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Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs

K. Hanáková, O. Bernatík, M. Kravec, M. Micka, J. Kumar, J. Harnoš, P. Ovesná, P. Paclíková, M. Rádsetoulal, D. Potěšil, K. Tripsianes, L. Čajánek, Z. Zdráhal, V. Bryja,

. 2019 ; 17 (1) : 170. [pub] 20191223

Language English Country Great Britain

Document type Comparative Study, Journal Article, Research Support, Non-U.S. Gov't

Persistent link   https://www.medvik.cz/link/bmc20028702

BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.

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$a Comparative phosphorylation map of Dishevelled 3 links phospho-signatures to biological outputs / $c K. Hanáková, O. Bernatík, M. Kravec, M. Micka, J. Kumar, J. Harnoš, P. Ovesná, P. Paclíková, M. Rádsetoulal, D. Potěšil, K. Tripsianes, L. Čajánek, Z. Zdráhal, V. Bryja,
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$a BACKGROUND: Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported (CK1ε, NEK2, PLK1, CK2α, RIPK4, PKCδ) and newly identified (TTBK2, Aurora A) DVL kinases. METHODS: Shotgun proteomics including TiO2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy. RESULTS: We used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630-643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization. CONCLUSIONS: We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome. Video Abtract.
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$a Bernatík, Ondřej $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic. Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
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$a Kravec, Marek $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
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$a Micka, Miroslav $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
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$a Kumar, Jitender $u CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
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$a Harnoš, Jakub $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
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$a Ovesná, Petra $u Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
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$a Paclíková, Petra $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
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$a Rádsetoulal, Matěj $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic.
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$a Potěšil, David $u CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
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$a Tripsianes, Konstantinos $u CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
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$a Čajánek, Lukáš $u Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
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$a Zdráhal, Zbyněk $u CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic. zdrahal@sci.muni.cz. National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno, Czech Republic. zdrahal@sci.muni.cz.
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$a Bryja, Vítězslav $u Department of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37, Brno, Czech Republic. bryja@sci.muni.cz. Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic. bryja@sci.muni.cz.
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