JBC Ideal method for primary cell transfection

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Volume 272, Number 40, Issue of October 3, 1997 pp. 25310-25318
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Tyrosine Phosphorylation of the CD3-epsilon Subunit of the T Cell Antigen Receptor Mediates Enhanced Association with Phosphatidylinositol 3-Kinase in Jurkat T Cells

(Received for publication, May 9, 1997, and in revised form, July 17, 1997)

Isabel de Aós Dagger , Markus H. Metzger , Mark Exley par , Charles E. Dahl ** , Suniti Misra Dagger Dagger , Dexian Zheng §§ , Lyuba Varticovski Dagger Dagger , Cox Terhorst and Jaime Sancho Dagger

From the Dagger  Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Ventanilla 11, 18001 Granada, Spain, the  Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, par  Cancer Biology Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, the ** Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02215, §§ Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Beijing 10005, People's Republic of China, and the Dagger Dagger  Department of Biomedical Research, St. Elizabeth's Hospital, Tufts University School of Medicine, Boston, Massachusetts 02135

T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta  chain (zeta A-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon ) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon , respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon , p85alpha , and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon -ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon -ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.


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