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.

Purification and Characterization of Human ZAP-70 Protein-tyrosine Kinase from a Baculovirus Expression System

“…Further analysis of the importance of the individual tyrosine residues were performed using site-directed mutagenesis approaches and evaluation of the activity of the mutated ZAP-70 in vivo. These studies demonstrated that tyrosine 493 is critical for ZAP-70 activation and that phosphorylation of this residue is most likely the critical event that immediately follows ZAP-70 association with tyrosine phosphorylated ITAMs on TCR chains upon receptor engagement (48,49). Additional studies suggested that tyrosine phosphorylation of ZAP-70 may function to serve as a docking site for SH2-containing molecules and that through this recruiting mechanism ZAP-70 controls the activation process (42).…”

“…Recent studies with site-directed mutagenized ZAP-70 (48,49) have shown that phosphorylation of ZAP-70 by Lck on tyrosine 493 greatly increases ZAP-70 catalytic activity. Another proposed model for ZAP-70 activation is based on studies with Syk, a ZAP-70 homologous PTK with an overall similar structure, including the tandem SH2 domains.…”

“…Although Fyn is constitutively associated with nonpolymorphic components of TCR/CD3 subunits (6,7), Lck is noncovalently associated with the cytoplasmic domain of the CD4 and CD8 co-receptor molecules (8,9). Both Fyn and Lck can also directly associate with the inner leaflet of the plasma membrane via their Nterminal myristylation/palmitylation signals (10), whereas ZAP-70, which lacks this sequence, is thought to reside in the cytosol in resting cells and then becomes rapidly recruited to the TCR upon its activation and phosphorylation (11,12). Following interaction of the TCR with peptide-bound MHC molecules on the surface of antigen presenting cells, CD4 or CD8 interact with the MHC molecules, thus recruiting Lck into close proximity with the cytoplasmic tails of the TCR/CD3 subunits.…”

Cloning and characterization of APS, an adaptor molecule containing PH and SH2 domains that is tyrosine phosphorylated upon B-cell receptor stimulation

“…Iga chain may physically associate with Lyn or Fyn (Clark et al, 1992). After tyrosine-phosphorylation by these Src-family PTKs, the ITAMs recruit the Syk and ZAP70 PTKs to BCR and TCR complexes respectively (Wange et al, 1993;Iwashima et al, 1994;Kimura et al, 1996). Activated PTKs phosphorylate numerous cellular proteins, including phospholipase C (PLC)g1, Vav proto-oncogene product and adaptor proteins such as Shc and Crk (Carter et al, 1991;Bustelo and Barbacid, 1992;Panchamoorthy et al, 1996;Tezuka et al, 1996).…”

Gadd45α Regulates p38-Dependent Dendritic Cell Cytokine Production and Th1 Differentiation

“…Recombinant proteins and Abs were purchased from Sigma-Aldrich, Cell Signaling, and BD Pharmingen except anti-IL-4 (11B.11), which was obtained from the National Institutes of Health Biological Resources Branch repository, and anti-ZAP70 Ab, which was kindly provided by Dr. Larry Samelson (National Cancer Institute, National Institutes of Health, Bethesda, MD) (23). Anti-rabbit and anti-mouse Abs conjugated to AlexaFluor680 or IRDye800 were obtained from Molecular Probes or Rockland Chemicals, respectively.…”