Mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but not kinase activity.
The protein tyrosine kinase Syk plays a pivotal role in mediating the high-affinity IgE receptor (Fc epsilonRI)-induced degranulation of mast cells. To examine the mechanism of Syk regulation, the two tyrosine residues at 519 and 520 in the putative activation loop of rat Syk were mutated to phenylalanine either singly or in combination. The various mutants were expressed in a Syk-negative variant of the RBL-2H3 (rat basophilic leukemia 2H3) mast cell line. In these transfected cell lines, mutant Syk did show increased tyrosine phosphorylation in vivo and increased enzymatic activity in vitro after Fc epsilonRI aggregation. There were conformational changes detected by an Ab when the wild-type and mutant Syk were either tyrosine phosphorylated or bound to tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these mutant Syk were incapable of transducing Fc epsilonRI signaling. In cells in which the expression level of mutant Syk was similar to that of the wild-type Syk, Fc epsilonRI cross-linking induced no increase in cellular protein tyrosine phosphorylation, no increase in tyrosine phosphorylation of phospholipase C-gamma2 and mitogen-activated protein kinase, and no histamine release. Overexpression of Y519F or Y520F Syk mutants partially reconstituted the signaling pathways. These results indicate that these tyrosines in the putative activation loop are not essential for the enzymatic activity of Syk or for the conformational changes induced by binding of tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif peptides. However, these tyrosines are necessary for Syk-mediated propagation of Fc epsilonRI signaling.
Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA. lzhang@yoda.nidr.nih.gov
J
J
Zhang
T
T
Kimura
R P
RP
Siraganian
D000818:Animals; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D015550:Cell Degranulation / Q000276:immunology; D004792:Enzyme Precursors / Q000235:genetics / Q000276:immunology*; D047908:Intracellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000276:immunology*; D009154:Mutation; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology*; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D000072377:Syk Kinase
D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat