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Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-as...

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Autores principales: Venkat, Aarya, Watterson, Grace, Byrne, Dominic P., O’Boyle, Brady, Shrestha, Safal, Gravel, Nathan, Fairweather, Emma E., Daly, Leonard A., Bunn, Claire, Yeung, Wayland, Aggarwal, Ishan, Katiyar, Samiksha, Eyers, Claire E., Eyers, Patrick A., Kannan, Natarajan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081240/
https://www.ncbi.nlm.nih.gov/pubmed/37034755
http://dx.doi.org/10.1101/2023.03.29.534689
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author Venkat, Aarya
Watterson, Grace
Byrne, Dominic P.
O’Boyle, Brady
Shrestha, Safal
Gravel, Nathan
Fairweather, Emma E.
Daly, Leonard A.
Bunn, Claire
Yeung, Wayland
Aggarwal, Ishan
Katiyar, Samiksha
Eyers, Claire E.
Eyers, Patrick A.
Kannan, Natarajan
author_facet Venkat, Aarya
Watterson, Grace
Byrne, Dominic P.
O’Boyle, Brady
Shrestha, Safal
Gravel, Nathan
Fairweather, Emma E.
Daly, Leonard A.
Bunn, Claire
Yeung, Wayland
Aggarwal, Ishan
Katiyar, Samiksha
Eyers, Claire E.
Eyers, Patrick A.
Kannan, Natarajan
author_sort Venkat, Aarya
collection PubMed
description Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory ‘tail’ segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to ‘supercharge’ the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a ‘Swiss-army’ assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome–wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.
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spelling pubmed-100812402023-04-08 Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases Venkat, Aarya Watterson, Grace Byrne, Dominic P. O’Boyle, Brady Shrestha, Safal Gravel, Nathan Fairweather, Emma E. Daly, Leonard A. Bunn, Claire Yeung, Wayland Aggarwal, Ishan Katiyar, Samiksha Eyers, Claire E. Eyers, Patrick A. Kannan, Natarajan bioRxiv Article Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory ‘tail’ segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to ‘supercharge’ the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a ‘Swiss-army’ assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome–wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders. Cold Spring Harbor Laboratory 2023-07-18 /pmc/articles/PMC10081240/ /pubmed/37034755 http://dx.doi.org/10.1101/2023.03.29.534689 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
Venkat, Aarya
Watterson, Grace
Byrne, Dominic P.
O’Boyle, Brady
Shrestha, Safal
Gravel, Nathan
Fairweather, Emma E.
Daly, Leonard A.
Bunn, Claire
Yeung, Wayland
Aggarwal, Ishan
Katiyar, Samiksha
Eyers, Claire E.
Eyers, Patrick A.
Kannan, Natarajan
Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title_full Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title_fullStr Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title_full_unstemmed Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title_short Mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in DCLK family kinases
title_sort mechanistic and evolutionary insights into isoform-specific ‘supercharging’ in dclk family kinases
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10081240/
https://www.ncbi.nlm.nih.gov/pubmed/37034755
http://dx.doi.org/10.1101/2023.03.29.534689
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