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Updated: Aug 21, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
The Y105 phosphosite between the SH3.1 and SH3.2 domains of Nck1 regulates its binding to CD3ϵ
Jatuporn Ngoenkam1, Piyamaporn Wipa2, Pussadee Paensuwan3
1Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand.
Abstract:
In αβ T cells, ligand binding to T cell receptor (TCR) triggers conformational changes at CD3ε subunits to expose the proline-rich sequence (PRS), which binds to the non-catalytic region of tyrosine kinase (Nck) via its Src homology 3 (SH3).1 domain. Recruitment of Nck to CD3ε initiates phosphorylation of various signaling proteins that activate T cells. However, the mechanisms by which Nck associates with and dissociates from the TCR remain poorly understood. This study identifies the tyrosine 105 (Y105) phosphosite between the SH3.1 and SH3.2 domains of Nck1 that regulates the Nck1-CD3ϵ association. Notably, a Y105 mutation resulted in prolonged association of Nck1 with TCR, impaired the phosphorylation of CD3ε, TCRζ, ζ-chain-associated protein kinase 70, and extracellular signal-regulated kinase 1/2, and abrogated the recruitment of lymphocyte-specific kinase (Lck) to TCR. These findings revealed the function of Y105 as a molecular switch that controls the assembly and disassembly of Nck1 and CD3ε.
Insights
Tyrosine 105 (Y105) on Nck1 acts as a molecular switch, controlling how Nck1 binds and releases from the T cell receptor (TCR) complex. This regulation is crucial for T cell activation signaling pathways.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- T cell receptor (TCR) activation involves CD3ε conformational changes, exposing a proline-rich sequence (PRS).
- The PRS binds to the Src homology 3 (SH3).1 domain of Nck, initiating T cell signaling cascades.
- Mechanisms governing Nck's dynamic association with the TCR remain unclear.
Purpose of the Study:
- To elucidate the regulatory mechanisms of Nck association and dissociation from the TCR complex.
- To identify specific phosphosites on Nck that control its interaction with CD3ε.
Main Methods:
- Site-directed mutagenesis of Nck1, specifically targeting tyrosine 105 (Y105).
- Analysis of Nck1-TCR complex formation and dissociation dynamics.
- Phosphorylation assays for key signaling molecules including CD3ε, TCRζ, ZAP70, and ERK1/2.
- Assessment of lymphocyte-specific kinase (Lck) recruitment to the TCR.
Main Results:
- Identification of Y105 as a critical phosphosite regulating Nck1-CD3ε interaction.
- A Y105 mutation led to sustained Nck1 binding to the TCR.
- Impaired phosphorylation of CD3ε, TCRζ, ZAP70, and ERK1/2 in Y105 mutants.
- Abrogation of Lck recruitment to the TCR upon Y105 mutation.
Conclusions:
- Y105 functions as a molecular switch governing Nck1 assembly and disassembly with CD3ε.
- This phosphosite is essential for proper TCR signaling initiation and propagation.
- Understanding Y105 regulation offers insights into controlling T cell activation.
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