title: Activating and inhibitory mutations in adjacent tyrosines in the kinase domain of ZAP-70.
abstract: ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is is positively regulated by tyrosine phosphorylation. Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement. To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for the tyrosine phosphorylation-induced activation of ZAP-70. The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation. These results reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.
authors.affiliation: Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: R L
authors.initials: RL
authors.lastname: Wange
authors.forename: R
authors.initials: R
authors.lastname: Guitián
authors.forename: N
authors.initials: N
authors.lastname: Isakov
authors.forename: J D
authors.initials: JD
authors.lastname: Watts
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
authors.forename: L E
authors.initials: LE
authors.lastname: Samelson
mesh_terms: D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase