David A. Fruman

Immunophilins in protein folding and immunosuppression.

Lymphocyte activation requires the transmission of signals from molecules at the plasma membrane to nuclear signals that regulate gene expression. In recent years, several immunosuppressive compounds have been used as probes to identify important and potentially novel molecules involved in lymphocyte signal transduction processes. The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin have been studied in particular detail. Two distinct classes of immunosuppressant binding proteins have been identified, and collectively termed immunophilins. The cyclophilin family of immunophilins binds CsA, whereas the FK506‐binding protein (FKBP) family binds FK506 and rapamycin. This review will discuss both the endogenous functions of immunophilins as well as their roles in mediating immunosuppression.—Fruman, D. A., Burakoff, S. J., Bierer, B. E. Immunophilins in protein folding and immunosuppression. FASEB J. 8: 391‐400; 1994.

Reduced expression of the murine p85α subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes

A critical component of insulin action is the enzyme phosphoinositide (PI) 3-kinase. The major regulatory subunits of PI 3-kinase, p85alpha and its splice variants, are encoded by the Pik3r1 gene. Heterozygous disruption of Pik3r1 improves insulin signaling and glucose homeostasis in normal mice and mice made insulin-resistant by heterozygous deletion of the Insulin receptor and/or insulin receptor substrate-1 (IRS1) genes. Reduced expression of p85 modulates the molecular balance between this protein, the p110 catalytic subunit of PI 3-kinase, and the IRS proteins. Thus, despite the decrease in p85alpha, PI 3-kinase activation is normal, insulin-stimulated Akt activity is increased, and glucose tolerance and insulin sensitivity are improved. Furthermore, Pik3r1 heterozygosity protects mice with genetic insulin resistance from developing diabetes. These data suggest that regulation of p85alpha levels may provide a novel therapeutic target for the treatment of type 2 diabetes.

Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology

The microbial products cyclosporin A (CsA), FK506 and rapamycin are potent immunosuppressive agents. The introduction of CsA in the early 1970s significantly improved the outcome of organ and bone marrow allograft transplantation and advanced therapeutic options in autoimmune diseases. FK506 appears to have a higher therapeutic index than CsA, and has been used with encouraging results in clinical transplantation trials. FK506 and CsA, although structurally unrelated, appear to target similar signal transduction pathways in hematopoietic cells by inhibiting the action of calcineurin, a serine/threonine phosphatase. A structural analog of FK506, rapamycin, inhibits cellular function by a different molecular mechanism. These agents have advanced our understanding of signal transmission pathways in lymphocyte activation.

Identification of a Physical Interaction between Calcineurin and Nuclear Factor of Activated T Cells (NFATp)

In T lymphocytes, the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, plays a pivotal role in transducing membrane-associated signals to the nucleus. One of the putative targets of calcineurin is the pre-existing, cytosolic component of the nuclear factor of activated T cells (NFATp; also referred to as NFAT1), which is one of several transcription factors required for the expression of interleukin 2. Inhibition of calcineurin by the immunosuppressive drugs cyclosporin A and FK506 prevents dephosphorylation of NFATp and its translocation to the nucleus. However, a physical interaction between calcineurin and NFATp has not been demonstrated. Here we demonstrate the binding of NFATp from lysates of T cells to immobilized calcineurin. Stimulation of T cells with calcium ionophore induced a shift in the molecular weight of NFATp that is due to its dephosphorylation. This dephosphorylation was inhibited by treatment of T cells with cyclosporin A or FK506 prior to stimulation. Of note, both the phosphorylated and the dephosphorylated form of NFATp bound to calcineurin. Furthermore, the binding of both forms of NFATp to calcineurin was inhibited by pretreatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of calcineurin with NFATp and that this interaction does not depend upon the phosphorylation sites of NFATp affected by activation.

Characterization of a mutant calcineurin A alpha gene expressed by EL4 lymphoma cells.

The calmodulin-stimulated phosphatase calcineurin plays a critical role in calcium-dependent T-lymphocyte activation pathways. Here, we report the identification of a missense mutation in the calcineurin A alpha gene expressed by EL4 T-lymphoma cells. This mutation changes an evolutionarily conserved aspartic acid to asparagine within the autoinhibitory domain of the calcineurin A alpha protein. A comparison of wild-type and mutant autoinhibitory peptides indicates that this amino acid substitution greatly reduces inhibition of calcineurin phosphatase activity. Additional peptide inhibition studies support a pseudosubstrate model of autoinhibitory function, in which the conserved aspartic acid residue may serve as a molecular mimic of either phosphoserine or phosphothreonine. Expression of the mutant calcineurin appears to affect cellular signal transduction pathways, as EL4 cells can be activated by suboptimal concentrations of calcium ionophore in the presence of phorbol esters. Moreover, this phenotype can be transferred to Jurkat T cells by transfection of the mutated calcineurin gene. These findings implicate a conserved aspartic acid in the mechanism of calcineurin autoinhibition and suggest that mutation of this residue is associated with aberrant calcium-dependent signaling in vivo.

T cell receptor (β chain) transgenic mice have selective deficits in γδ T cell subpopulations

Abstract TCR-β (T cell receptor-β-chain) transgenic mice have altered lymphocyte development. TCR-β transgenic mice are hyporesponsive to alloantigens in vivo and are deficient in γδ T cells. In order to begin a study of the relationship between a deficiency of alloreactive γδ cells and the defective function of in vivo alloantigen recognition, we analysed the γδ T cell development in TCR-β mice. The presence of the TCR-Vβ8.2 chain transgene is associated with inhibition of γ chain gene rearrangement. In order to determine how the presence of the TCR-β transgene affects γδ T cell development, γδ T cells were studied in the skin, intestine and spleen. TCR-β mice have dramatically reduced numbers of γδ T cells in the spleen and moderately reduced numbers of γδ T cells among intestinal intraephithelial lymphocytes. In contrast, these mice have normal numbers of γδ dendritic epidermal cells (DEC). These selective deficits could be due to the developmental regulation of transgene transcription during fetal life. We examined transcription of the TCR-β transgene in the fetal thymus and found that the TCR-β transgene is first transcribed at high levels on day 16 of fetal life, after DEC have already migrated from the thymus to the epidermis. Furthermore, mRNA from the transgene was detected in DEC, ruling out the formal possibility that DEC bear a γδ receptor only because they are incapable of expressing the transgene. The expression of the transgene is temporally associated with inhibition of further TCR gene rearrangement and this developmental sequence may be responsible for the selective tissue deficits of γδ T cells.