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Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade
S C Bunnell1, M Diehn, M B Yaffe
1Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Abstract:
Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.
Insights
Itk kinase is recruited to T cell receptor signaling complexes through multiple interactions, enabling T cell activation. Kinase-inactive Itk can block T cell activation by inhibiting SLP-76 signaling.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Itk (Interleukin-2-inducible T-cell kinase) is a Tec family tyrosine kinase crucial for T cell receptor (TCR) signaling.
- Itk functions downstream of Lck and PI3K, regulating calcium influx and ERK activity.
- Understanding Itk's interactions is key to deciphering TCR-mediated immune responses.
Purpose of the Study:
- To elucidate the specific molecular interactions of Itk within the TCR signaling pathway.
- To identify the domains of Itk responsible for its recruitment and function in T cells.
- To investigate the role of Itk in regulating T cell activation, specifically NF-AT signaling.
Main Methods:
- Co-immunoprecipitation and in vitro binding assays to demonstrate protein-protein interactions.
- Analysis of Itk domain functions using kinase-inactive mutants.
- Assessment of NF-AT activation in response to TCR stimulation.
Main Results:
- Itk constitutively associates with buoyant membranes via its pleckstrin homology domain, interacting with Lck and LAT.
- The proline-rich region of Itk binds Grb2 and LAT, while SH3 and SH2 domains interact with SLP-76.
- Kinase-inactive Itk inhibits SLP-76-dependent NF-AT activation, with the PH, proline-rich, and SH2 domains being essential for this inhibition.
Conclusions:
- Multivalent interactions involving Itk's domains recruit it to LAT-signaling complexes.
- Itk facilitates the activation of phospholipase Cgamma1 (PLCγ1) by LAT-associated proteins.
- Itk plays a critical role in TCR-dependent T cell activation, partly through modulation of SLP-76 and NF-AT pathways.