SIGNAL TRANSDUCTION IN HEMATOPOIETIC CELLS 
Roger M. Perlmutter, M.D., Ph.D., Associate Investigator 
Dr. Perlmutter's research is focused on the devel- 
opmental regulation of antibody repertoire diversi- 
fication and on the mechanisms responsible for sig- 
nal transduction in hematopoietic cells. During the 
past year, emphasis has been placed on the study of 
transgenic mice bearing altered signaling mole- 
cules. Disturbances in T cell function in such ani- 
mals provide insight into normal lymphocyte signal 
transduction pathways. 
I. Development of the Human Antibody Repertoire. 
Antibodies are encoded by discontinuous germ- 
line gene segments that are juxtaposed through so- 
matically propagated gene rearrangement events. 
Assembly of the adult antibody repertoire is devel- 
opmentally programmed: a small number of germ- 
line antibody gene segments contribute preferen- 
tially to the generation of antibodies in fetal and 
neonatal B lymphocytes. Previous studies estab- 
lished that heavy-chain gene segments involved in 
these early rearrangements are concentrated near 
the 3' end of the locus. Detailed analysis of the pat- 
tern of expression of antibody light chains in the 
human fetus revealed a hierarchical pattern of vari- 
able region gene segment rearrangement, indicat- 
ing that the set of combining sites produced early 
in immune system ontogeny is limited. One curious 
feature of fetal antibody repertoires in general is 
the high frequency of autoreactive elements. This 
observation has led to the proposal that disease- 
inducing autoantibodies of the type encountered in 
rheumatologic illness result from inappropriate ex- 
pansion of preexisting self-reactive B cell clones. At 
the same time, considerable evidence supports the 
view that most autoantibodies arise through anti- 
gen-driven somatic mutation. These alternative 
views of the pathogenesis of autoimmunity can 
now be productively addressed, using the large 
database of fetal antibody sequences developed in 
Dr. Perlmutter's laboratory and new microscale 
methods for determining the sequences of patho- 
logic autoantibodies. 
II. Protein Tyrosine Kinase Signaling Elements in 
Lymphocytes. 
Although steady progress has been made in iden- 
tifying the receptors on lymphocytes that permit 
recognition of foreign macromolecules, no consen- 
sus has emerged regarding the signaling process 
that permits antigen recognition to induce lympho- 
cyte activation. Members of Dr. Perlmutter's labora- 
tory have identified two membrane-associated pro- 
tein tyrosine kinases (p56''^* and p5S^"^) that are 
lymphocyte-specific signal transduction elements. 
Previous studies defined the Ick gene by virtue of its 
overexpression in a murine lymphoma cell line and 
demonstrated that, like several other protein tyro- 
sine kinases, the activity of p56''^^ is regulated by 
phosphorylation on a carboxyl-terminal tyrosine 
residue (Tyr505). Substitution of phenylalanine for 
tyrosine at this position generates a potent trans- 
forming element. 
Two observations suggest that p56''^* is intimately 
involved in T cell signal transduction. First, p56''^* is 
physically associated with the CD4 and CDS mole- 
cules that form part of the T cell antigen recogni- 
tion complex. Second, T cell activation results in 
rapid conversion of p56''^* to a p60 form and in the 
downregulation of the Ick transcriptional unit. 
To elucidate the function of p56''^^. Dr. Perlmut- 
ter and his colleagues are attempting to alter p56''^* 
activity directly in otherwise normal T lymphocytes. 
Two distinct Ick promoter elements that are sepa- 
rated by 35 kb of intervening sequence have been 
defined. Both elements behave in a lymphocyte- 
specific fashion in transgenic mice. The most 3' of 
these promoter elements have been used to gener- 
ate mice that bear an activated version of p56''^* 
containing phenylalanine at position 505. The T 
cells from these animals have an altered T cell sig- 
naling profile, in that they can no longer be acti- 
vated appropriately by mitogens. These observa- 
tions focus attention on p56''^^ as a mediator of 
signals from the T cell antigen receptor complex. 
In related studies, a second lymphocyte-specific 
protein tyrosine kinase has been identified. The 
product of alternative splicing of transcripts from 
the fyn gene, p59^"^ contains an unusual catalytic 
domain structure, suggesting that the substrates for 
this kinase will differ from those phosphorylated by 
p56''^^. In addition, p5^^"^ does not interact with 
CD4 or CDS. Interestingly p5S^^"^ is greatly over- 
expressed in lymphocytes from Ipr/lpr mice that 
suffer from a lymphoproltferative abnormality. This 
observation suggests that p59^^'"^ will also assist in 
regulating T cell activation. 
A third kinase under study is p59*'^^, which, al- 
though present at low levels in B lymphocytes, is 
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