The T-cell antigen receptor (TCR) complex is the key structure involved in signal transduction in T cells. To analyze associations between the TCR complex and other molecules, immunoprecipitations were carried out, followed by phosphorylation of molecules in vitro by tyrosine kinases associated with the precipitated molecules. This provided a sensitive assay for molecular complexes, and associations were demonstrated between the TCR complex and the surface antigens CD2, CD4, or CD8 and CD5 in normal rat T cells. The complexes were readily seen in immunoprecipitates from BrU 96 but not Nonidet P40 detergent extracts. The multimolecular complexes are associated with the internal tyrosine kinases p56kk and p59VY. The presence of p56kk associated with CD4 or CD8 was also examined in early thymocytes, natural killer cells, and macrophages. The kinase was present in all cases except that of normal macrophages.The earliest known events in T-cell activation result in phosphorylation of tyrosine residues of cytoplasmic proteins by tyrosine kinases (Tyr kinases) (1). It is believed that phospholipase C,1 is activated by phosphorylation (2,3) and that another substrate is the ' chain of the T-cell antigen receptor (TCR) complex (4, 5). Tyr kinases of the src family that are particularly associated with T cells include p56lck and p59fyn (6,7). The p56lck protein binds to the cytoplasmic domains of the CD4 and CD8 molecules (4,(8)(9)(10), and this association is required for efficient signal transduction via the TCR complex (11,12). A key role for p59f1n in T-cell signal transduction is also indicated by the findings that this protein is associated with the TCR (13) and that transgenic expression of p59fyn in thymocytes leads to enhanced signaling in these cells in response to anti-CD3 monoclonal antibodies (mAbs) (6). Taken together, the data suggest that the TCR and the CD4/CD8 coreceptors interact together to transmit signals by coupling to the internal Tyr kinases p56lck and p59fyn.Crosslinking of the CD2 and CD5 T-cell antigens can also have effects on T-cell activation (14). Certain combinations of anti-CD2 mAbs induce mitogenesis in T cells, and the same activation of signal transduction parameters occurs as is seen with activation via the TCR (15, 16). For these events, the cytoplasmic domain of CD2 is required (15,17), and it also seems that a functional T-cell receptor is required for cells to be efficiently activated via CD2 (15,18 MATERIALS AND METHODS Animals and Antibodies. Thymuses, cervical lymph nodes, and peritoneal exudate cells were from AO-RT1u rats. The rat NK cell line A181 arose in a Fischer rat (18). mAbs used (refs. 22-24 unless otherwise stated) were W3/25 (IgG1) and OX-70 (IgG2a) noncompetitive anti-rat CD4 mAbs; OX-1 (IgG1) anti-rat CD45 mAb; OX-2 (IgG1) mAb against an immunoglobulin-related molecule of rat thymocytes and brain (25); OX-8 (IgG1) anti-rat CD8 a-chain mAb; OX-18 (IgG1) anti-rat RT1A mAb (nonpolymorphic); anti-rat CD5 mAb; (IgG1) 2945The publication costs of this artic...

CD5 is a glycoprotein expressed on thymocytes, T cells, and a subset of B cells. Antibody-mediated cross-linking studies or studies on CD5 knockout mice implicate CD5 as a co-stimulatory or negative regulatory molecule. CD5 is rapidly phosphorylated on tyrosine (Y) residues following Tcell activation. Y429 and Y441 occur in an imperfect immunoreceptor tyrosine-based activation motif (ITAM)-like sequence. We investigated whether phosphatidylinositol (PI) 3-kinase, which binds to tyrosine-phosphorylated ITAM, interacts with CD5 following T cell activation. PI 3-kinase activity and the regulatory p85 subunit of PI 3-kinase associated with CD5 in pervanadate-stimulated, but not in unstimulated thymocytes. Cellular p85 as well as the recombinant Src homology 2 (SH2) domains of p85 bound a tyrosine-phosphorylated peptide encompassing Y463 with approximately threefold greater affinity than a doubly tyrosine-phosphorylated Y429-Y441 peptide. Binding of the C-SH2 domain to the Y463 phosphopeptide, together with preferential binding of the N-SH2 domain to the Y429-Y441 phosphopeptide, suggests a bivalent interaction. A 120-kDa phosphoprotein (pp120) associated with CD5 and specifically with the Y429-Y441 phosphopeptide in stimulated thymocytes. We conclude that stimulation of thymocytes with pervanadate induces the recruitment of PI 3-kinase and pp120 to CD5.

SummaryCrosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca 2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR-can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the ~'/'y heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that ~" chains may provide the link between signaling on NK cells and T cells. This could be tested on TCK-cells since when CD16 is transfected into T cells it is expressed in a complex with TCK ~" homodimer or the ~'/'g heterodimer. At first, potentiation of CD2 signaling was seen on TCP,-Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCP, complex and that TCR ~" chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.