dictyNews
Electronic Edition
Volume 35, number 14
Nov 19, 2010

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=========
Abstracts
=========


Autophosphorylation activates Dictyostelium Myosin II Heavy Chain Kinase A 
by providing a ligand for an allosteric binding site in the alpha-kinase domain

Scott W. Crawley1, Mojdeh Samimi Gharaei 1, Qilu Ye1, Yidai Yang1, 
Barak Raveh2, Nir London2, Ora Schueler-Furman2, Zongchao Jia1 
and Graham P. Côté1

1Department of Biochemistry, Queen’s University, Kingston, Ontario, 
Canada K7L 3N6
2Department of Microbiology and Molecular Genetics, Institute for Medical 
Research Israel-Canada, Hadassah Medical School, The Hebrew University, 
Jerusalem, 91120 Israel


The Journal of Biological Chemistry, in press

Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member 
of the atypical alpha-kinase family, phosphorylates sites in the myosin II tail that 
block filament assembly. Here we show that the catalytic activity of A-CAT, the 
alpha-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 Thr825, which was found to be 
constitutively autophosphorylated. Dephosphorylation of Thr825 using shrimp 
alkaline phosphatase decreased A-CAT activity. The activity of a truncated 
ACAT lacking Thr825 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 Pi-binding 
pocket located in the C-terminal lobe of ACAT. 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 FlexPepDock protocols 
showed that it is feasible for the phosphorylated Thr825 to dock intramolecularly 
into the Pi-pocket. Allosteric activation is predicted to involve a conformational 
change in Arg734, which bridges the bound Pi to Asp762 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.


Submitted by Scott Crawley [crawleys@queensu.ca]
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[End dictyNews, volume 35, number 14]