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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

The STIL-CC domain is essential for STIL oligomerization.(A) Schematic illustration of STIL constructs used for the co-immunoprecipitation experiments shown in (B–D). On the right, the relative strengths of the interactions are indicated (+, strong; ±, weak; -, not detected). (B–D) Western blot analysis of co-immunoprecipitation experiments to map the region required for STIL self-association. HEK293T cells were transfected with the indicated plasmids to co-express the corresponding STIL constructs for 24–36 hr. Subsequently, cells were lysed and co-immunoprecipitations were performed with anti-myc or anti-FLAG antibodies.DOI:http://dx.doi.org/10.7554/eLife.07888.008
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fig3s1: The STIL-CC domain is essential for STIL oligomerization.(A) Schematic illustration of STIL constructs used for the co-immunoprecipitation experiments shown in (B–D). On the right, the relative strengths of the interactions are indicated (+, strong; ±, weak; -, not detected). (B–D) Western blot analysis of co-immunoprecipitation experiments to map the region required for STIL self-association. HEK293T cells were transfected with the indicated plasmids to co-express the corresponding STIL constructs for 24–36 hr. Subsequently, cells were lysed and co-immunoprecipitations were performed with anti-myc or anti-FLAG antibodies.DOI:http://dx.doi.org/10.7554/eLife.07888.008

Mentions: As STIL has been shown to self-associate (Tang et al., 2011), we also tested a possible involvement of the STIL-CC motif in self-interaction. We found that STIL-MD, and, more precisely, the CC motif, is indeed strictly required for STIL self-association, whereas the STAN domain is not (Figure 3—figure supplement 1). We conclude that the CC motif is critical for the function of STIL in centriole duplication through its role in PLK4 binding, STIL self-interaction, and STIL centriolar recruitment.


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)

The STIL-CC domain is essential for STIL oligomerization.(A) Schematic illustration of STIL constructs used for the co-immunoprecipitation experiments shown in (B–D). On the right, the relative strengths of the interactions are indicated (+, strong; ±, weak; -, not detected). (B–D) Western blot analysis of co-immunoprecipitation experiments to map the region required for STIL self-association. HEK293T cells were transfected with the indicated plasmids to co-express the corresponding STIL constructs for 24–36 hr. Subsequently, cells were lysed and co-immunoprecipitations were performed with anti-myc or anti-FLAG antibodies.DOI:http://dx.doi.org/10.7554/eLife.07888.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530586&req=5

fig3s1: The STIL-CC domain is essential for STIL oligomerization.(A) Schematic illustration of STIL constructs used for the co-immunoprecipitation experiments shown in (B–D). On the right, the relative strengths of the interactions are indicated (+, strong; ±, weak; -, not detected). (B–D) Western blot analysis of co-immunoprecipitation experiments to map the region required for STIL self-association. HEK293T cells were transfected with the indicated plasmids to co-express the corresponding STIL constructs for 24–36 hr. Subsequently, cells were lysed and co-immunoprecipitations were performed with anti-myc or anti-FLAG antibodies.DOI:http://dx.doi.org/10.7554/eLife.07888.008
Mentions: As STIL has been shown to self-associate (Tang et al., 2011), we also tested a possible involvement of the STIL-CC motif in self-interaction. We found that STIL-MD, and, more precisely, the CC motif, is indeed strictly required for STIL self-association, whereas the STAN domain is not (Figure 3—figure supplement 1). We conclude that the CC motif is critical for the function of STIL in centriole duplication through its role in PLK4 binding, STIL self-interaction, and STIL centriolar recruitment.

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