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

doi:10.1074/jbc.m110.177014

Scott W Crawley, Mojdeh Samimi Gharaei + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m110.177014

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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 MHCK A (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 Thr(825) using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr(825) could be rescued by P(i), 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 P(i)-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr(825) activates ACAT by providing a covalently tethered ligand for the P(i)-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr(825) to dock intramolecularly into the P(i)-pocket. Allosteric activation is predicted to involve a conformational change in Arg(734), which bridges the bound P(i) to Asp(762) in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.

Highlights

Dictyostelium discoideum MHCK A3 is a highly specialized protein kinase that targets three threonine residues located in
To model the C-terminal tail sequence (C-tail) at the active site a soft constraint was imposed in the initial low resolution step that required that the C␤ atom of Thr825 be within 3– 6 Å of the C␤ atom of Asp756, which is presumed to act as the catalytic base in the phosphotransferase reaction [23]
myosin II heavy chain kinase A (MHCK A) is potently activated by autophosphorylation but the identity of the autophosphorylation sites and the mechanisms involved in the activation process have remained obscure

Summary

EXPERIMENTAL PROCEDURES

Materials—Peptides used in this study were synthesized by the Sheldon Biotechnology Facility, McGill University. Autophosphorylation assays contained 14.5 ␮M A-CAT-5xA and were carried out in kinase buffer containing [␥-32P]ATP at a specific activity of 500 cpm/pmol of ATP. Crystallization Procedures and Data Collection and Analysis—A-CAT truncated at Leu809 (A-CAT-⌬809) was concentrated to 230 ␮M using a 10,000 molecular weight cutoff Ultrafree-4 Centrifugal Filtration Unit (Millipore) The His tag was cleaved off by incubation overnight at 4 °C with AcTEV protease (Invitrogen), and the protease was removed by chromatography over a His-Bind column. To model the C-tail at the active site a soft constraint was imposed in the initial low resolution step that required that the C␤ atom of Thr825 be within 3– 6 Å of the C␤ atom of Asp756, which is presumed to act as the catalytic base in the phosphotransferase reaction [23]. An ATP molecule at the active site was included, starting from its orientation in the A-CAT-D766A structure (PDB code 3LMI)

RESULTS

DISCUSSION

In several experiments the kinase and ATPase activities of