co-expression with PLK4-ND, indicating that FLAG-STIL is phosphorylated by myc-PLK4-ND.DOI:http://dx.doi.org/10.7554/eLife.07888.005

Mentions: As 3D-SIM imaging of U2OS cells revealed extensive co-localization of STIL and PLK4 at the proximal end of daughter centrioles (Figure 1A), we asked whether the two proteins depend on each other for recruitment to this site. Upon depletion of PLK4, localization of STIL to centrioles was drastically reduced (1.7 ± 2.3% residual intensity compared to untreated cells, Figure 1B), suggesting that PLK4 is essential for STIL centriolar targeting and/or maintenance. On the other hand, PLK4 localization to centrioles was not abrogated in STIL depleted cells. On the contrary, centriolar PLK4 levels were strongly elevated and PLK4 localized in a ring-, rather than a spot-like pattern to the outer wall of centrioles (Ohta et al., 2014) (Figure 1C). Western blot analysis confirmed significant elevation of PLK4 levels in STIL depleted cells (Figure 1—figure supplement 1A–D). This increase in PLK4 levels was comparable to that observed after depletion of βTrCP, which is known to interfere with PLK4 degradation (Cunha-Ferreira et al., 2009; Rogers et al., 2009; Guderian et al., 2010; Holland et al., 2010) (Figure 1—figure supplement 1). These data suggest that PLK4 degradation is strongly reduced in the absence of STIL, which then results in its accumulation around centrioles. In further support of a functional interaction between PLK4 and STIL, we also observed that STIL was phosphorylated by a recombinant GST-PLK41-430 fusion protein in vitro (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015) (Figure 1—figure supplement 2A).10.7554/eLife.07888.003Figure 1.STIL is an interaction partner of PLK4.


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)

STIL is a phosphorylation target of PLK4 and PLK4-ND.(A) FLAG-STIL is phosphorylated by GST-PLK41–430 in vitro. HEK293T cells were transfected with FLAG-STIL for 48 hr. After cell lysis, FLAG-STIL was purified using anti-FLAG antibodies and subjected to an in vitro kinase assay with recombinant GST-PLK41–430 or, as a control, with a kinase-inactive version of GST-PLK41–430 (D154A). On the left, the Western blot probed with anti-FLAG antibody shows the amounts of FLAG-STIL used for the reactions. The autoradiograph (right side) shows the phosphorylation of FLAG-STIL by GST-PLK41–430 (upper band) as well as the autophosphorylation of GST-PLK41–430 (lower band). (B) HEK293T cells were transfected with the indicated plasmids and cell extracts were subjected to anti-myc co-immunoprecipitations followed by Western blot analysis. PLK4-ND—the non-degradable PLK4 mutant used throughout this study (S285A and T289A [Guderian et al., 2010])—exhibits enhanced stabilization and thus facilitates visualization of STIL binding (higher amounts of FLAG-STIL were detected in the precipitate of myc-PLK4-ND compared to wild-type myc-PLK4). Note the upshift of the STIL band upon co-expression with PLK4-ND, indicating that FLAG-STIL is phosphorylated by myc-PLK4-ND.DOI:http://dx.doi.org/10.7554/eLife.07888.005
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Related In: Results  -  Collection

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fig1s2: STIL is a phosphorylation target of PLK4 and PLK4-ND.(A) FLAG-STIL is phosphorylated by GST-PLK41–430 in vitro. HEK293T cells were transfected with FLAG-STIL for 48 hr. After cell lysis, FLAG-STIL was purified using anti-FLAG antibodies and subjected to an in vitro kinase assay with recombinant GST-PLK41–430 or, as a control, with a kinase-inactive version of GST-PLK41–430 (D154A). On the left, the Western blot probed with anti-FLAG antibody shows the amounts of FLAG-STIL used for the reactions. The autoradiograph (right side) shows the phosphorylation of FLAG-STIL by GST-PLK41–430 (upper band) as well as the autophosphorylation of GST-PLK41–430 (lower band). (B) HEK293T cells were transfected with the indicated plasmids and cell extracts were subjected to anti-myc co-immunoprecipitations followed by Western blot analysis. PLK4-ND—the non-degradable PLK4 mutant used throughout this study (S285A and T289A [Guderian et al., 2010])—exhibits enhanced stabilization and thus facilitates visualization of STIL binding (higher amounts of FLAG-STIL were detected in the precipitate of myc-PLK4-ND compared to wild-type myc-PLK4). Note the upshift of the STIL band upon co-expression with PLK4-ND, indicating that FLAG-STIL is phosphorylated by myc-PLK4-ND.DOI:http://dx.doi.org/10.7554/eLife.07888.005
Mentions: As 3D-SIM imaging of U2OS cells revealed extensive co-localization of STIL and PLK4 at the proximal end of daughter centrioles (Figure 1A), we asked whether the two proteins depend on each other for recruitment to this site. Upon depletion of PLK4, localization of STIL to centrioles was drastically reduced (1.7 ± 2.3% residual intensity compared to untreated cells, Figure 1B), suggesting that PLK4 is essential for STIL centriolar targeting and/or maintenance. On the other hand, PLK4 localization to centrioles was not abrogated in STIL depleted cells. On the contrary, centriolar PLK4 levels were strongly elevated and PLK4 localized in a ring-, rather than a spot-like pattern to the outer wall of centrioles (Ohta et al., 2014) (Figure 1C). Western blot analysis confirmed significant elevation of PLK4 levels in STIL depleted cells (Figure 1—figure supplement 1A–D). This increase in PLK4 levels was comparable to that observed after depletion of βTrCP, which is known to interfere with PLK4 degradation (Cunha-Ferreira et al., 2009; Rogers et al., 2009; Guderian et al., 2010; Holland et al., 2010) (Figure 1—figure supplement 1). These data suggest that PLK4 degradation is strongly reduced in the absence of STIL, which then results in its accumulation around centrioles. In further support of a functional interaction between PLK4 and STIL, we also observed that STIL was phosphorylated by a recombinant GST-PLK41-430 fusion protein in vitro (Dzhindzhev et al., 2014; Ohta et al., 2014; Kratz et al., 2015) (Figure 1—figure supplement 2A).10.7554/eLife.07888.003Figure 1.STIL is an interaction partner of PLK4.

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