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.
Gravenhorst, P., Berner, F., Qadota, H., Dorendorf, T., Zhou, T., Williams, R., Kovermann, M., Benian, G., Mayans, O.
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