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-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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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 18, 2023. ; https://doi.org/10.1101/2023.03.29.534689 doi: bioRxiv preprint

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 18, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  Quantitative LC-MS/MS data showing tryptic phosphopeptides identified from DCLK1.1 and 1.2 that were directly comparable between isoforms. Detailed are peptide sequences, identified sites of phosphorylation (red), the site of phosphorylation within the protein polypeptide and the ptmRS score relevant to confidence of phosphosite localisation, as well as the Mascot score for peptide identification. Fold-changes in the relative abundance of the two phosphopeptides in DCLK 1.2 are computed with reference to these same two phosphopeptides in DCLK1.1, normalising against 3 non-modified peptides to account for potential difference in the amount analysed. C) As described in B, quantitative LC-MS/MS data for sites directly comparable between DCLK1.2 and its variants. Fold change in abundance could not be calculated for the peptide containing pThr 395 given the presence of the inserted amino acid mutations and the differences in relative ionisation efficiency for the resulting tryptic peptide.
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