AI Insight
This study investigates twitchin kinase (TwcK), a mechanosensory protein found in muscle sarcomeres, using a combination of crystallography, NMR spectroscopy, biochemical assays, and CRISPR-edited C. elegans models. The researchers found that TwcK is activated through a distinct conformational mechanism controlled by an N-terminal tail sequence, a feature conserved in sarcomeric but not cytoskeletal kinases. Although TwcK can target myosin light chain proteins in vitro, consistent with evolutionarily conserved substrate targeting across related kinases, the enzyme and its candidate substrate MLC-4 are physically segregated in living muscle cells, meaning TwcK does not function as a conventional kinase in its native cellular context and instead fulfills an alternative, so-called moonlighting role.
Why it matters
Understanding how sarcomeric kinases adopt non-canonical functions in their cellular environment may shed light on the molecular basis of muscle diseases linked to titin and twitchin-related proteins. This work also has broader implications for how structural context shapes the functional evolution of signaling enzymes, which is relevant to drug target identification in musculoskeletal disorders.
⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
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 physiological 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.