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Functional cooperation between c-Cbl and Src-like adaptor protein 2 in the negative regulation of T-cell receptor
Michael P Loreto1, Donna M Berry, C Jane McGlade
1Department of Medical Biophysics, University of Toronto and Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
Abstract:
Adaptor proteins assemble multiprotein signaling complexes, enabling the transduction of intracellular signals. While many adaptor proteins positively regulate signaling in this manner, a subgroup of adaptors function as negative regulators. Here we report the identification of a hematopoiesis-specific adaptor protein that we have designated Src-like adaptor protein 2 (SLAP-2). SLAP-2 is most closely related to SLAP and contains a Src homology 3 (SH3) domain and an SH2 domain, as well as an amino-terminal myristoylation site that mediates SLAP-2 association with membranes. Following stimulation of primary thymocytes with anti-CD3 and anti-CD28, SLAP-2 coimmunoprecipitates with tyrosine-phosphorylated c-Cbl and an unidentified protein of approximately 72 kDa. In activated Jurkat T cells, SLAP-2 also binds an additional 70-kDa phosphoprotein, identified as ZAP-70. Binding of SLAP-2 to both p72 and ZAP-70 is dependent on its SH2 domain, while c-Cbl interacts with the carboxy-terminal region. Overexpression of wild-type SLAP-2 alone or in combination with c-Cbl in Jurkat T cells leads to inhibition of T-cell antigen receptor-induced activation of nuclear factor of activated T cells. The inhibitory effect of SLAP-2 requires the carboxy-terminal c-Cbl binding region. Expression of SLAP-2 with SYK or ZAP-70 in COS cells or Jurkat T cells causes the degradation of these kinases, and SLAP-2 overexpression in Jurkat T cells reduces the surface expression of CD3. These results suggest that the mechanism of action of SLAP-2 and the related protein SLAP is to promote c-Cbl-dependent degradation of the tyrosine kinases SYK and ZAP-70 and down-regulation of CD3 at the cell surface.
Insights
A newly identified protein, Src-like adaptor protein 2 (SLAP-2), negatively regulates T-cell signaling by promoting the degradation of key tyrosine kinases and reducing CD3 surface expression.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- Adaptor proteins are crucial for intracellular signal transduction.
- Some adaptor proteins act as negative regulators of signaling pathways.
Purpose of the Study:
- To identify and characterize a novel hematopoiesis-specific adaptor protein, SLAP-2.
- To elucidate the mechanism by which SLAP-2 regulates T-cell receptor signaling.
Main Methods:
- Co-immunoprecipitation assays to identify binding partners of SLAP-2.
- Overexpression studies in T-cell lines (Jurkat) and COS cells.
- Analysis of T-cell activation markers and kinase degradation.
Main Results:
- SLAP-2 binds to c-Cbl and the tyrosine kinases ZAP-70 and an unknown 72 kDa protein.
- SLAP-2 overexpression inhibits T-cell receptor-induced NFAT activation.
- SLAP-2 promotes the degradation of SYK and ZAP-70 and reduces surface CD3 expression.
Conclusions:
- SLAP-2 functions as a negative regulator of T-cell signaling.
- SLAP-2 mediates c-Cbl-dependent degradation of SYK and ZAP-70.
- SLAP-2 down-regulates T-cell activation by reducing surface CD3 expression.