Autophosphorylation Is a Mechanism of Inhibition in Twitchin Kinase. Issue 6 (16th March 2018)

• Record Type: Journal Article
• Authors: Williams, Rhys M.; Franke, Barbara; Wilkinson, Mark; Fleming, Jennifer R.; Rigden, Daniel J.; Benian, Guy M.; Eyers, Patrick A.; Mayans, Olga
• Is Part Of: Journal of molecular biology. Volume 430:Issue 6(2018)
• Page Start: 793
• Page End: 805
• Publication Date: 2018-03-16
• Subjects: kinase regulation -- mass spectrometry -- site-directed mutagenesis -- phosphotransfer catalysis; TwcK twitchin kinase -- NL N-terminal linker -- CRD C-terminal regulatory domain -- MS mass spectrometry -- SEC size exclusion chromatography -- MDS molecular dynamics simulation -- ZIPK Zipper Interacting Protein Kinase -- TK titin kinase; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2018.01.020
• Languages: English
• ISSNs: 0022-2836

Abstract

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 . Graphical abstract: Highlights: TwcK is inhibited by an intrasteric mechanism, but its regulation by phosphorylation is unknown. We find that TwcK undergoes hyper-phosphorylation in mechanistic motifs that leads to inhibition. Thus, TwcK has a triple mechanism of inhibition, where regulation uses both mechanical and biochemical cues. The findings challenge current models of kinase mechanical sensing, where stretch correlates with catalysis.