T he earliest documented events following engagement of TCRs by their specific ligands, peptide͞MHC complexes, involves the activity of Src and Syk͞ZAP-70 family protein tyrosine kinases and the phosphorylation of a number of their cellular substrates (1, 2). These include the immunoreceptor, tyrosine-based activation motifs (ITAM) of the TCR and CD3 chains (3), the adaptor proteins LAT (4) and SLP-76 (5), and the proto-oncogene product, Vav (6), as well as proteins of lessdefined function, such as SLAP-130͞FYB (7, 8). Vav, a guanine nucleotide exchange factor for Rho-like small GTPases such as Rac, plays an important role in T cell activation (9). One of its functions is related to the reorganization of the T cell actin cytoskeleton after TCR stimulation, and studies with Vav Ϫ/Ϫ T cells have established the requirement for Vav in actindependent TCR͞CD3 cap formation after TCR crosslinking (10). The upstream protein tyrosine kinase(s) responsible for the phosphorylation and activation of Vav have not been well delineated, since previous studies have shown that Vav can serve as a substrate for all four of the proximal tyrosine kinases: Lck, Fyn, Syk,. In contrast, individual PTKs appear to preferentially phosphorylate some of the other important downstream substrates, e.g., LAT is a specific substrate for ZAP-70 (4), ZAP-70 itself is a substrate for Lck (15), and SLP-130͞FYB and PYK-2 are substrates for Fyn (16,17).Some of these activation-induced tyrosine phosphorylation events appear to be exquisitely sensitive to the affinity of interaction between the TCR and its MHC͞peptide ligand. Studies on single amino acid-substituted antigenic peptide variants, so-called altered peptide ligands (APL), have indicated that low-affinity interactions often lead to an incomplete pattern of tyrosine phosphorylation. Initial studies indicated that stimulation by APL led to a preponderance of an incompletely phosphorylated chain, with the resulting accumulation of a low molecular weight mass TCR. Furthermore, antagonist APL failed to phosphorylate and activate . More recently, we have demonstrated that antagonist peptides are capable of inducing the signaling pathway that is required for cytoskeletal reorganization in T cells, the formation of stable T cell͞APC conjugates, and the localization of certain T cell proteins to the areas of contact between APC and T cell. This signaling pathway is characterized by the phosphorylation and activation of Vav guanine nucleotide exchange factor activity and subsequent activation of the small G protein, Rac (21). This finding indicated the presence of a hierarchy of T cell signal transduction that is sensitive to the affinity of TCR͞ligand interaction. To characterize further this signaling pathway, we wished to determine which PTK(s) is activated by antagonist peptide͞MHC engagement of the TCR and is responsible for initiating the signaling cascade that leads to the partial activation of T cells and the subsequent formation of APC͞T cell conjugates. In this report, we provide evidence ...