Regulation of Cellular Processes 
by Protein-Tyrosine Phosphorylation 
Joan S. Brugge, Ph.D. — Investigator 
Dr. Brugge is also Professor of Microbiology at the University of Pennsylvania School of Medicine. She 
received her B.A. degree in biology from Northwestern University and her Ph.D. degree in virology from 
Baylor College of Medicine. Her postdoctoral research was done with Raymond Erikson at the University 
of Colorado School of Medicine, Denver. Before moving to the University of Pennsylvania, Dr. Brugge 
was a member of the Department of Microbiology at the State University of New York at Stony Brook. 
THE modification of intracellular proteins by 
the reversible addition of phosphate groups 
(phosphorylation/dephosphorylation) is the most 
common mechanism for regulating their activity. 
Enzymes that specifically transfer phosphate to 
tyrosine residues of proteins (protein-tyrosine ki- 
nases) play critical roles in intracellular signal 
transduction events — biochemical processes 
that transform interactions at the surface of the 
cell into an intracellular response. Our labora- 
tory is interested in investigating the role of tyro- 
sine phosphorylation in these processes. We have 
chosen for these studies two model systems that 
offer unique advantages: platelets, which are ide- 
ally suited for studies of cell adhesion, secretion, 
and cytoskeletal rearrangements, and PCI 2 cells, 
which allow dissection of events that are in- 
volved in the differentiation of neuronal cells 
after treatment with nerve growth factors. 
Tyrosine Phosphorylation in Platelets 
Platelets are small, anucleate peripheral blood 
cells that contain many intracellular vesicles 
whose components are released upon activation 
by cellular hormones. Platelet aggregation and 
the released products from platelets are responsi- 
ble for the formation of blood clots and wound 
healing. We have found that hormones that acti- 
vate platelet aggregation and secretion cause 
rapid changes in the phosphorylation of multiple 
proteins on tyrosine. In collaboration with San- 
ford Shattil (University of Pennsylvania School of 
Medicine) , we have shown that the phosphoryla- 
tion of several of these proteins requires platelet 
aggregation. 
This process is mediated by interactions be- 
tween the serum adhesion protein fibrinogen and 
its receptor, GP Ilb-IIIa, on the platelet surface. 
GP Ilb-IIIa is a member of the integrin family of 
receptors that bind to extracellular matrix and 
adhesion proteins. These receptors are believed 
to be important in causing changes in cell behav- 
ior that are mediated by cell adhesion. Our stud- 
ies demonstrating that inhibition of GP Ilb-IIIa- 
induced cell aggregation prevents tyrosine 
phosphorylation of cellular proteins suggest that 
this receptor may regulate the activation of tyro- 
sine kinases, which play a role in aggregation- 
dependent events. 
We have recently found that a cytoplasmic tyro- 
sine protein kinase, pi 25-FAK, is phosphorylated 
on tyrosine and activated following platelet ag- 
gregation. This kinase was first identified by Tom 
Parsons (University of Virginia School of Medi- 
cine) as a substrate phosphorylated in Rous sar- 
coma virus-transformed cells. The enzyme is lo- 
calized in focal adhesion plaques — sites where 
integrin receptors couple with extracellular ma- 
trix proteins and intracellular microfilaments. It 
is not phosphorylated on tyrosine or activated in 
platelets from patients with Glanzmann's throm- 
bocytopenia, a disorder caused by the absence of 
a functional GP Ilb-IIIa. These results suggest 
that pi 25-FAK may be activated by events follow- 
ing GP Ilb-IIIa-dependent platelet aggregation. 
It is also phosphorylated on tyrosine in fibro- 
blasts following activation of other integrin re- 
ceptors and could play a critical role in integrin- 
mediated activation of intracellular signal 
transduction events mediated by cell-cell and 
cell-matrix interactions. 
GP Ilb-IIIa may also be important for the assem- 
bly of intracellular cytoskeletal complexes that 
follow thrombin-induced platelet aggregation. 
Together with Joan Fox (Gladstone Foundation), 
we have found that the assembly of these cytoskel- 
etal proteins with enzymes involved in signal 
transduction is dependent on GP Ilb-IIIa and 
platelet aggregation. We have found that four 
members of the Src family of protein-tyrosine ki- 
nases — Src, Fyn, Lyn, and Yes — are part of these 
assemblies, as are the cytoskeletal proteins actin, 
vinculin, talin, and GPIV. In platelets from 
Glanzmann's thrombocytopenia patients, these 
proteins do not associate with the detergent- 
insoluble cell fraction, indicating that GP Ilb-IIIa 
is critical for these cytoskeletal rearrangements. 
Thus platelet aggregation mediated by fibrinogen 
binding to GP Ilb-IIIa appears to play an impor- 
tant role in nucleating the assembly of cytoskele- 
tal signaling complexes that may be important for 
activation of intracellular processes, and these 
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