Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.
Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.
Smith
J L
JL
Department of Biochemistry, Beckman Center, Stanford University Medical Center, CA 94305-5307, USA.
Silveira
L A
LA
Spudich
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D003208:Concanavalin A / Q000494:pharmacology; D004023:Dictyostelium / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013912:Threonine / Q000378:metabolism*
D010710:Phosphates; D011994:Recombinant Proteins; D003208:Concanavalin A; D010769:Phosphothreonine; D013912:Threonine; D018698:Glutamic Acid; D009219:Myosin-Light-Chain Kinase