Disrupting the intermolecular self-association of Itk enhances T cell signaling.
The Tec family tyrosine kinase (Itk), is a key component of the TCR signaling pathway. Biochemical studies have shown that Itk activation requires recruitment of Itk to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. However, the regulation of Itk enzymatic activity by Itk domain interactions is not yet well understood. In this study, we show that full-length Itk self-associates in an intermolecular fashion. Using this information, we have designed an Itk variant that exhibits reduced self-association but maintains normal binding to exogenous ligands via each of its regulatory domains. When expressed in insect cells, the Itk substrate phospholipase Cgamma1 is phosphorylated more efficiently by the Itk variant than by wild-type Itk. Furthermore, expression of the Itk variant in primary murine T cells induced higher ERK activation and increased calcium flux following TCR stimulation compared with that of wild-type Itk. Our results indicate that the Tec kinase Itk is negatively regulated by intermolecular clustering and that disruption of this clustering leads to increased Itk kinase activity following TCR stimulation.
Department of Biochemistry, Iowa State University, Ames, IA 50011, USA.
Lie
L
Min
Wenfang
W
Wu
Raji E
RE
Joseph
D Bruce
DB
Fulton
Leslie
L
Berg
Amy H
AH
Andreotti
D000818:Animals; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000201:enzymology* / Q000276:immunology*; D002454:Cell Differentiation / Q000235:genetics / Q000276:immunology; D019070:Cell Lineage / Q000235:genetics / Q000276:immunology; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology; D004789:Enzyme Activation / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors / Q000276:immunology; D051379:Mice; D018448:Models, Immunological; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics / Q000276:immunology
D011948:Receptors, Antigen, T-Cell; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase