Cell, Vol.

70, 365-366,

August

7, 1992, Copyright

0 1992 by Cell Press

ImmunophilinSensitive
Protein Phosphatase Action
in Cell Signaling Pathways
Stuart L. Schreiber
Department of Chemistry
Harvard University
Cambridge, Massachusetts

02138

Signaling pathways involved in GO and Gl phases of the

cell cycle are dependent on the phosphorylation
states
of distinct proteins. In lymphocytes, these states can be
modulated by complexes of three microbial natural products with their endogenous
immunophilins.
(The latter
term refers to a family of proteins that bind immunosuppressants.)
The immunophilins
are cyclophilin and
FK506binding
protein (FKBP). The natural products are
cyclosporin A, FK506, and rapamycin, which were discovered by their ability to suppress immune function (Seghal
et al., 1975; Borel, 1978; Tanaka et al., 1987). Alteration
of phosphorylation
patterns specifically blocks cell cycle
progression (Schreiber, 1991). Recent molecular insights
into these processes are the subject of this review.
To date, the only characterized target of immunophilinligand complexes
is the Ca*+, calmodulin-dependent
protein phosphatase calcineurin, also known as PPPB.
Remarkably,
calcineurin
binds to both cyclophilincyclosporin A and FKBP-FK506,
but not the free ligands
or proteins; despite no obvious similarity in their threedimensional structures, this binding is mutually exclusive
(Liu et al., 1991). As will become evident later in this review,
the existence of a distinct target of an FKBP-rapamycin
complex can be inferred. The data are consistent with
several possibilities, including that the target may be a
new member of the family of cytoplasmic protein phosphatases. The high affinity interactions between members of
the immunophilin
family and their signaling targets is dependent upon the immunophilin
ligand. Thus, the small
molecule can be viewed as a form of molecular glue, akin
to the short peptides that mediate interactions between
MHC molecules and T cell receptors (Bjorkman et al.,
1987).
The ease with which the natural products penetrate the
cell and their ability to interfere with intracellular signal
transmission in a variety of cell types has resulted in their
use over the past several years as probes of drug-sensitive
signaling pathways (Sigal and Dumont, 1992). Cyclosporin
A and FK506 exhibit a nearly identical spectrum of actions,
but they differ in their responses to rapamycin and 506BD,
which is a designed, synthetic variant of FK506 (Bierer et
al., 1990). These compounds inhibit the actions of FK506
by competing for intracellular
FK5Obbinding
sites on
FKBPs. They do not bind to cyclophilin and, in turn, do not
inhibit the actions of cyclosporin A. These are some of the
results suggesting that the natural products confer a new
activity to their cognate immunophilin.
While cyclophilins or FKBPs alone might not be involved in signal transmission, complexes of the immunophilins with their natural product ligands have profound

Minireview

consequences.
Both the cyclophilin-cyclosporin
A and
FKBP-FK506
complexes were shown to inhibit a previously unrecognized step in a family of Caz+-dependent
signal transduction
pathways that emanate from the
multichain immune receptors (IgE receptor on mast cells
and the antigen receptors on T and B cells). Shortly thereafter, an FKBP-rapamycin
complex was shown to interfere with a previously unrecognized
step in a family of
growth factor-activated
signal transduction pathways that
emanate from, for example, cytokine receptors in lymphocytes and the insulin receptor in hepatocytes. To characterize the events inferred from immunophilin-ligand
action, the molecular basis for the ligand-induced
gain in
immunophilin
function required definition.
An insight into this problem came from the identification
of calcineurin
as a common target of cyclophilincyclosporin A and FKBP-FK506
(but not FKBP-rapamycin) complexes (Liu et al., 1991; McKeon, 1991). Several new papers have linked these binding events with
functional properties of T lymphocytes and, in so doing,
have confirmed that calcineurin is a necessary and lateacting cytoplasmic mediator of the signal arising from the
T cell receptor. Following propagation by calcineurin, the
signal continues its journey into the nucleus, where early
T cell activation genes (for example, 11-2) are transcribed.
Whereas cyclosporin A and FK506 block this T cell receptor-mediated,
calcineurin-dependent
transition from a
resting GO state to the Gl phase of the cell cycle (activation), rapamycin acts later by inhibiting an IL-2 receptorinitiated transition from the Gl phase to the S phase of
the cell cycle (progression). Several recent papers have
provided insights into this latter process.
OKeefe et al. (1992) and Clipstone and Crabtree (1992)
have tested the hypothesis that calcineurin propagates the
T cell receptor-initiated
signal by overexpressing
the A
and B calcineurin subunits in the T cell line Jurkat. Although there are differences in the experimental details,
their conclusions are concordant. The calcineurin transfectants are hypersensitive to T cell receptor signaling,
indicating that there is a calcineurin-mediated
step in the
signaling pathway and that this step may be rate determining. Increased intracellular levels of calcineurin also result
in a decreased sensitivity of the transfectants to both
cyclosporin A and FK506, indicating the drugs cause a
loss of calcineurin function in vivo. These findings are consistent with those of Fruman et al. (1992) who report that
cyclosporin A and FK506, as their immunophilin
complexes, inhibit the phosphatase activity of calcineurin in
cellular extracts.
Thus, an outline of the events that constitute
membrane-to-nucleus
signaling along this pathway has
emerged (Schreiber and Crabtree, 1992). Following T cell
receptor occupancy, a kinase cascade causes an increase
in the concentration
of intracellular Ca*+ and activation
of calcineurin. This is followed by translocation into the
nucleus of the cytoplasmic subunit of the T cell-restricted

Cell
366

Figure

1. Cross

Sections

of Three

Immunophilin-Ligand

Complexes

lmmunophilins
are indicated by a blue dot surface; ligands by a yellow
dot surface. (A) Cyclophilin-cyclosporin
A composite surface (Spitzfaden et al., 1992). Orange dot surface indicates the isobutyl side chain
at the 6MeLeu position of cyclosporin
A, which is important for binding
to calcineurin
but not cyclophilin A. Replacing the isobutyl group with a
methylgroupabrogates
theabilityofthecomplex
to bind tocalcineurin.
and therefore,
to inhibit signal transduction
(Liu et al., 1992). (B)
FKSP12-FK506
composite surface (Van Duyne et al., 1991a). Orange
dot surfaces
indicate methoxyl and ally1 groups, which are important
for binding to calcineurin
but not FKBPlP. Replacing
the methoxyl
group (lower orange dot surface) with a hydroxyl group abrogates the
ability of the complex to bind to calcineurin,
and therefore, to inhibit
signal transduction
(Liu et al., 1992). (C) FKBP12-rapamycin
composite surface (Van Duyne et al., 1991 b). Note the difference
in geometry
of the composite
surface, which is not a calcineurin
ligand, relative to
the analogous
region in (B).

transcription factor NF-AT. Although the relationship between calcineurin and the cytoplasmic subunit of NF-AT
is not yet known, studies of the yeast transcription factor
SWI-5 (Mall et al., 1991) suggest the possibility that the
cytoplasmic subunit of NF-AT is a substrate of calcineurin.
Nuclear translocation of SWI-5 is cell cycle regulated and
follows dephosphorylation
of its cytoplasmic form (by an
unidentified phosphatase).
Analogs of cyclosporin A and FK506 have provided insights into the structural bases for their glue-like effects
and independent confirmation of calcineurin as their common, downstream signaling target (Liu et al., 1992). Figure
1A illustrates the cyclophilin-cyclosporin
A composite surface that constitutes one of the calcineurin ligands. Investigations of cyclosporin A structural variants provided
results inconsistent with a simple model of drug action involving a loss in function of cyclophilin (e.g., inhibition of its

rotamase [peptidyl-prolyl
cis-trans isomerase] activity).
Replacing the isobutyl side chain of the g-position of cyclosporin A with a methyl substituent (GMeLeu+GMeAla)
has little effect on cyclophilin binding yet abrogates the
ability of the corresponding
cyclophilin complex to bind to
calcineurin in vitro (Liu et al., 1992). Also, GMeAla-cyclosporin A does not inhibit T cell receptor-mediated
signal
transduction.
Similar results were obtained with FK506 variants; replacing the methoxyl group at Cl 5 of FK506 (Figure 16)
with hydroxyl had little effect on FKSP binding, yet abrogated the ability of the complex to bind to calcineurin and
to inhibit signal transduction. Whereas structural investigations of immunophilin-ligand
complexes
revealed
immunophilin-binding
sites on the natural products (Van
Duyne et al., 1991a; Spitzfaden et al., 1992), the latter
studies havedelineated
theircalcineurin-binding
moieties.

Minireview
367

Together, they provide a clearer picture of these receptorligand-receptor

interactions. The combination of methods
used to characterize the role of calcineurin in T cell receptor-mediated
signaling appears promising for efforts to
define calcineurins possible role in other signaling pathways, such as those involved in mast cell degranulation,
6 cell activation, thymocyte apoptosis, and ion channel
activity.
What then does the composite FKBP-rapamycin
surface (Figure 1C) (Van Duyne et al., 1991 b) recognize? The
ability of rapamycin to induce a gain in function of this
particular FKBP (FKBP12) has been clearly demonstrated
in Saccharomyces
cerevisiae (Koltin et al., 1991; Heitman
et al., 1991). Mutant strains lacking FKBP12 are completely viable yet resistant to the inhibitory actions of rapamycin, which normally results in Gl cell cycle arrest at
nanomolar concentrations.
Transfection of FKBP12 restored rapamycin sensitivity. Although the direct target
has yet to be identified, recent studies in T lymphocytes,
fibroblasts, hepatocytes, and transfected COS cells have
provided illuminating insights into the downstream targets
and the nature of the rapamycin-sensitive
step that appears to be common to a family of mitogen-activated
signaling pathways (Chung et al., 1992; Kuo et al., 1992;
Calvo et al., 1992; Price et al., 1992).
The binding of IL-2 to its receptor initiates intracellular
signals shared by many pathways mediated by insulin,
mitogens, or growth factors. One of the most rapid sequelae is the hyperphosphorylation
of the 40s ribosomal
protein S6, which is mediated by the p70 and ~65 classes
of S6 kinases (SGKs). Several recent reports demonstrate
that FKBP-rapamycin
results in the inactivation of basal
and receptor-enhanced
activity of ~70~~~ and that these
receptor-mediated
S6 phosphorylations
are due to p70SjK
and not MAP kinase-activated
~65~~ (rsk). Particularly
intriguing are the findings that IL-2 receptor-mediated
phosphorylation
(and concomitant activation) of ~70~~
can be subsequently reversed by rapamycin, which was
shown to act through binding to an endogenous
FKBP,
and that rapamycin has no effect on any of the kinase
activities known to be involved in p70SsK activation. Although the FKBP-rapamycin
complex might inhibit an unknown p70SBK kinase, an equally attractive possibility is
that FKBP-rapamycin
activates a ~70~~~ phosphatase. If
this putative phosphatase shared structural homology with
other protein phosphatases, including calcineurin, the remarkable ability of FKBP to interact with distinct protein
targets in a ligand-dependent
fashion would be put into a
more manageable context.
Unlike protein kinases, which recognize short, specific
amino acid sequences, protein phosphatases exhibit substrate specificity governed by sequences remote from the
dephosphorylation
site, presumably by recognizing threedimensional patches on the phosphoprotein.
To achieve
this patch-based selectivity, thefourwell-characterized
cytoplasmic serine/threonine
protein phosphatases
(PPl ,
PP2A, PPPB, and PPPC) are equipped with regulatory subunits (Cohen, 1969). It is of interest that the smallT antigen
of SV40 is able to replace the regulatory B subunit of PP2A

and thereby alter its substrate specificity and/or its cellular

localization
(Mumby and Walter, 1991). Cyclophilincyclosporin A and FKBP-FK506
serve to alter the substrate specificity of calcineurin as well. Although these
complexes inhibit the actions of calcineurin toward phosphopeptide and phosphoprotein
substrates, they moderately activate calcineurin toward a small substrate, p-nitrophenyl phosphate (Liu et al., 1991). Like SV40 small T
antigen, immunophilin-ligand
complexes can be thought
of as phosphatase regulatory subunits.
It will be exciting to learn if FKBP-rapamycin
acts in a
similar manner, thereby activating a phosphatase toward
~70%~. Likewise, it will be important to determine if endogenous, small molecules exist that can combine with immunophilins tocause the same gain in function induced by the
binding of the natural products. These issues constitute
some of the challenges that are being addressed in current
investigations of immunophilins,
their natural product ligands, and the cell signaling pathways they combine to
modulate.
References
Bierer, B. E., Somers, P. K., Wandless,
T. J., Burakoff.
Schreiber,
S. L. (1990). Science 250, 556-559.

S. J., and

Bjorkman,
P. J., Saper. M. A., Samraoui. B., Bennett, W. S., Strominger, J. L., and Wrley, D. C. (1967). Nature 329, 512-518.
Borel,

J. F. (1976).

Immunology

37, 631-641.

Calvo, V., Crews, C. M., Vik, T. A., and Bierer,

Acad. Sci. USA 89, in press.
Chung,
1227-l

J., Kuo, C. J., Crabtree,

236.

Clipstone,
Cohen,

N. A., and Crabtree,

P. (1989).

Annu.

B. E. (1992).

G. Ft., and Blenis, J. (1992).

G. Ft. (1992).

Rev. Biochem.

Nature

J., Mowa.

Cell 69,

357, 695-697.

58, 453-508.

Fruman, D. A., Klee. C. B., Bierer, B. E.. and Burakoff,

Proc. Natl. Acad. Sci. USA 89, 3686-3690.
Heitman,
909.

Proc. Natl.

N. R.. and Hall, M. N. (1991).

S. J. (1992).

Science

253, 905-

Koltin, Y., Faucette,

L., Bergsma,
D. J., Levy, M. A., Cafferkey,
R.,
Koses, P. L., Johnson,
R. K., and Livi, G. P. (1991). Mol. Cell. Biol. 7 1,
1718-1723.
Kuo, C. J., Chung, J.. Fiorentino,
D. F., Flanagan,
and Crabtree,
G. R. (1992). Nature 358, 70-73.
Liu, J., Farmer, J. D., Lane, W. S., Friedman,
Schreiber,
S. L. (1991). Cell 66, 807-815.

W. M., Blenis,

J., Weissman.

J.,

I,, and

Liu, J., Albers, M. W., Wandless,

T. J., Luan, S., Alberg, D. G., Belshaw. P. J., Cohen, P., Macintosh,
C.. Klee, C. B., and Schreiber,
S. L. (1992). Biochemistry
37, 3896-3901,
McKeon,

F. (1991).

Cell 66, 823-826.

Mall, T., Tebb, G., Surana,

Cell 86, 743-758.
Mumby,

U., Robitsch,

H., and Nasmyth,

G. (1991).

Cell Reg. 2. 589-598.

M. C., and Walter,

OKeefe, S. J., Tamura, J., Kincaid,

E. A. (1992). Nature 357, 692-694.
Price, D. J., Grove,
Science, in press.

J. R.. Calvo, V., Avruch,

Schreiber,

S. L. (1991).

Schreiber,
142.

S. L.. and Crabtree.

Seghal,
729.

R. L., Tocci,

S. N., Baker,

Science

M. J., and ONeill,

J., and Bierer,

B. E. (1992).

257, 283-287.
G. R. (1992).

H., and Vezina,

Sigal, N. H., and Dumont,

560.

K. (1991).

F. J. (1992).

Immunol.

C. (1975).
Annu.

Today

73,136-

J. Antibiot.

28, 727-

Rev. Immunol.

IO, 519-

Cell
366

Spitzfaden, C., Weber, H.-B., Braun,

H., Walkinshaw,
M. D., and Wiithrich,
300.

W., Kallen, J., Wider, G., Widner,

K. (1992). FEBS Lett. 300,291-

Tanaka, H., Kuroda, A., Marusawa,

Ii., Hatanaka,
H., Kino, T., Goto,
T., Hashimoto,
M., and Taga, T. (1987). J. Am. Chem. Sot. 109,50315033.
Van Duyne,
and Clardy,

G. D., Standaert,
Ft. F., Karplus, P. A., Schreiber,
J. (1991a). Science 251, 639-642.

Van Duyne, G. D., Standaert,

Ft. F., Schreiber,
(1991b). J. Am. Chem. Sot. 173. 7433-7434.

S. L..

S. L., and Clardy,

J.