Autophosphorylation activates Dictyostelium myosin II heavy chain kinase A by providing a ligand for an allosteric binding site in the alpha-kinase domain.
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.
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Scott W
SW
Crawley
Mojdeh Samimi
MS
Gharaei
Qilu
Q
Ye
Yidai
Y
Yang
Barak
B
Raveh
Nir
N
London
Ora
O
Schueler-Furman
Zongchao
Z
Jia
Graham P
GP
Côté
D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D009154:Mutation; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase