Regulatory mechanism of Dictyostelium myosin light chain kinase A.
In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.
Tokumitsu
Hiroshi
H
Department of Signal Transduction Sciences, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@kms.ac.jp
Hatano
Naoya
N
Inuzuka
Hiroyuki
H
Ishikawa
Yumi
Y
Uyeda
Taro Q P
TQ
Smith
Janet L
JL
Kobayashi
Ryoji
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology; D004023:Dictyostelium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D015800:Protozoan Proteins; D011994:Recombinant Proteins; C115459:MLCK-A protein, Dictyostelium discoideum; D009219:Myosin-Light-Chain Kinase