Identification by mass spectrometry of the phosphorylated residue responsible for activation of the catalytic domain of myosin I heavy chain kinase, a member of the PAK/STE20 family.
Myosin I heavy chain kinase from Acanthamoeba castellanii is activated in vitro by autophosphorylation (8-10 mol of P per mol). The catalytically active C-terminal domain produced by trypsin cleavage of the phosphorylated kinase contains 2-3 mol of P per mol. However, the catalytic domain expressed in a baculovirus-insect cell system is fully active as isolated without autophosphorylation in vitro. We now show that the expressed catalytic domain is inactivated by incubation with acid phosphatase and regains activity upon autophosphorylation. The state of phosphorylation of all of the hydroxyamino acids in the catalytic domain were determined by mass spectrometry of unfractionated protease digests. Ser-627 was phosphorylated in the active, expressed catalytic domain, lost its phosphate when the protein was incubated with phosphatase, and was rephosphorylated when the dephosphorylated protein was incubated with ATP. No other residue was significantly phosphorylated in any of the three samples. Thus, phosphorylation of Ser-627, which is in the same position as the Ser and Thr residues that are phosphorylated in many other kinases, is necessary and sufficient for full activity of the catalytic domain. Ser-627 is also phosphorylated when full-length, native kinase is activated by autophosphorylation.
Szczepanowska
J
J
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
X
X
Herring
C J
CJ
Qin
J
J
Korn
E D
ED
Brzeska
H
H
D000048:Acanthamoeba; D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D015800:Protozoan Proteins
D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase