Promoter studies have revealed that sequences related to the GT-1 binding site, known as GT elements, are conserved in plant nuclear genes of diverse functions. In this work, we addressed the issue of whether GT elements are involved in cell type-specific transcriptional regulation. We found that the inactivation of GT-1 site-mediated transcription in roots is correlated with the absence of the GT-1 binding activity in root extracts. In addition, the mutation of the related GT-1 (from the pea rbcs-3A) and the S1F (from the spinach rps1) sites resulted in an increase of their transcriptional activity in roots that contain a distinct GT element-binding factor, referred to as RGTF. Although specific to GT elements, RGTF has a different sequence requirement and a lower sequence specificity than GT-1. Interestingly, RGTF has a higher binding affinity to the mutant GT-1 and S1F sites than to the wild-type sequences. This correlation suggests that RGTF may have some role in transcriptional regulation in roots. Furthermore, root cellular protein extracts contain an inhibitory activity that prevents GT-1 from binding to DNA. This helps to explain the absence of the GT-1 binding activity in roots in which the gene of GT-1 is expressed. Together, these data suggest that the cell type-specific transcription modulation by GT elements is achieved by using two different strategies.

Human immunodeficiency virus type 1 (HIV-1) infection triggers a cytotoxic T-lymphocyte (CTL) response mediated by CD8؉ and perhaps CD4 ؉ CTLs. The mechanisms by which HIV-1 escapes from this CTL response are only beginning to be understood. However, it is already clear that the extreme genetic variability of the virus is a major contributing factor. Because of the well-known ability of altered peptide ligands (APL) to induce a T-cell receptor (TCR)-mediated anergic state in CD4؉ helper T cells, we investigated the effects of HIV-1 sequence variations on the proliferation and cytotoxic activation of a human CD4؉ CTL clone (Een217) specific for an epitope composed of amino acids 410 to 429 of HIV-1 gp120. We report that a natural variant of this epitope induced a functional anergic state rendering the T cells unable to respond to their antigenic ligand and preventing the proliferation and cytotoxic activation normally induced by the original antigenic peptide. Furthermore, the stimulation of Een217 cells with this APL generated altered TCR-proximal signaling events that have been associated with the induction of T-cell anergy in CD4 ؉ T cells. Importantly, the APL-induced anergic state of the Een217 T cells could be prevented by the addition of interleukin 2, which restored their ability to respond to their nominal antigen. Our data therefore suggest that HIV-1 variants can induce a state of anergy in HIV-specific CD4 ؉ CTLs. Such a mechanism may allow a viral variant to not only escape the CTL response but also facilitate the persistence of other viral strains that may otherwise be recognized and eliminated by HIV-specific CTLs.

Regulation of protein tyrosine kinases (PTKs) by tyrosine phosphorylation is well recognized; in fact, nearly all PTKs require phosphorylation of tyrosine residues in their "activation loop" for catalytic activity. In contrast, the phosphorylation of PTKs on serine and threonine residues has not been studied nearly as much. We report that the ZAP-70 PTK contains predominately phosphoserine in normal T lymphocytes as well as in Jurkat T leukemia cells. We have identified one site of phosphorylation as Ser-520 and find this site to be important for the recruitment and activation of ZAP-70 in T cells. Mutant ZAP-70-S520A had reduced ability to autophosphorylate and to mediate antigen receptor-induced interleukin 2 gene activation and was not enriched at the plasma membrane. These defects were rescued by addition of a myristylation signal to the N terminus of ZAP-70-S520A to force its plasma membrane and lipid raft localization. We conclude that phosphorylation of ZAP-70 at Ser-520 plays an important role in the correct localization of ZAP-70 and in priming ZAP-70 for its acute recruitment and activation upon antigen receptor ligation.The ZAP-70 protein tyrosine kinase (PTK) plays a critical role in T-cell-antigen-receptor (TCR) signaling (5,6,28). The lack of ZAP-70 expression causes a severe immunodeficiency (2,14), characterized by the absence of CD8 ϩ T cells and TCR-unresponsive mature CD4 ϩ T cells. Mice lacking ZAP-70 are also deficient in the production of CD4 ϩ T cells, while the natural killer cells are unaffected (30). Perhaps the most important feature of ZAP-70 is its recruitment and high-affinity association with the phosphorylated immune receptor tyrosine-based activation motifs (ITAMs) of the TCR receptor complex. The crystal structure of the complex of the N terminus of ZAP-70 bound to a doubly phosphorylated ITAM peptide (18) showed that the second SH2 domain binds the first phosphorylated tyrosine of the peptide in the usual SH2-ligand manner, while the second phosphorylated tyrosine interacts with both SH2 domains in a unique manner due to the presence of an incomplete phosphotyrosine (PTyr)-binding pocket in the N-terminal SH2 domain, which is made functional by the close proximity of the other SH2 domain (17). This feature of the solved structure explains the strongly synergistic binding of ZAP-70 to doubly phosphorylated ITAMs (18,20,29,31). Binding of ZAP-70 to ITAMs alone is insufficient to activate the kinase (20, 30), and inactive ZAP-70 can be associated with TCR-, as observed with thymocytes (29).Activation of the receptor-associated ZAP-70 is accomplished by the phosphorylation of Tyr-493 of ZAP-70, a reaction catalyzed by the Src family kinase Lck (4,26,28,36). Tyr-493 corresponds to the positive regulatory phosphorylation site in the activation loop in all protein kinases, except Csk. In contrast to Src family PTKs and many other kinases, ZAP-70 is unable to autophosphorylate at this site. However, once ZAP-70 has been activated by phosphorylation at Tyr-493, the kinase becomes able t...

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