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 catalyti cally active C-terminal domain produced by trypsin cleavage of the pho sphorylated kinase contains 2-3 mol of P per mol. However, the catalyt ic domain expressed in a baculovirus-insect cell system is fully activ e as isolated without autophosphorylation in vitro. We now show that t he expressed catalytic domain is inactivated by incubation with acid p hosphatase and regains activity upon autophosphorylation, The state of phosphorylation of all of the hydroxyamino acids in the catalytic dom ain were determined by mass spectrometry of unfractionated protease di gests. Ser-627 was phosphorylated in the active, expressed catalytic d omain, lost its phosphate when the protein was incubated with phosphat ase, and was rephosphorylated when the dephosphorylated protein was in cubated with ATF, 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 phosphorylat ed in many other kinases, is necessary and sufficient for full activit y of the catalytic domain, Ser-627 is also phosphorylated when full-le ngth, native kinase is activated by autophosphorylation.