Protein kinase C-theta (PKC-theta) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide-major histocompatibility complex (MHC) induces translocation of PKC-theta to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-theta regulatory domain and required Lck but not ZAP-70. In addition, PKC-theta was associated with Lck in the rafts. An isolated PKC-straight theta catalytic fragment did not partition into rafts or activate the transcription factor NF-kappa B, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-theta to rafts, which localize to the T cell synapse; this PKC-theta translocation is important for its function.

Abstract. Botulinum C3 exoenzyme specifically ADPribosylates a group of ms-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32p]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADPribosylated in situ in a time-and concentrationdependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-l-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzymeinduced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells. R as and ras-related genes constitute a gene family encoding a series of closely related proteins with guanine nucleotide-binding activities. To date, ~40 ras and ras-related GTP-binding proteins are known, and they are divided into four subfamilies: ms, rho, tab, and others (Hall, 1990;Bourne et al., 1991). These proteins exist in two interconvertible functional states; one in an inactive GDPbound form and the other in an active GTP-bound form. In resting cells, they are present in an inactive GDP-bound form, and upon cell stimulation, converted to the active GTP-bound form and work as molecular switches linking external stimuli to various cellular responses such as growth, differentiation, and secretion. Among the ras-related GTPbinding proteins, rho proteins are believed to be involved in organization of cytoskeleton and maintenance of cell shape. There are at least three members in this family in human, which are called rhoA, B, and C (Yeramian et al., 1987;Chardin et al., 1988). rho proteins are unique among the small GTP-binding proteins in being substrates for ADP ribosylation by the exoenzyme C3 from Clostridium botulinum (Aktories et al., 1987;Morii et al., 1988;Narumiya et al., 1988;Kikuchi et al., 1988). This enzyme ADPribosylates the proteins at an asparagine residue in the putative effector domain and inhibits their biological activities presumably by interfering with their interaction ...

T cell helper type 2 (Th2) differentiation is driven by a source of IL-4 receptor (IL-4R) that mobilizes IL-4R signaling pathways and the transcription factor GATA-3. Naïve CD4 cells can secrete IL-4 independently of IL-4R signals, but how this secretion is regulated is not understood. Here we demonstrate that costimulation through the tumor necrosis factor receptor family molecule OX40, in synergy with CD28, is essential for high levels of nuclear factor of activated T cells c1 to accumulate in the nucleus of a recently activated naïve T cell. This action is not dependent on either IL-4R or IL-2R signals and results in OX40 controlling initial naïve T cell IL-4 transcription. OX40 signals subsequently enhance nuclear GATA-3 accumulation through an IL-4R-dependent action, leading to Th2 differentiation. These data show that, in the absence of an exogenous source of IL-4, OX40 provides a critical synergistic and temporal signal with other noncytokine receptors to modulate nuclear factor of activated T cells c1 and to promote optimal Th2 generation.IL-4 ͉ GATA-3

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 ...

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