Syk family kinases are essential for lymphocyte development and activation. Therefore the identification of their direct effectors is of critical importance. Here, we report that Syk interacts in the yeast two-hybrid system with Vav, a proto-oncogene product exclusively expressed in hematopoietic cells. This interaction was direct, required the catalytic activity of Syk, the SH2 domain of Vav, and tyrosine residues in the linker domain of Syk. Vav also associated with Syk and Zap in antigen receptor-stimulated B or T cells, respectively. Functionally, Vav was phosphorylated by Syk family kinases both in vivo and in vitro. Furthermore, Syk and Vav cooperated to activate NF-AT synergistically. These results indicate that the interaction between Syk family kinases and Vav plays an important role in coupling immune recognition receptors to signaling pathways involved in lymphokine production.

Activation of resting T lymphocytes by ligands to the T-celI antigen receptor (TCR)/CD3 complex is initiated by rapid tyrosine phosphorylation of cellular proteins.

Src homology 2 (SH2) domains bind to phosphotyrosine (Tyr(P)) residues in specific sequence contexts in other proteins and thereby mediate tyrosine phosphorylationdependent protein-protein interactions. The SH2 domain of the Src family kinase Lck is phosphorylated at tyrosine 192 in T cells upon T cell antigen receptor triggering. We have studied the consequences of this phosphorylation on the properties of the SH2 domain and on the function of Lck in T cell activation. We report that phosphorylation at SH21 domains are independently folded hemispherical units of ϳ100 amino acid residues, which are found in many signaling proteins (1-5). Their physiological function is to bind Tyr(P) residues in specific sequence contexts in other cellular proteins, thereby facilitating the formation of tyrosine phosphorylationinduced multimeric protein complexes (1, 3).The ligand-binding surface of the SH2 domain of the Lck nonreceptor protein tyrosine kinase contains two pockets, one for the Tyr(P) residue and another for the amino acid residue three positions C-terminal to it, the ϩ3 amino acid (2, 5). Although the first pocket is well conserved among SH2 domains, the residues of the SH2 domain that form and surround the second pocket vary more. These differences determine the depth and properties of the pocket and thereby the preferred amino acid ligand (6, 7). In the case of the Lck SH2 domain, the optimal ligand is a Tyr(P) followed by two acidic residues followed by an isoleucine at position ϩ3, a specificity largely determined by amino acid residues in ␤ strands D and E and in the EF loop adjacent to the second pocket (6, 8). Recently, it was shown that a single amino acid substitution in the EF loop in the SH2 domain of c-Src (a T215W mutation) changed the ligand selection of the SH2 domain to that of the Grb2 SH2 domain (9), which has a tryptophan residue at the corresponding location in its EF loop. Conversely, a switch from tryptophan to threonine in the SH2 domain of Grb2 changed its ligand preference to that of the c-Src SH2 domain (9).Here we describe a more physiological regulation of the function of an SH2 domain, namely the effect of phosphorylation of a highly conserved tyrosine residue, Tyr 192 , in the end of ␤ strand E in the Lck SH2 domain. This phosphorylation event, which can be catalyzed by Syk and possibly Zap nonreceptor kinases, leads to a profound down-regulation of the ligand binding capacity of the SH2 domain. The consequences for the function of Lck as a signal transducer in T cell activation were studied. MATERIALS AND METHODSCells and Reagents-Jurkat human T leukemia cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, L-glutamine, and antibiotics. COS-1 cells were kept at logarithmic growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, L-glutamine, and antibiotics. The rabbit antiserum against the protein tyrosine kinase Syk (residues 253-365) was described earlier (10). The anti-Lck antiserum, raised against residues 39 -64 of...

Activation of resting T lymphocytes is initiated by rapid but transient tyrosine phosphorylation of a number of cellular proteins. Several protein tyrosine kinases and protein tyrosine phosphatases are known to be important for this response. Here we report that normal T lymphocytes express the B isoform of low molecular weight protein tyrosine phosphatase B (LMPTP-B). The cDNA was cloned from Jurkat T cells, and an antiserum was raised against it. LMPTP immunoprecipitated from resting Jurkat T cells was found to be tyrosine phosphorylated. On stimulation of the cells through their T cell antigen receptor, the phosphotyrosine content of LMPTP-B declined rapidly. In co-transfected COS cells, Lck and Fyn caused phosphorylation of LMPTP, whereas Csk, Zap, and Jak2 did not. Most of the phosphate was located at Tyr-131, and some was also located at Tyr-132. Incubation of wild-type LMPTP with Lck and adenosine 5-O-(thiotriphosphate) caused a 2-fold increase in the activity of LMPTP. Site-directed mutagenesis showed that Tyr-131 is important for the catalytic activity of LMPTP, and that thiophosphorylation of Tyr-131, and to a lesser degree Tyr-132, is responsible for the activation.One of the earliest biochemical events seen in T lymphocytes triggered through the T cell antigen receptor complex is the enhanced phosphorylation of a number of cellular proteins on tyrosine residues (1, 2). Inhibition of this event by pharmacological agents prevents T cell activation as measured by both functional readouts and biochemical assays (3, 4). It has become evident that several protein tyrosine kinases (PTKs) 1 and the CD45 protein tyrosine phosphatase (PTPase) play crucial roles (reviewed in Refs. 5-7), and that the T cell antigen receptor-induced cascade of transient tyrosine phosphorylation events depends on a dynamic interplay between these and, presumably, many additional PTKs and PTPases. In addition to CD45 (8 -10), only two other PTPases have been implicated in T cell activation, namely SHP1 (11) and SHP2 (12).The low molecular weight PTPases LMPTP-A and LMPTP-B constitute a class of PTPases with limited sequence homology to the other PTPases (13-16). Nevertheless, these enzymes are highly specific for PTyr (14). Chemical modifications and mutagenesis experiments have shown that their catalytic mechanism involves a cysteine residue, Cys-12, which participates in phosphoenzyme intermediate formation (15), as in other PTPases. The recent crystallization of LMPTP (16) showed that the catalytic center is quite similar to that of "classical" PTPases, with Cys-12 residing in the bottom of the catalytic pocket.The physiological functions of LMPTP are unknown. Overexpression of LMPTP in cells transformed by PTK oncogenes leads to decreased proliferation and the ability to form colonies in soft agar (17). Thus, a potential physiological function of the LMPTPs is to control normal cell growth by interacting directly or indirectly with the PTK signaling network. Reportedly, LMPTP can interact directly with the platelet-derived grow...

The p56ik and p590M protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and phairmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCRICD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56kk. Here we examine the regulation of p72yk and its interaction with p56"k in transfected COS-1 cells. p72'k was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by -90%.Coexpression of Ick did not alter the catalytic activity of p72syk, but the expressed p56"k was much more active in the presence of p72yk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56kk was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56kk was no longer phosphorylated by p72A"k. Phosphate was also detected in p56"k at in lymphoid cells. These findings suggest that p56kk is positively regulated by the p723* kinase.T-cell activation by ligands to the T-cell antigen receptor (TCR)/CD3 complex depends on the rapid ligand-induced tyrosine phosphorylation of cellular substrates, such as phospholipase C-yl, the t chain of the TCR, phosphatidylinositol 3-kinase, and the guanine nucleotide exchange factor p95vav (reviewed in reference 27). Inhibition of protein tyrosine kinases (PTKs) by pharmacological agents prior to TCR/CD3 stimulation blocks T-cell activation (19, 31), and overexpression of some PTKs (especially Src-related PTKs) in T cells renders them hypersensitive to TCR/CD3 stimulation (1, 10).Two members of the Src family of nonreceptor PTKs, p56ck and p59fy', which associate with the CD4/CD8 coreceptors and the TCR/CD3, respectively, are crucial in T-cell maturation (26) (2,25). In vivo, the phosphorylation of Tyr-505 is probably mediated by p5Ocsk, a PTK known to phosphorylate Src family PTKs at their conserved C-terminal regulatory site in vitro (7,33,37