Autophosphorylation is a mechanism of inhibition in twitchin kinase

Williams, Rhys, Franke, Barbara, Wilkinson, Mark, Fleming, Jennifer, Rigden, DJ ORCID: 0000-0002-7565-8937, Benian, Guy, Eyers, Patrick ORCID: 0000-0002-9220-2966 and Mayans, Olga (2018) Autophosphorylation is a mechanism of inhibition in twitchin kinase. Journal of Molecular Biology, 430 (6). pp. 793-805.

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Abstract

Titin-like kinases are muscle-specific kinases that regulate mechanical sensing in the sarcomere. Twitchin kinase (TwcK) is the best-characterized member of this family, both structurally and enzymatically. TwcK activity is auto-inhibited by a dual intrasteric mechanism, in which N- and C-terminal tail extensions wrap around the kinase domain, blocking the hinge region, the ATP binding pocket and the peptide substrate binding groove. Physiologically, kinase activation is thought to occur by a stretch-induced displacement of the inhibitory tails from the kinase domain. Here, we now show that TwcK inhibits its catalysis even in the absence of regulatory tails, by undergoing auto-phosphorylation at mechanistically important elements of the kinase fold. Using mass spectrometry, site-directed mutagenesis and catalytic assays on recombinant samples, we identify residues T212, T301, T316 and T401 as primary auto-phosphorylation sites in TwcK in vitro. Taken together, our results suggest that residue T316, located in the peptide substrate binding P + 1 loop, is the dominantly regulatory site in TwcK. Based on these findings, we conclude that TwcK is regulated through a triple-inhibitory mechanism consisting of phosphorylation and intrasteric blockage, which is responsive not only to mechanical cues but also to biochemical modulation. This implies that mechanically stretched conformations of TwcK do not necessarily correspond to catalytically active states, as previously postulated. This further suggests a phosphorylation-dependent desensitization of the TwcK-mediated mechanoresponse of the sarcomere in vivo.

Item Type:	Article
Uncontrolled Keywords:	kinase regulation, mass spectrometry, site-directed mutagenesis, phosphotransfer catalysis
Depositing User:	Symplectic Admin
Date Deposited:	01 Feb 2018 08:22
Last Modified:	19 Jan 2023 06:42
DOI:	10.1016/j.jmb.2018.01.020
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3017277