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Opening a window on immunosuppression
SHIRISH SHENOLIKAR
T-CELL ACTIVATION
Opening a window on immunosuppression Immunosuppressants rapamycin and FK506 bind the same receptor, but act differently to prevent T-cell activation. Recent results shed light on the basis of this difference. When T cells are activated they synthesize and secrete cytokines, which coordinate the functions of other cell types involved in the immune response. The immunosuppressive drugs cyclosporin A (CsA) and FK506, although structurally unrelated, both inhibit a Ca 2 +-regulated step in T-cell activation and block cytokine synthesis [ 1]. These drugs work by binding specific intracellular receptors, known as immunophilins. Complexes between CsA and its receptor, cyclophilin, and between FK506 and the FK506-binding protein (FKBP) are thought to mediate the therapeutic and toxic effects of these drugs. Although the immunosuppressive drug rapamycin competes with FK506 for binding to FKBP, it has no effect on cytokine synthesis by T cells - - instead it prevents T cells from responding to the autocrine action of the crucial cytokine interleukin-2 (IL-2). The primary target of CsA~zyclophilin and FK506-FKBP complexes is thought to be calcineurin, a Ca2+-reg ulated protein phosphatase [2]. Interaction of the drug-receptor complexes with calcineurin leads to inhibition of transcription of the gene for IL-2. Rapamycin, however, has recently been found to block activation of the protein kinase called p70 $6 kinase, or pp70 s6K (as its molecular weight is 70 kD and its best-known substrate is the ribosomal protein $6) [3-6]. These new results imply that pp70 s6K lies on a signalling pathway essential for T-cell proliferation in response to IL-2. A number of cyclophilins and FKBPs have been identified, and these and the targets of the drug-receptor complexes appear to be expressed in many cell types, opening a new avenue of investigation into the functions of calcineurin and pp70 s6K in non-lymphoid cells. Studies in non-lymphoid cells may prove vital in working out the key events responsible for both immunosuppression and the toxicity of the immunosuppressant drugs. CsA and FK506 block the transcription of several genes in T cells, but our ctirrent understanding of how these drugs work comes primarily from studies of their inhibition of IL-2 gene transcription. The FK506-FKBP and CsA~Tclophilin complexes bind tightly to calcineurin [2], altering its function in a way that has been postulated to prevent the activation and/or nuclear translocation of transcription factors such as NFAT (Fig. 1). The evidence that calcineurin is important in T-cell activation and immunosuppressant action has been strengthened recently by experiments in which calcineurin was overexpressed in transfected T cells [7,8]. T cells can be activated by a combination of a Ca 2+ ionophore and a phorbol ester. Expression of a mutant Volume 2
Number 10
1992
Fig. 1. Inhibition of IL-2 gene
transcription by CsA and FK506. Binding of the drug-receptor complexes to calcineurin prevents the .activation of a transcription factor, NFAT, as well as its translocation to the nucleus where it binds specific enhancer sequences to activate IL-2 gene transcription. The activity of other transcription factors, such as OAP (and possibly Oct-l) may also be regulated by calcineurin.
calcineurin catalytic subunit, predicted to be constitutively active, can replace the requirement for elevated intracellular Ca 2 + levels and act in concert with phorbol esters to promote IL-2 expression [7]. IL 2 transcription induced by either elevating intracellular Ca2 + levels or overexpressing calcineurin can be inhibited by FK506, and this inhibition is antagonized by rapamycin. Overexpression of both calcineurin subunits (A and B) results in increased resistance of T cells to FK506 [8], and also augments the Ca2+-induced transcription of reporter genes regulated by enhancer elements NFIL2E (bound by NFAT) and NFIL2A (bound by OAP/Oct 1), taken from the IL 2 gene. These and other recent results suggest that both calcineurin subunits are required for optimal binding of the drug-receptor complexes. Thus, a new role for calcineurin in the control of gene expression in T cells has been identified. Whether calcineurin directly catalyses dephosphorylation of trans cription factors, and how the cytosolic protein phosphatase 'communicates' with nuclear proteins is unclear. Although CsA and FK506 have been shown to inhibit dephosphorylation of a synthetic phosphopeptide 549
substrate, calcineurin activity against a non-protein substrate (p-nitrophenyl phosphate) was found to be increased by FK506-FKBP [2]. If the drug-receptor complexes alter the substrate specificity of calcineurin [9], then the use of CsA or FK506 to elucidate the physiological functions of calcineurin may be fraught with difficulty. Thus, analysis of the effects of the CsA/FK506-receptor complexes o n calcineurin's activity with its known protein substrates is essential. As mentioned above, rapamycin inhibits the induction of T-cell proliferation by IL-2 but has no effect on IL-2 synthesis. Growth factors, such as 1L-2, activate a number of protein phosphorylation cascades in the cells that they stimulate. Phosphorylation of ribosomal protein $6 is a highly conserved response in mammalian cells, and is stimulated in T cells by IL-2. Two families of $6 Mnases have been identified: the pp70 s6K family, and a distinct family of 85-90 kD kinases called pp90 rsk (or simply RSK, for ribosomal $6 kinase). Whereas pp90 rsk have a wide range of protein substrates and have been implicated in the control of transcription and translation, the only substrate identified for pp70 s6K is ribosomal protein $6 and their role in growth regulation is largely unexplored. The recent discovery that rapamycin, but not FK506, inhibits the IL 2-stimulated phosphorylation and activation of pp70 s6K has thus generated considerable excitement [3,4]. The new studies also show that IL-2 does not activate the pp90 rsk. By contrast, signalling via the T-ceil re ceptor (Fig. 2) selectively activates the pp90 rsk. Whereas many of the steps leading to pp90 rsk activation have been elucidated (several involve serine/threonine protein kinases), the pathway of pp70 s6K activation remains u n k n o w n . The complete segregation of these pathways implies that the two types of $6 kinase have independent roles in the regulation of T-cell growth. Activation of pp70 s6~ is, however, more sensitive than IL-2-stimulated DNA synthesis to inhibition by rapamycin [3], indicating that pp70 s6K inhibition may not be sufficient to block cell growth and that other rapamycin-sensitive pathways must also be inhibited to prevent T-cell proliferation. Whereas CsA and FK506, at concentrations that block T-cell activation, have little effect on the proliferation of other cell types, rapamycin induces cell-cycle arrest in both yeast and mammalian ceils. Recent studies show that rapamycin blocks pp70 s6K activation in non-lymphoid cells [5,6]. In Swiss 3T3 fibroblasts, mpamycin inhibited $6 phosphorylation in response to a wide variety of mitogenic stimuli, and delayed DNA synthesis [5]. In rat hepatoma cells, rapamycin inhibits both basal and insulin-stimulated DNA synthesis [6]. The effects of rapamycin in these two sets of experiments were antagonized by FK506, indicating that FKBP is involved and confirming .that FK506-FKBP complexes have no effect on the proliferation of non-lymphoid cells. In all cells ex: amined, the activation of other growth factor-stimulated protein kinases, such as Raf-1, MAP kinases and pp90 rsk, was unaffected by rapamycin. Although the direct target of rapamycin-FKBP complexes has not yet been identified, that rapamycin blocks pp70 s6K activation in many cell types suggests it inhibits a highly conserved step in the activation pathway. Mutations in several yeast genes 550
Fig. 2. Inhibition of pp70s6K activation by rapamycin, blocking signa} transduction from the IL-2 receptor and }eading to growth arrest. Antigen recognition by the T-cell receptor, by contrast, activates pp90 rsk in a way not affected by rapamycin.
have been shown to confer rapamycin resistance, suggesting there may be muhiple targets for rapan~ycin that can lead to growth arrest. Although progress has been made in our understanding of the molecular basis of immunosuppression by CsA, FK506 and rapamycin, many key questions remain unanswered. For example immobilized CsA analogues [101, which cannot penetrate cell membranes, cause therapeutically useful immunosuppression, suggesting that interactions with cyclophilins or calcineurin may not entirely explain the mechanism of action of these drugs. Although the peptidyl-prolyl isomerase activity of immunophilins, which is inhibited by binding of the drugs, appears to have no role in immunosuppression, whether the enzyme activity has any biological significance in nonlymphoid cells remains unclear. Although experimental data suggest cellular concentrations of calcineurin may play a part in determining the sensitivity of some cells to CsA and FK506, no correlation can presently be made between the tissues affected by these drugs and the tissue levels of calcineurin. The biological effects of these dmgs in non-lymphoid cells are difficult to predict as they may depend on the multiple roles of calcineurin, such as regulation of the cytoskeleton, ion channels and other proteins in these cells rather than simply the levels of the enzyme [9]. By contrast, rapamycin inhibits pp70 s6~ in many different cells but it is the unusual reliance of the IL2 receptor on this signalling pathway that may account for the sensitivity of T ceils to rapamycin. Rapamycin may be an excellent tool for investigating the process by which growth factors signal to one or both types of $6 kinases and for identifying the signals initiated by pp70 s6K that lead to cell growth. Thus, a window has been opened on the mechanism of action of immunosuppressive drugs. The identification of © 1992 Current Biology
targets of these drugs provides a lead that should facilitate research into signal transduction pathways in many cell types, not just T ceils.
6.
PEACEDJ, GROVEJR, CALVOV, AVRUCHJ, BIERER BE: Rapamycininduced inhibition of t h e 70-kilodalton S6 protein kinase. Science 1992, 257:973-977.
7.
O'KEEFESJ, TAMURAJ, KINCAIDEL, TOCCI MJ~ O'NEILL EA: FK506and CsA-sensitive activation of the intefleukin-2 p r o m o t e r by calcineurin. Nature 1992, 357:692~594.
References 1.
SCEIREIBERSL, CRABTREEGR: The m e c h a n i s m of action of cyclosporin A and FK506. Immunol Today 1992, 13:136~142.
8.
2.
l~u J, FARMERJD, LANEWS, FRIEDMANJ, WEISSMANl, SCHREIBERSL: Calcineurin is a c o m m o n target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66:807-815.
CLIPSTONE NA, CRABTREE GR: Identification of calcineurin as a key enzyme in T-lymphocyte activation. Nature 1992, 357:695497.
9.
SHENOLIKAR S, NAIRN AC: Protein phosphatases: recent progress. Adv Second Messenger Phoaphoprotein Res 1990, 23:1-121.
10.
CACALANONA, CHEN BX, CLEVELANDXyfL,ERLANGERBF: Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes. Proc Natl Acad Sci USA 1992, 89:4353-4357.
3.
Kuo CJ, CHUNG J, FIORENTINO DF, FLANAGAN WM, BLENIS
J, CRABTREE GR: Rapamycin selectively inhibits interleukin2 activation of pT0 S6 kinase. Nature 1992, 358:70 73. 4.
CALVOV, CREWS CM, glK TA, BIERER BE: Interleukin-2 stimulation of p70 S6 kinase is inhibited by the i m m u n o s u p p r e s s a n t rapamycin. Proc Natl Acad Sci USA 1992, 89:7571-7575.
5.
CI-IUNGJ, Kuo CJ, CRABTREE GR, BLENIS J: Rapamycin-FKBP specifically blocks g r o w t h - d e p e n d e n t activation of and signalling by the 70 kD $6 protein kinases. Cell 1992, 69:122~1236.
Shirish Shenolikar, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 2771O, USA.
On pages 545 547, Dagmar Ringe makes the case for cyclosporin A, rapamycin and FK506 as molecular schatchens, the Yiddish word for a marriage broker, as in: Having patiently endured a schatchen's spiel, the young man said. "But you left out just one little fact, didn't you?" "No/What?" "She limps". "Well, yes'; said the schatchen, "but only when she walks'[
Volume2
Number 10
1992
SSl
T-CELL ACTIVATION
Opening a window on immunosuppression Immunosuppressants rapamycin and FK506 bind the same receptor, but act differently to prevent T-cell activation. Recent results shed light on the basis of this difference. When T cells are activated they synthesize and secrete cytokines, which coordinate the functions of other cell types involved in the immune response. The immunosuppressive drugs cyclosporin A (CsA) and FK506, although structurally unrelated, both inhibit a Ca 2 +-regulated step in T-cell activation and block cytokine synthesis [ 1]. These drugs work by binding specific intracellular receptors, known as immunophilins. Complexes between CsA and its receptor, cyclophilin, and between FK506 and the FK506-binding protein (FKBP) are thought to mediate the therapeutic and toxic effects of these drugs. Although the immunosuppressive drug rapamycin competes with FK506 for binding to FKBP, it has no effect on cytokine synthesis by T cells - - instead it prevents T cells from responding to the autocrine action of the crucial cytokine interleukin-2 (IL-2). The primary target of CsA~zyclophilin and FK506-FKBP complexes is thought to be calcineurin, a Ca2+-reg ulated protein phosphatase [2]. Interaction of the drug-receptor complexes with calcineurin leads to inhibition of transcription of the gene for IL-2. Rapamycin, however, has recently been found to block activation of the protein kinase called p70 $6 kinase, or pp70 s6K (as its molecular weight is 70 kD and its best-known substrate is the ribosomal protein $6) [3-6]. These new results imply that pp70 s6K lies on a signalling pathway essential for T-cell proliferation in response to IL-2. A number of cyclophilins and FKBPs have been identified, and these and the targets of the drug-receptor complexes appear to be expressed in many cell types, opening a new avenue of investigation into the functions of calcineurin and pp70 s6K in non-lymphoid cells. Studies in non-lymphoid cells may prove vital in working out the key events responsible for both immunosuppression and the toxicity of the immunosuppressant drugs. CsA and FK506 block the transcription of several genes in T cells, but our ctirrent understanding of how these drugs work comes primarily from studies of their inhibition of IL-2 gene transcription. The FK506-FKBP and CsA~Tclophilin complexes bind tightly to calcineurin [2], altering its function in a way that has been postulated to prevent the activation and/or nuclear translocation of transcription factors such as NFAT (Fig. 1). The evidence that calcineurin is important in T-cell activation and immunosuppressant action has been strengthened recently by experiments in which calcineurin was overexpressed in transfected T cells [7,8]. T cells can be activated by a combination of a Ca 2+ ionophore and a phorbol ester. Expression of a mutant Volume 2
Number 10
1992
Fig. 1. Inhibition of IL-2 gene
transcription by CsA and FK506. Binding of the drug-receptor complexes to calcineurin prevents the .activation of a transcription factor, NFAT, as well as its translocation to the nucleus where it binds specific enhancer sequences to activate IL-2 gene transcription. The activity of other transcription factors, such as OAP (and possibly Oct-l) may also be regulated by calcineurin.
calcineurin catalytic subunit, predicted to be constitutively active, can replace the requirement for elevated intracellular Ca 2 + levels and act in concert with phorbol esters to promote IL-2 expression [7]. IL 2 transcription induced by either elevating intracellular Ca2 + levels or overexpressing calcineurin can be inhibited by FK506, and this inhibition is antagonized by rapamycin. Overexpression of both calcineurin subunits (A and B) results in increased resistance of T cells to FK506 [8], and also augments the Ca2+-induced transcription of reporter genes regulated by enhancer elements NFIL2E (bound by NFAT) and NFIL2A (bound by OAP/Oct 1), taken from the IL 2 gene. These and other recent results suggest that both calcineurin subunits are required for optimal binding of the drug-receptor complexes. Thus, a new role for calcineurin in the control of gene expression in T cells has been identified. Whether calcineurin directly catalyses dephosphorylation of trans cription factors, and how the cytosolic protein phosphatase 'communicates' with nuclear proteins is unclear. Although CsA and FK506 have been shown to inhibit dephosphorylation of a synthetic phosphopeptide 549
substrate, calcineurin activity against a non-protein substrate (p-nitrophenyl phosphate) was found to be increased by FK506-FKBP [2]. If the drug-receptor complexes alter the substrate specificity of calcineurin [9], then the use of CsA or FK506 to elucidate the physiological functions of calcineurin may be fraught with difficulty. Thus, analysis of the effects of the CsA/FK506-receptor complexes o n calcineurin's activity with its known protein substrates is essential. As mentioned above, rapamycin inhibits the induction of T-cell proliferation by IL-2 but has no effect on IL-2 synthesis. Growth factors, such as 1L-2, activate a number of protein phosphorylation cascades in the cells that they stimulate. Phosphorylation of ribosomal protein $6 is a highly conserved response in mammalian cells, and is stimulated in T cells by IL-2. Two families of $6 Mnases have been identified: the pp70 s6K family, and a distinct family of 85-90 kD kinases called pp90 rsk (or simply RSK, for ribosomal $6 kinase). Whereas pp90 rsk have a wide range of protein substrates and have been implicated in the control of transcription and translation, the only substrate identified for pp70 s6K is ribosomal protein $6 and their role in growth regulation is largely unexplored. The recent discovery that rapamycin, but not FK506, inhibits the IL 2-stimulated phosphorylation and activation of pp70 s6K has thus generated considerable excitement [3,4]. The new studies also show that IL-2 does not activate the pp90 rsk. By contrast, signalling via the T-ceil re ceptor (Fig. 2) selectively activates the pp90 rsk. Whereas many of the steps leading to pp90 rsk activation have been elucidated (several involve serine/threonine protein kinases), the pathway of pp70 s6K activation remains u n k n o w n . The complete segregation of these pathways implies that the two types of $6 kinase have independent roles in the regulation of T-cell growth. Activation of pp70 s6~ is, however, more sensitive than IL-2-stimulated DNA synthesis to inhibition by rapamycin [3], indicating that pp70 s6K inhibition may not be sufficient to block cell growth and that other rapamycin-sensitive pathways must also be inhibited to prevent T-cell proliferation. Whereas CsA and FK506, at concentrations that block T-cell activation, have little effect on the proliferation of other cell types, rapamycin induces cell-cycle arrest in both yeast and mammalian ceils. Recent studies show that rapamycin blocks pp70 s6K activation in non-lymphoid cells [5,6]. In Swiss 3T3 fibroblasts, mpamycin inhibited $6 phosphorylation in response to a wide variety of mitogenic stimuli, and delayed DNA synthesis [5]. In rat hepatoma cells, rapamycin inhibits both basal and insulin-stimulated DNA synthesis [6]. The effects of rapamycin in these two sets of experiments were antagonized by FK506, indicating that FKBP is involved and confirming .that FK506-FKBP complexes have no effect on the proliferation of non-lymphoid cells. In all cells ex: amined, the activation of other growth factor-stimulated protein kinases, such as Raf-1, MAP kinases and pp90 rsk, was unaffected by rapamycin. Although the direct target of rapamycin-FKBP complexes has not yet been identified, that rapamycin blocks pp70 s6K activation in many cell types suggests it inhibits a highly conserved step in the activation pathway. Mutations in several yeast genes 550
Fig. 2. Inhibition of pp70s6K activation by rapamycin, blocking signa} transduction from the IL-2 receptor and }eading to growth arrest. Antigen recognition by the T-cell receptor, by contrast, activates pp90 rsk in a way not affected by rapamycin.
have been shown to confer rapamycin resistance, suggesting there may be muhiple targets for rapan~ycin that can lead to growth arrest. Although progress has been made in our understanding of the molecular basis of immunosuppression by CsA, FK506 and rapamycin, many key questions remain unanswered. For example immobilized CsA analogues [101, which cannot penetrate cell membranes, cause therapeutically useful immunosuppression, suggesting that interactions with cyclophilins or calcineurin may not entirely explain the mechanism of action of these drugs. Although the peptidyl-prolyl isomerase activity of immunophilins, which is inhibited by binding of the drugs, appears to have no role in immunosuppression, whether the enzyme activity has any biological significance in nonlymphoid cells remains unclear. Although experimental data suggest cellular concentrations of calcineurin may play a part in determining the sensitivity of some cells to CsA and FK506, no correlation can presently be made between the tissues affected by these drugs and the tissue levels of calcineurin. The biological effects of these dmgs in non-lymphoid cells are difficult to predict as they may depend on the multiple roles of calcineurin, such as regulation of the cytoskeleton, ion channels and other proteins in these cells rather than simply the levels of the enzyme [9]. By contrast, rapamycin inhibits pp70 s6~ in many different cells but it is the unusual reliance of the IL2 receptor on this signalling pathway that may account for the sensitivity of T ceils to rapamycin. Rapamycin may be an excellent tool for investigating the process by which growth factors signal to one or both types of $6 kinases and for identifying the signals initiated by pp70 s6K that lead to cell growth. Thus, a window has been opened on the mechanism of action of immunosuppressive drugs. The identification of © 1992 Current Biology
targets of these drugs provides a lead that should facilitate research into signal transduction pathways in many cell types, not just T ceils.
6.
PEACEDJ, GROVEJR, CALVOV, AVRUCHJ, BIERER BE: Rapamycininduced inhibition of t h e 70-kilodalton S6 protein kinase. Science 1992, 257:973-977.
7.
O'KEEFESJ, TAMURAJ, KINCAIDEL, TOCCI MJ~ O'NEILL EA: FK506and CsA-sensitive activation of the intefleukin-2 p r o m o t e r by calcineurin. Nature 1992, 357:692~594.
References 1.
SCEIREIBERSL, CRABTREEGR: The m e c h a n i s m of action of cyclosporin A and FK506. Immunol Today 1992, 13:136~142.
8.
2.
l~u J, FARMERJD, LANEWS, FRIEDMANJ, WEISSMANl, SCHREIBERSL: Calcineurin is a c o m m o n target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66:807-815.
CLIPSTONE NA, CRABTREE GR: Identification of calcineurin as a key enzyme in T-lymphocyte activation. Nature 1992, 357:695497.
9.
SHENOLIKAR S, NAIRN AC: Protein phosphatases: recent progress. Adv Second Messenger Phoaphoprotein Res 1990, 23:1-121.
10.
CACALANONA, CHEN BX, CLEVELANDXyfL,ERLANGERBF: Evidence for a functional receptor for cyclosporin A on the surface of lymphocytes. Proc Natl Acad Sci USA 1992, 89:4353-4357.
3.
Kuo CJ, CHUNG J, FIORENTINO DF, FLANAGAN WM, BLENIS
J, CRABTREE GR: Rapamycin selectively inhibits interleukin2 activation of pT0 S6 kinase. Nature 1992, 358:70 73. 4.
CALVOV, CREWS CM, glK TA, BIERER BE: Interleukin-2 stimulation of p70 S6 kinase is inhibited by the i m m u n o s u p p r e s s a n t rapamycin. Proc Natl Acad Sci USA 1992, 89:7571-7575.
5.
CI-IUNGJ, Kuo CJ, CRABTREE GR, BLENIS J: Rapamycin-FKBP specifically blocks g r o w t h - d e p e n d e n t activation of and signalling by the 70 kD $6 protein kinases. Cell 1992, 69:122~1236.
Shirish Shenolikar, Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 2771O, USA.
On pages 545 547, Dagmar Ringe makes the case for cyclosporin A, rapamycin and FK506 as molecular schatchens, the Yiddish word for a marriage broker, as in: Having patiently endured a schatchen's spiel, the young man said. "But you left out just one little fact, didn't you?" "No/What?" "She limps". "Well, yes'; said the schatchen, "but only when she walks'[
Volume2
Number 10
1992
SSl