Autophosphorylation activates Dictyostelium myosin II heavy chain kinase A by providing a ligand for an allosteric binding site in the α-kinase domain

Scott W. Crawley, Mojdeh Samimi Gharaei, Qilu Ye, Yidai Yang, Barak Raveh, Nir London, Ora Schueler-Furman, Zongchao Jia, Graham P. Côté

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20 Scopus citations

Abstract

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCKA (residues 552-841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806-841). The key residue in the C-tail was identified as Thr 825, which was found to be constitutively autophosphorylated. Dephosphorylation of Thr825 using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr 825 could be rescued by Pi, phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a P i-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the Pi-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr825 activates ACAT by providing a covalently tethered ligand for the Pi-pocket. Ab initio modeling studies using the Rosetta FloppyTail and Flex-PepDock protocols showed that it is feasible for the phosphorylated Thr825 to dock intramolecularly into the P i-pocket. Allosteric activation is predicted to involve a conformational change in Arg734, which bridges the bound P i to Asp762 in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCKB-D and metazoan eukaryotic elongation factor-2 kinases.

Original languageAmerican English
Pages (from-to)2607-2616
Number of pages10
JournalJournal of Biological Chemistry
Volume286
Issue number4
DOIs
StatePublished - 28 Jan 2011

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