Synergism between the calmodulin-binding and autoinhibitory domains on calcineurin is essential for the induction of their phosphatase activity

Elevation of the intracellular calcium concentration is necessary for cell growth and the activation of several lymphokine genes. The immunosuppressive drugs cyclosporin A and FK506 profoundly inhibit the calcium-dependent signaling pathway in T lymphocytes by interfering with the activity of the calcium/calmodulin (CaM)-dependent serine/threonine phosphatase, calcineurin (CN). Little is known, however, about how activation of CN enzyme activity or interaction with its substrate, nuclear factor of activated T cell (NF-AT), is regulated. We show here that the binding of CaM to CN may affect the conformation of CN at both the CaM-binding and the autoinhibitory (AI) domains and that this is critical for activation of CN to dephosphorylate NF-AT. Dissociation of the AI domain from the enzyme active site on CN leads to expose a binding site for NF-AT at the N terminus of CN and allows the CN binding to NF-AT. Since the cytoplasmic form of NF-AT can interact with CN mutants lacking enzyme activity, the interaction of the two molecules is independent of CN enzyme activity and occurs prior to the dephosphorylation of NF-AT. Dephosphorylation converts NF-AT from the cytoplasmic to the nuclear form by exposing the DNA-binding domain on NF-AT, and the nuclear form of NF-AT brings CN together into the nuclei. We therefore propose that the activation of CN by the CaM binding independently regulates the interaction with NF-AT and the dephosphorylation of NF-AT.     

A T cell nuclear factor resembling NF-AT binds to an NF-kappa B site and to the conserved lymphokine promoter sequence "cytokine-1".

Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B-binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin-sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor.     

The yeast hnRNP-Like proteins Yra1p and Yra2p participate in mRNA export through interaction with Mex67p

Yra1p is an essential nuclear protein which belongs to the evolutionarily conserved REF (RNA and export factor binding proteins) family of hnRNP-like proteins. Yra1p contributes to mRNA export in vivo and directly interacts with RNA and the shuttling mRNP export receptor Mex67p in vitro. Here we describe a second nonessential Saccharomyces cerevisiae family member, called Yra2p, which is able to complement a YRA1 deletion when overexpressed. Like other REF proteins, Yra1p and Yra2p consist of two highly conserved N- and C-terminal boxes and a central RNP-like RNA-binding domain (RBD). These conserved regions are separated by two more variable regions, N-vr and C-vr. Surprisingly, the deletion of a single conserved box or the deletion of the RBD in Yra1p does not affect viability. Consistently, neither the conserved N and C boxes nor the RBD is required for Mex67p and RNA binding in vitro. Instead, the N-vr and C-vr regions both interact with Mex67p and RNA. We further show that Yra1 deletion mutants which poorly interact with Mex67p in vitro affect the association of Mex67p with mRNP complexes in vivo and are paralleled by poly(A)(+) RNA export defects. These observations support the idea that Yra1p promotes mRNA export by facilitating the recruitment of Mex67p to the mRNP.     

PPARgamma1 attenuates cytosol to membrane translocation of PKCalpha to desensitize monocytes/macrophages

Recently, we provided evidence that PKCalpha depletion in monocytes/macrophages contributes to cellular desensitization during sepsis. We demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists dose dependently block PKCalpha depletion in response to the diacylglycerol homologue PMA in RAW 264.7 and human monocyte-derived macrophages. In these cells, we observed PPARgamma-dependent inhibition of nuclear factor-kappaB (NF-kappaB) activation and TNF-alpha expression in response to PMA. Elucidating the underlying mechanism, we found PPARgamma1 expression not only in the nucleus but also in the cytoplasm. Activation of PPARgamma1 wild type, but not an agonist-binding mutant of PPARgamma1, attenuated PMA-mediated PKCalpha cytosol to membrane translocation. Coimmunoprecipitation assays pointed to a protein-protein interaction of PKCalpha and PPARgamma1, which was further substantiated using a mammalian two-hybrid system. Applying PPARgamma1 mutation and deletion constructs, we identified the hinge helix 1 domain of PPARgamma1 that is responsible for PKCalpha binding. Therefore, we conclude that PPARgamma1-dependent inhibition of PKCalpha translocation implies a new model of macrophage desensitization.     

A common factor regulates both Th1- and Th2-specific cytokine gene expression.

Murine T helper cell clones are classified into two distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), on the basis of cytokine secretion patterns. Th1 clones produce interleukin-2 (IL-2), tumor necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), while Th2 clones produce IL-4, IL-5, IL-6 and IL-10. These subsets differentially promote delayed-type hypersensitivity or antibody responses, respectively. The nuclear factor NF-AT is induced in Th1 clones stimulated through the T cell receptor-CD3 complex, and is required for IL-2 gene induction. The NF-AT complex consists of two components: NF-ATp, which pre-exists in the cytosol and whose appearance in the nucleus is induced by an increase of intracellular calcium, and a nuclear AP-1 component whose induction is dependent upon activation of protein kinase C (PKC). Here we report that the induction of the Th2-specific IL-4 gene in an activated Th2 clone involves an NF-AT complex that consists only of NF-ATp, and not the AP-1 component. On the basis of binding experiments we show that this 'AP-1-less' NF-AT complex is specific for the IL-4 promoter and does not reflect the inability of activated Th2 cells to induce the AP-1 component. We propose that NF-ATp is a common regulatory factor for both Th1 and Th2 cytokine genes, and that the involvement of PKC-dependent factors, such as AP-1, may help determine Th1-/Th2-specific patterns of gene expression.