Signal Transduction Pathways 
in B Lymphocytes 
Sankar Ghosh, Ph.D. — Assistant Investigator 
Dr. Ghosh is also Assistant Professor of Immunobiology and of Molecular Biophysics and Biochemistry 
at Yale University School of Medicine. After receiving his Ph.D. degree from the Albert Einstein College 
of Medicine, Bronx, New York, he conducted postdoctoral work at the Whitehead Institute 
in Cambridge, Massachusetts, under the supervision of David Baltimore. 
THE focus of research in our laboratory is to 
understand the signals that regulate B cell de- 
velopment and the pathways through which they 
are transduced. We propose to address these ques- 
tions by studying the regulation of developmen- 
tal-stage-specific expression of the immunoglob- 
ulin K light-chain gene. Primarily, we wish to find 
the signal that initiates k gene expression during 
B cell development and determine whether ex- 
pression of the gene is necessary for rearrange- 
ment of the K locus. 
The expression of the k gene in mature B cells 
is driven by an enhancer located in the intron 
between the J/c and Ck genes. The activity of this 
enhancer depends on the binding of a transcrip- 
tion factor, called NF-/cB, to a specific site in the 
element. In keeping with its role as a regulator for 
developmental-stage-specific expression of the k 
gene, NF-/cB can only be detected in an active 
form in the nucleus of mature B cells. However, it 
is present in the cytoplasm of pre-B cells and 
other cell types as an inactive precursor, bound 
to an inhibitory protein called I/cB. 
NF-kB activity can be induced by treatment of 
cells with agents such as phorbol esters or bacte- 
rial lipopolysaccharide. These agents activate sig- 
nal transduction pathways that result in the modi- 
fication of I/cB and the subsequent dissociation of 
the NF-/cB:IkB complex. The free NF-zcB then 
enters the nucleus and activates gene expression. 
Therefore the inducible system of NF-/cB behaves 
like a second messenger, transducing cell surface 
signals to the nucleus. 
To understand the change in IkB that results in 
the dissociation of the NF-kB:IkB complex, we es- 
tablished an in vitro system to look at the involve- 
ment of protein kinases in the activation process. 
The results strongly suggested phosphorylation 
of I/cB as a key event in the activation of NF-kB. 
However, further analysis of the activation pro- 
cess was hampered by the lack of reagents, such 
as antibodies, or clones for the genes encoding 
NF-kB. Therefore we undertook to clone the 
genes; the information we obtained has resulted 
in significant new insights into the function of 
these proteins. 
NF-kB is composed of two subunits of 50 kDa 
(p50) and 65 kDa (p65) and exists as a hetero- 
dimer. Cloning the cDNA encoding p50 revealed 
that it was actually synthesized as a larger protein 
of 105 kDa, which was processed to the mature 
50-kDa size. The full-length protein did not bind 
to DNA, but removal of the carboxyl-terminal re- 
gion revealed the DNA-binding activity. How- 
ever, the most interesting feature of the clone was 
the homology for over 300 amino acids in the 
amino-terminal half (i.e., the region encoding 
p50) to the oncogene \-rel, its cellular counter- 
part c-rel, and the Drosophila morphogen 
dorsal. 
Subsequent cloning of the p65 subunit re- 
vealed a similar re/-homology region, which con- 
tains both DNA-binding and dimerization do- 
mains. In fact, with the recent cloning of other 
re/-homologous proteins, a picture has begun to 
emerge in which the different members associate 
with one another to form transcription factors 
that can activate different genes. 
The homology between NF-kB and dorsal also 
has interesting implications for understanding 
the biology of both proteins. The developmental 
morphogen dorsal is responsible for the estab- 
lishment of the dorsoventral polarity in Drosoph- 
ila embryos. The basis for its action is the estab- 
lishment of a gradient of dorsal protein that is 
progressively localized to the nucleus. The cyto- 
plasmic localization of dorsal was genetically de- 
termined to be due to the action of a gene called 
cactus, which behaves like IkB, and recent clon- 
ing of the gene has shown that it is a homologue 
of mammalian IkB. 
The signal for the nuclear localization of dor- 
sal goes through a pathway involving eight up- 
stream maternal-effect genes. However, for those 
of us studying NF-kB, what has been of great inter- 
est is the finding that one of these upstream 
genes, toll, is a membrane receptor with a cyto- 
plasmic domain that is homologous to one of the 
mammalian interleukin-1 (IL-1) receptors. This 
is significant because, among the different inter- 
leukins, only IL-1 can activate NF-kB. Therefore 
the two genes that lie between toll and dorsal 
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