Cell, Vol. 66, 823-826,

September

6, 1991, Copyright

0 1991 by Cell Press

When Worlds Collide: lmmunosuppressants Meet Protein Phosphatases Frank McKeon Cellular and Molecular Physiology Harvard Medical School Boston, Massachusetts 02115

In the early 1970s Bore1 and his colleagues discovered a cyclic undecapeptide from a fungal isolate that suppressed T cell-mediated immune responses in mice (Borel, 1976). The potency and specificity of this cyclic peptide, called cyclosporin A, promised new therapies for allograft rejection and autoimmune diseases, as well as fundamental insights into mechanisms of signal transduction. This first promise has been realized already: since its clinical introduction in the late 197Os, cyclosporin A has been credited with the dramatic increase in survival rates of patients receiving kidney, heart, and liver transplants. In addition, experimental trials are yielding positive results in the treatment of graft versus host disease and other autoimmune diseases. In contrast to the clinical advances with cyclosporin A, an understanding of the biochemical events perturbed by this drug has been elusive. Regardless, it is now apparent that cyclosporin A, as well as newer immunosuppressants such as FK506 and rapamycin, will be important tools for probing signal transduction, much as the ADP-ribosyl transferase toxins have been for G-protein function and brefeldin A for elucidating membrane trafficking. Here the present concepts and paradoxes of immunosuppression by cyclosporin A and FK506, the striking new findings that focus these drugs into common signal transduction pathways, and the implications of these findings for immunosuppression and intracellular signaling pathways are discussed. Intracellular Receptors for Cyclosporin A, FK506, and Rapamycin In 1984, Handschumacher and coworkers identified the first of a family of conserved proteins with high affinity for cyclosporin A (Handschumacher et al., 1984; Harding et al., 1986). This 17 kd candidate receptor for cyclosporin A, named cyclophilin A, displayed high affinities for immunosuppressing analogs of cyclosporin A and low affinities for inactive analogs. Subsequently the role of cyclophilin A in the suppression of T cell activation by cyclosporin A has been called into question because it is present in all cells and tissues examined (Harding et al., 1986), and it is now only one member of an increasingly diverse family of general (Price et al., 1991) and tissue-specific cyclophilins (Stamnes et al., 1991). More recently, an unrelated fungal product with inhibitory effects on T cell activation surprisingly similar to those produced by cyclosporin Awas discovered. This molecule, known as FK506, is a macrolide (cyclic ester) that binds a separate group of conserved, abundant proteins, termed FK-binding proteins (FKBPs) (Harding et al., 1989; Siekierka et al., 1989, and references therein). Neither cy-

M inireview

closporin A nor FK506 appears to interact with the receptors of the other. Since either drug effectively blocks the induction of cytokine gene transcription at the early stages of antigen-induced helper T cell activation, it was assumed that T cell activation required the separate activity of both cyclophilin(s) and FKBP(s). This functional similarity was underscored by the findings that both cyclophilins and FKBPs were active as peptidyl-prolyl cis-trans isomerases (rotamases), and that this activity was blocked by the binding of their respective drug (Fischer et al., 1989; Takahashi et al., 1989; Siekierka et al., 1989; Harding et al., 1989). These observations suggested that immunosuppression resulted from improper folding of a transcription factor required for cytokine mRNA expression. In fact, data from Crabtree and associates imply that these immunosuppressants block the nuclear translocation of a cytoplasmic subunit of NF-AT, a transcription factor implicated in the activation of cytokine genes during T cell activation (Flanagan et al., 1991). However, the disruption of cytokine gene activation may occur at a step downstream of the immunosuppressant targets. Receptor-Drug Complex as a Functional Mediator of lmmunosuppression Recently, evidence from a number of unrelated sources has begun to erode the belief that proline isomerase inhibition is the basis of the T cell inhibition by cyclosporin A and FK506. The idea emerging is that these drugs become active as a complex with their respective intracellular receptors by imparting new effector properties to these receptors. Thus, rather than simply inhibiting functions of these receptors, they induce a “gain-of-function” transformation in which the complex promotes immunosuppression through associations with targets in the cell. This major conceptual change came with the data from yet another immunosuppressant, the macrolide rapamycin. Rapamycin not only shares structural similarity with FK506, but also binds FKBP with high affinity and in doing so blocks its proline isomerase activity (Bierer et al., 199Oa). Despite these activities, rapamycin does not block cytokine transcription at the early stages of T cell activation. Rather, rapamycin appears to block the later proliferative step in T cell activation. Since both rapamycin and FK506 are immunosuppressants and inhibit FKBP rotamase activity, these two effects appeared mechanistically linked. Yet, these two effects were uncoupled by a synthetic FK506 analog known as 506BD (Bierer et al., 1990b). 506BD has the FKBP-binding motif common to both FK506 and rapamycin and, as expected, bind; FKBP and inhibits its rotamase activity. Strikingly, 50680 fails to suppress T cell activation. Furthermore, nonimmunosuppressing analogs of cyclosporin A that still bind to and inhibit rotamase activity of cyclophilin also have been described (Sigal et al., 1991). The inability of rotamase inhibition to explain the immunosuppressive effects of CsA and FK506 forced a fundamental reconsideration of how these fungal toxins are

Cdl 024

Phvsioloaical

Function

1. Endogenous Ligands for Cellular Regulation?

I

lmmunosutwression

J CYP L3 I

2. General Protein foldases Involved in Protein Synthesis and Trafficking? 3. Other Biological Functions?

Figure 1. Schematic

Representation

of Proposed

Drug Complex

Interactions

Cyclophilins (CyP) bind cyclosporin A (CsA) to form a complex in which both components change in structure. This complex binds to, and inhibits, calcineurin (CnA, A subunit; CnB. B subunit; CaM, calmodulin) in a calcium-dependent manner. FK506-binding protein (FKBP) complexes with FK506 or rapamycin (Rapa). FKBP-FK506 also binds calcineurin. The target of FKBP-rapamycin is unknown, but presumed to be different from

subverting cellular regulatory pathways. This led to experiments to test the notion that the drug-receptor complex was acting as a single effector. Friedman and Weissman (1991) showed that cyclophilin C bound a 77 kd protein in the absence of cyclosporin A, while the cyclophilin-drug complex bound a completely different protein migrating at 55 kd. Liu et al. (1991a) approached the same problem using an array of the drug complexes and came up with results that not only explain the parallel effects of cyciosporin A and FK506, but also provide the first hint of the steps in the signal transduction pathways that are abrogated by these drugs. They showed that complexes between cyclosporin A and cyclophilin A or C, as well as FK506-FKBP complexes, bind to the same set of proteins (M, 61 K, 17K, and 15K). Neither drug nor their receptors alone bind to these proteins. Initial efforts to identify these binding proteins were frustrated by blocked amino-termini, which made their amino acid sequencing impossible. However, when EGTA was added to chelate the requisite calcium in the binding reaction, p17 underwent a dramatic shift in electrophoretic mobility. This protein had the molecular weight and calcium-dependent mobility consistent with those of calmodulin. This, in turn, suggested that the other proteins that bound the immunosuppressant-receptor complexes were the calciumlcalmodulin-dependent phosphatase 28, known as calcineurin, which has a catalytic A subunit of 61 kd and a regulatory B subunit of 19 kd (M, 14-19K) (Klee et al., 1988). Antibodies against calcineurin confirmed the identities of these common targets of the immunosuppressant-receptor complexes, all made

without a single cycle of Edman degradation. More important still was the finding that these drug-receptor complexes strongly inhibit the phosphatase activity of calcineurin in vitro (Liu et al., 1991a). Calcineurin: Into the Limelight The specific inhibition of calcineurin by immunosuppressant-receptor complexes has focused attention on these highly conserved, calciumlcalmodulin-activated protein phosphatases. Although first characterized in skeletal muscle and brain, it is now clear that they are found in a wide range of tissues. The 61 kd calcineurin A subunit possesses the catalytic activity and has a binding site for calmodulin as well as for the 19 kd regulatory calcineurin B subunit, which also binds calcium (Klee et al., 1988). Efforts to define a specific role for calcineurins in calciumactivated signal transduction pathways have been hampered, until now, by the lack of specific inhibitors. To make matters more complex, three genes encoding the catalytic A subunit have been identified, some of which yield transcripts that are differentially processed (Guerini et al., 1989; Wadzinski et al., 1990). The.,yeast Saccharomyces cerevisiae has two genes encoding proteins homologous to mammalian calcineurin, CMM and CMP2 (Liu et al., 1991 b). Surprisingly, single or double disruption mutations have no effect on growth rate, cell morphology, heat shock responses, sensitivity to nitrogen starvation, a factor sensitivity, or sporulation. While one could propose a system redundancy in which calcineurin function is complemented by other phosphatases, one phenotype has been uncovered by an independent analysis of yeast calcineur-

Minireview 825

ins. Although a factor arrest is unaffected in the calcineurin double mutant strain, the cells never reenter the cell cycle after extended exposure to a factor and therefore appear defective in a factor tidaptation (Cyert et al., 1991). The value of this observation is to tie calcineurin (and presumably calcium) to the mating factor response, a field that has yielded a myriad of discoveries in signal transduction and cell Cycle COntrOl.

Rapamycin-FKBP Separate Pathway

Complex

Focuses

on

lf cyclosporin A and FK506 act through calcineurin, what targets are recognized by rapamycin-FKBP? With the exception of the “effector” portion of rapamycin, complexes between rapamycin and FKBP must appear somewhat similar to FK506-FKBP (Van Duyne et al., 1991 b). Possible candidates include other phosphatases, which, as a group, share significant homology. One caution, however, is found in thefindingof Liu et al. (1991a)thatverydifferent complexes can bind to the same target. Insights into targets of the rapamycin-FKBP complex will likely come from yeast, which is highly sensitive to rapamycin. The toxicity of rapamycin can be removed by mutations in the gene encoding FKBP (RBP7), and restored by expression of the human FKBP72 cDNA (Koltin et al., 1991). These data imply that the rapamycin-RBPl complex is mediating the lethality of rapamycin in yeast, presumably though interactions with critical cellular components. The high toxicity of rapamycin toward yeast has been exploited to screen for other mutations that could confer resistance to this drug (Heitman et al., 1991). Mutations in two genes, TORI and TOR2, which map outside RBP7, confer resistance to rapamycin. The identification of the gene products encoded by TO/37 and TOR2 might reveal an immediate relationship to signal transduction pathways blocked by rapamycin-FKBP complexes.

Perspectives The recent advances in understanding the mechanism of immunosuppression by cyclosporin A and FK506, including the molecular structures of FKBPs (Michnick et al., 1991; Van Duyne et al., 1991b; Moore et al., 1991) and cyclophilins (Ke et al., 1991), still leave thefield in a quandary. The growing roster of immunosuppressant receptor proteins and their downstream targets will demand precise biochemical, genetic, and cell biological analyses for understanding those interactions relevant to immunosuppression. An even more vexing question is what do these data suggest about the normal functions of the cyclophilins and FKBPs? Do endogenous ligands exist for regulating cyclophilin and FKBP interactions with cellular proteins? If so, what do they look like and what is their complexity? As for the endogenous targets of cyclophilins and putative ligands, we will have to await the identity and specificity of the 77 kd protein that associates with cyclophilin C in the absence of cyclosporin A. All of this returns attention to the common peptidyl-prolyl isomerase activities of cyclophilins and FKBPs. While inhibition of this activity probably has no causative role in immunosuppression, it may play a role in ligand presentation clinically as well as with putative endogenous ligands.

It should be noted that both cyclosporin A and FK506 undergo cis-trans isomerization about particular bonds upon binding their respective receptors, an action that likely results in significant structural changes at the surface of these complexes (Fesik et al., 1990; Van Duyne et al., 1991 a). The conformational changes experienced by ras upon binding GTP, and its subsequent interaction with GAP, may be the appropriate paradigm for the proposed regulated presentation of effector surfaces. Alternatively, these diverse toxins may have subverted proteins whose normal role is more along the lines of protein folding and trafficking.

Bierer, B. E., Mattila, P.S., Standaert, R. F., Herzenberg, L. A., Burakoff, S. J., Crabtree, G., and Schreiber, S. L. (1990a). Proc. Natl. Acad. Sci. USA 87, 9231-9235. Bierer, B. E., Somers, P. K., Wandless, T. J., Burakoff. Schreiber, S. L. (1990b). Science 250, 556-559. Borel, J. F. (1976). Immunology

S. J., and

37, 631-641,

Cyert, M. S., Kunisawa, R., Kaim, D., and Thorner, J. W. (1991). Proc. Natl. Acad. Sci. USA, in press. Fesik, S. W., Gampe, R. T.. Holzman, T. F., Egan, D. A., Edalji, R., Luly, J. R., Simmer, R., Helfrich, R.. Kishore, V., and Rich, D. H. (1990). Science 250, 1406-1409. Fischer, G., Wittmann-Liebold, B., Lang, Schmid, F. X. (1989). Nature 337, 476-478.

K., Kiefhaber,

T., and

Flanagan, W. M., Corthesy, B.. Bram, R. J., and Crabtree, G. R. (1991). Nature, in press. Friedman,

J., and Weissman,

I. (1991). Cell 66, 799-805.

Guerini, D., and Klee, C. B. (1989). Proc. Natl. Acad. Sci. USA 86, 9183-9187. Handschumacher, R. E., Harding, M. W., Rice, J., and Drugge, R. J. (1984). Science 226, 544-547. Harding, M. W., Handschumacher, J. Biol. Chem. 261, 8547-8555.

R. E., and Speicher, D. W. (1986).

Harding, W. H., Galat, A., Uehling, D. E., and Schreiber, Nature 347, 758-760. Heitman, J., Mowa,

S. L. (1989).

N. R., and Hall, M. N. (1991). Science, in press.

Ke, H., Zydowsky, L. D., Liu, J., and Walsh, C. T. (1991). Proc. Natl. Acad. Sci. USA, in press. Klee, C. B.. Draetta, G. F., and Hubbard, 67, 149-200.

M. J. (1988). Adv. Enzymol.

Koltin, Y., Faucette, L., Bergsma, D. J., Levy, M. A., Cafferkey, R.. Koser, P. L., Johnson, R. K., and Livi, G. P. (1991). Mol. Cell Biol. 7 7, 1718-l 723. Liu, J., Farmer, J. D.. Lane, W. S., Friedman, Schreiber, S. L. (1991a). Cell 66, 807-815.

J., Weissman,

I., and

Liu, Y., Ishii, S., Tokai, M., Tsutsumi, H., Ohki, O., Akada, R., Tanaka, K., Tsuchiya, E., Fukui, S., and Miyakawa, T. (1991 b). Mol. Gen. Genet. 227, 52-59. Michnick, S. W., Rosen, M. K.. Wandless, T. J., Karplus, Schreiber, S. L. (1991). Science 252, 836-839.

M., and

Moore, J. M., Peattie, D. A., Fitzgibbon, M. J., and Thomson, J. A. (1991). Nature 357, 248-250. Price, E. R., Zydowsky, L. D., Jin, M., Baker, C. H., McKeon, F. D., and Walsh, C. T. (1991). Proc. Natl. Acad. Sci. USA 88, 1903-1907. Siekierka, J. J., Hung, S. H. Y., Poe, M., Lin, C. S., and Sigal, N. H. (1989). Nature 347, 755-757. Sigal, M. H., Dumont, F., Durette, P., Siekierka, J. J., Peterson, L., Rich, D. H., Dunlap, B. E., Staruch, M. J., Melino, M. R., Koprak, S. L., Williams, D.. Witzel, B., and Pisano, J. M. (1991). J. Exp. Med. 7 73, 619-628.

Cell 826

Stamnes, M. A., Shieh, B.-H., Chuman, C. S. (1991). Cell 65, 219-227. Takahashi, 476.

L., Harris, G. L., and Zuker,

N., Hayano, T., and Suzuki, M. (1969). Nature 337, 473-

Van Duyne, G. D., Standaert, Ft. F., Karplus, P.A., Schreiber, and Clardy, J. (1991a). Science 252, 639-642.

S. L.,

Van Duyne, G. D., Standaert, Ft. F., Schreiber, (1991b). J. Am. Chem. Sot., in press.

S. L., and Clardy, J.

Wadzinski, B. E., Heasley, L. E., and Johnson, Chem. 265, 21504-21506.

G. L. (1990). J. Biol.

When worlds collide: immunosuppressants meet protein phosphatases.

Cell, Vol. 66, 823-826, September 6, 1991, Copyright 0 1991 by Cell Press When Worlds Collide: lmmunosuppressants Meet Protein Phosphatases Frank...
362KB Sizes 0 Downloads 0 Views