Biochemical identification of a direct physical interaction between the CD4: p56^lck and T_i(TcR)/CD3 complexes

Abstract

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein-tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co-modulate with TcR/CD3 suggesting that these receptor complexes may associated on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4:p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB-ALL results in the co-purification of p56lck with an additional polypeptide at 20 kDa. Re-precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3 epsilon chain. An alternative approach which involves the labeling of microsomal membranes with [gamma-32P]ATP revealed the presence of CD3 epsilon and zeta chains in anti-CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4:p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4:p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

“…Although previous studies have raised controversy about the effects of HIV-1 gp120-mediated CD4 cross-linking on p56lCk activity (32,35,37), data from four experiments indicated that the cross-linking of HIV-1 gp120 has a marked modulator effect on the activity of PI 3-and PI 4-kinases associated with the receptor. DISCUSSION CD4-pS6ck associates and synergizes with the TcRUICD3 complex in the optimal response of T cells to antigen (2,8,20,59). p561ck expression is also required for thymic differentiation and the ability of T cells to respond to foreign antigen (46).…”

“…Previous studies by us (53) have shown that the TcRC/CD3-p59'5n complex also associates with PI 3-kinase. In the context of a multimeric complex (7,8), CD4-p56lck could contribute to the overall level of PI 3-kinase activity. Alternatively, given that only a small percentage of TcRV/CD3 complexes associated with p59Yf (and hence, associated PI 3-kinase), CD4-p56lck may contribute the lipid kinase in coreceptor interactions involving TcRC/CD3-p59'5n negative complexes.…”

“…The association is required for an optimal response of certain T cells to antigen (1,27,65). CD4-p56lck complex can also physically associate with the TcRUICD3 complex (8), and as such synergize with the TcRC/CD3 complex in augmenting T-cell proliferation (2,20,21). Although anti-CD4 cross-linking may increase lck activity (43,71), the effects of HIV-1 gpl20 have been controversial (32,35,37).…”

Identification of a novel dimeric phosphoprotein (PP29/30) associated with signaling receptors in human T lymphocytes and natural killer cells.

“…Previous data had shown that the protein tyrosine kinases p56 lck and p59~, CD3-~, and ~" chains copurify with the accessory receptor molecules CD2, CD4, CD5, and CD8 as well as the TCR/CD3 complex in Brij lysates of rat and human T lymphocytes (24)(25)(26)(27)(28)(29). It was therefore important to investigate whether pp29/30 copurifies also with these receptors.…”

Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

“…Signaling is mediated by p56lck and p59fyn(T), which associate with the CD4/CD8 and TCRt/CD3 complexes, respectively (2)(3)(4)(5). CD4 and CD8 associate and functionally synergize with the TCRC/CD3 complex, augmenting T-cell proliferation (6)(7)(8). p59fyn(T), a form of p59fyn expressed in T cells, is generated by alternative splicing (9).…”