Genetic Regulatory Mechanisms in Cellular Differentiation 
presenting cell and a T lymphocyte is transient, 
all of the molecular events required for the deci- 
sion to proceed down this 1 0- to 1 4-day pathway 
of cellular differentiation must occur in the short 
period during which the T cell and the antigen- 
presenting cell interact. Our laboratory is seeking 
an understanding of the molecular basis of this 
cellular decision. 
Our approach to understanding the events that 
initiate this pathway was to begin by defining the 
molecules essential for the activation of genes 
that are known to be essential for T cell activation 
and difl'erentiation. This approach led us to de- 
fine two transcription factors that specifically re- 
ceive signals from the antigen receptor and are 
responsible for activation of early genes such as 
interleukin-2. One of these proteins, nuclear fac- 
tor of activated T cells (NFAT), is expressed al- 
most exclusively in T lymphocytes, and its tran- 
scriptional activity is under rigorous control by 
the antigen receptor. Furthermore, its transcrip- 
tional activity is induced by the antigen receptor 
immediately before the activation of most early 
genes. We thus focused our attention on this tran- 
scription factor rather than others that are acti- 
vated in many cell types and under many biologic 
circumstances. 
Immunosuppressants Cyclosporin A 
and FK-506 Block Nuclear Translocation 
of the Cytosolic Component of NFAT 
Cyclosporin A and FK-506 specifically inhibit 
T cell activation, a characteristic that underlies 
their clinical use as immunosuppressants to pre- 
vent transplant rejection. Although the mecha- 
nism of action of these drugs is unknown, they 
appear to work early during the commitment pe- 
riod for T cells. By acting early, they block the 
late functions of T cells and many of the functions 
of B lymphocytes and other hematopoietic cells 
that are directed by T cells. Other groups have 
found that these drugs bind and inhibit the func- 
tion of cis-trans prolyl isomerases. These en- 
zymes accelerate the folding of newly synthe- 
sized proteins. In studies with Stuart Schreiber 
(Harvard University) , we have shown that the iso- 
merase activity is not involved in the action of the 
cyclosporin and FK-506, but rather that an inhibi- 
tory complex formed between either cyclosporin 
A and cyclophilin or between FK-506 and FKBP 
blocks signal transduction. This inhibitory com- 
plex binds and blocks the activity of calcineurin, 
a heteromeric protein phosphatase also known as 
PP2B. This inhibition is likely to be related to the 
mechanism of action of the FK-506 and cyclo- 
sporin A, since immunosuppressive drugs form 
complexes that block the activity of calcineurin 
but inactive ones do not. We have found that the 
specific transcriptional activity of NFAT, but not 
its DNA-binding activity, is affected by cyclo- 
sporin A and FK-506. Mike Flanagan, Peter Kao, 
and Blaise Corthesy found that NFAT is a complex 
heterodimeric protein, one subunit of which is 
constitutive, T cell specific, and located in the 
cytoplasm of resting cells; the other subunit is 
located in the nucleus and is induced. The tran- 
scriptionally active protein forms when the cyto- 
plasmic component translocates to the nucleus in 
response to stimulation through the antigen re- 
ceptor of T cells. Cyclosporin A and FK-506 ap- 
pear to function by inhibiting the translocation 
but do not interfere with the induction of the 
nuclear component. The inhibition of nuclear 
import is specific to the extent that the drugs do 
not block nuclear import of NF-kB. Recently, 
Peter Kao and Blaise Corthesy have purified the 
proteins and isolated the genes for the two sub- 
units of NFAT. This should allow us to determine 
directly if calcineurin is involved in the signaling 
pathway initiating T cell activation. 
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