Biology subjects

Gravenhorst, P.

Publications and source records attributed to Gravenhorst, P..

2 recordsLinked to original sources

bioRxiv2026

Twitchin kinase, a mechanoreceptor in the muscle sarcomere, is a catalytically-primed moonlighting kinase

To explore conserved mechanisms and functions across mechanosensory kinases associated with the skeletal architectures of the cell, we investigated in vitro and in vivo the substrate targeting of twitchin kinase (TwcK), a mechanoreceptor from the muscle sarcomere. Specifically, we elucidated the crystal structure of TwcK in complex with substrates, used real-time 31P-NMR spectroscopy and luminescence-based assays to identify the phosphorylation site on a model peptide substrate, mined the C. elegans proteome to reveal the myosin regulatory protein MLC-4 as a substrate candidate and used CRISPR/Cas9 genome-edited and transgenic C. elegans strains to query the relation of twitchin and MLC-4 in muscle. Contrary to expectations, we find that TwcK undergoes activating conformational changes that are regulated by an N-terminal tail sequence that blocks hinge dynamics in the kinase fold. This distinct mechanism is conserved across sarcomeric, but not cytoskeletal, kinases. Functionally, cytoskeletal and sarcomeric kinases share an evolutionarily conserved phosphorylation targeting of myosin light chain (MLC) proteins. Yet, we find TwcK and its MLC4 substrate to segregate in vivo and not to constitute a functional kinase/substrate pair. Thus, canonical substrate targeting cannot be delivered by TwcK in its cellular context, where it has adopted a moonlighting role. We deduce this result to apply to other intrasarcomeric kinases. Our findings highlight how the cell context confers functional individuality to non-diffusible, otherwise conserved skeletal kinases.

biophysics↗

bioRxiv2025

Defining the molecular determinants of titin kinase by analysing distantly evolved fish orthologues

Titin kinase (TK) is a pseudokinase specific to the striated muscle of vertebrates. Embedded within the contractile sarcomere and flanked by extensible regulatory tails, TK is thought to sense mechanical signals arising from muscle function. Studies on TK to date have focused narrowly on the human representative. To investigate if a pseudokinase character is a hallmark of TK, we have studied sequences of distantly evolved fish representatives and rationalized conservation patterns by resolving the crystal structure of TK from medaka isoform b. We find that sequence alterations in catalytic motifs involved in ATP and magnesium binding, respectively {theta}xK ({theta}: bulky hydrophobic residue) and EFG, are evolutionarily conserved in TK. Beyond the kinase domain, N- and C-terminal flanking tails show remarkable structural similarity across human and medaka orthologues, even though sequence conservation is limited to individual residues and short motifs: a YD-motif in the N-terminal tail; a [R/K]H[R/K]RYY sequence, a R-7x-R motif and position -2 of the latter in the C-terminal tail. We observe that motifs in the C-terminal tail consistently covary with the divergent functional motifs of TK, being part of its pseudokinase signature. In contrast, the proposed inhibition of the catalytic aspartate by a tyrosine residue from the P+1 loop is not significantly conserved outside mammals. Finally, based on these features and sequence clustering analysis, we propose a classification of titin gene duplicates from fish into a and b isoforms (ttna and ttnb) that can assist future comparative studies. A curated genomic annotation is provided here.

bioinformatics↗