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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

Plk4 levels are elevated in STIL depleted cells.(A) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (B) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (A). Error bars represent SEM. (C) HeLa S3 cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (D) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (C). Error bars represent SEM. (E) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, fixed, stained with indicated antibodies and analysed in immunofluorescence microscopy. Representative images are shown. Scale bar: 1 µm. (F) Graph representing Plk4 levels measured in the centrosomal region of images as described in (E). 15 cells were measured for each condition in three independent experiments, error bars denote SEM.DOI:http://dx.doi.org/10.7554/eLife.07888.004
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fig1s1: Plk4 levels are elevated in STIL depleted cells.(A) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (B) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (A). Error bars represent SEM. (C) HeLa S3 cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (D) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (C). Error bars represent SEM. (E) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, fixed, stained with indicated antibodies and analysed in immunofluorescence microscopy. Representative images are shown. Scale bar: 1 µm. (F) Graph representing Plk4 levels measured in the centrosomal region of images as described in (E). 15 cells were measured for each condition in three independent experiments, error bars denote SEM.DOI:http://dx.doi.org/10.7554/eLife.07888.004

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)

Plk4 levels are elevated in STIL depleted cells.(A) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (B) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (A). Error bars represent SEM. (C) HeLa S3 cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (D) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (C). Error bars represent SEM. (E) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, fixed, stained with indicated antibodies and analysed in immunofluorescence microscopy. Representative images are shown. Scale bar: 1 µm. (F) Graph representing Plk4 levels measured in the centrosomal region of images as described in (E). 15 cells were measured for each condition in three independent experiments, error bars denote SEM.DOI:http://dx.doi.org/10.7554/eLife.07888.004
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Related In: Results  -  Collection

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fig1s1: Plk4 levels are elevated in STIL depleted cells.(A) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (B) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (A). Error bars represent SEM. (C) HeLa S3 cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, lysed and subjected to Western blot analysis using the indicated antibodies. (D) Graph representing Plk4 band intensities measured in Western blots from two independent experiments as described in (C). Error bars represent SEM. (E) U2OS cells were transfected with control (siGL2), three different STIL (siSTIL1-3), βTrCP and Plk4 siRNA oligonucleotides for 72 hr, fixed, stained with indicated antibodies and analysed in immunofluorescence microscopy. Representative images are shown. Scale bar: 1 µm. (F) Graph representing Plk4 levels measured in the centrosomal region of images as described in (E). 15 cells were measured for each condition in three independent experiments, error bars denote SEM.DOI:http://dx.doi.org/10.7554/eLife.07888.004
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