Abstract
Upon interaction with its ligand, B7, CD28 becomes phosphorylated on tyrosines. One tyrosine in particular (Y170 in mouse CD28, Y173 in human CD28) has received much attention. This is because it permits CD28 to recruit SH2-containing signaling molecules, including phosphoinositide 3 kinase, Grb2 and Gads. Using mice we employed a transgenic approach to express a tyrosine→phenylalanine mutant form of CD28 that uncouples these SH2-mediated interactions from CD28. The CD28 mutant is unable to up-regulate expression of the prosurvival protein Bcl-xL, rendering the T cells more susceptible to radiation-induced death. Nonetheless, this mutated form of CD28 still prevents the induction of anergy and promotes T cell proliferation, interleukin 2 secretion and B cell help. Thus, we describe a single point mutation within the CD28 cytoplasmic domain that uncouples signals required for proliferation and survival.
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Acknowledgements
We thank J. Ellis and A. Huang at the Hospital for Sick Children Transgenic Facility, Toronto for micro-injecting DNA and A. Veillette, S. Ilangumaran and B. Vanhaesebroeck for advice and for reviewing the manuscript.. Supported by grants from the Arthritis Society of Canada and the Medical Research Council (to R. R.) and the National Cancer Institute (to P. S. O.).
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Web Figure 1.
The YMNM motif is required for CD28-dependent up-regulation of Bcl-xL. Lymph node cells from C57BL/6, CD28-/-, WT Tg and Y170F Tg –1 were left unstimulated or stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 6, 10, 22, 34 and 46 h. Cells were collected, washed in PBS and lysed. Proteins were resolved by SDS-PAGE, transferred to PVDF membranes and probed with anti–Bcl-xL and anti-actin. (JPG 39 kb)
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Okkenhaug, K., Wu, L., Garza, K. et al. A point mutation in CD28 distinguishes proliferative signals from survival signals. Nat Immunol 2, 325–332 (2001). https://doi.org/10.1038/86327
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DOI: https://doi.org/10.1038/86327