REGULATION OF TYROSINE PHOSPHORYLATION 
Joan S. Brugge, Ph.D., Investigator 
Dr. Brugge's laboratory is examining the role of 
tyrosine phosphorylation in normal cells and the 
mechanism of oncogenic transformation by mu- 
tated variants of tyrosine kinases. As a model system 
for these studies, the cellular src protein, a 60 kDa 
protein tyrosine kinase, has been investigated. 
L Tyrosine Phosphorylation in Platelets. 
Platelets contain very high levels of the protein 
tyrosine kinase pp60'^"*'^'^. This regulatory enzyme 
represents 0.2-0.4% of total cellular protein in 
these cells. To determine whether changes in tyro- 
sine phosphorylation accompany events involved in 
platelet activation, Andy Golden and Dr. Brugge 
have examined the pattern of tyrosine phosphoryla- 
tion after treatment with agents that activate plate- 
let functions. Thrombin, a potent activator of plate- 
let secretion and aggregation, caused rapid changes 
in tyrosine phosphorylation of multiple platelet 
proteins, predominantly a 95-97 kDa protein 
doublet. Other agonists, including collagen, masto- 
paran, calcium ionophores, and ADP plus fibrino- 
gen also induced similar changes in tyrosine phos- 
phorylation; however, forskolin, which blocks 
thrombin-induced secretion and aggregation (by in- 
creasing the levels of intracellular cAMP) prevented 
the induction of tyrosine phosphorylation by 
thrombin and other agonists. These results sug- 
gested that the activation of tyrosine phosphoryla- 
tion was not specifically coupled to the thrombin 
receptor and that tyrosine phosphorylation may 
mediate some of the changes in cell physiology that 
are triggered by extracellular signals. 
To investigate the mechanism responsible for ac- 
tivation of tyrosine phosphorylation in platelets, 
Drs. Brugge and Golden used a variety of agonists 
and inhibitors of specific platelet functions to es- 
tablish whether phosphorylation of the 95-97 kDa 
protein doublet correlated with events associated 
with platelet aggregation (i.e., fibrinogen receptor 
occupancy) or with events that activate platelet se- 
cretion. Platelet aggregation was blocked with re- 
agents that interfere with the interaction between 
fibrinogen and its integrin-like receptor, gpIIb-IIIa 
[monoclonal antibodies to the fibrinogen receptor 
(provided by Dr. S. Shattil, University of Pennsylva- 
nia) or the peptide RGDS, which occupies the fi- 
brinogen-binding region of the receptor]. Platelet 
secretion was blocked using indomethacin in com- 
bination with the agonist ADP and fibrinogen. ADP 
induces fibrinogen binding to its receptor, leading 
to primary aggregation, which is followed by 
arachidonic acid metabolism and subsequent gran- 
ule secretion. Indomethacin inhibits secretion by 
blocking the oxidation of arachidonic acid metabo- 
lites. The effectiveness of each of these reagents 
was monitored using either a lumi-aggregometer or 
a fluorescence-activated cell sorter (FACS) (in col- 
laboration with Dr. Shattil) to monitor fibrinogen 
receptor exposure, occupancy, or granule fusion 
(secretion). Incubation with the peptide RGDS or 
antibodies to the fibrinogen receptor prevented 
platelet aggregation and blocked the phosphoryla- 
tion of the 95-97 kDa protein doublet. Incubation 
with indomethacin in the presence of ADP and fi- 
brinogen did not affect platelet aggregation but 
blocked platelet secretion. Under these conditions, 
phosphorylation of the 95-97 kDa doublet was not 
affected. These results indicate that tyrosine phos- 
phorylation of the 95-97 kDa doublet does not re- 
quire the process of secretion nor the products of 
secretion; however, this phosphorylation depends 
on events that are triggered by interaction between 
fibrinogen and its receptor. These results suggest 
that a tyrosine kinase(s) may be directly or indi- 
rectly coupled to the fibrinogen receptor in plate- 
lets. In addition, these findings also suggest that ty- 
rosine kinases may serve as second messengers to 
relay signals that are transmitted by interaction be- 
tween extracellular matrix proteins and/or cell ad- 
hesion molecules and their receptors. 
II. Functional Activity of a Neuron-specific Variant 
of the c-src Protein. 
Neurons express a structurally distinct form of 
the c-src gene product that possesses a higher spe- 
cific activity than the c-src protein expressed in 
other cell types. Dr. Joan Levy cloned c-src cDNA 
species from a chicken brain library. These cDNAs 
contained an 18-nucleotide insertion at the junc- 
tion between two exons, suggesting that the mes- 
senger RNA from neurons uses an alternate pattern 
of RNA splicing. Dr. Levy constructed a retroviral 
vector that allowed expression of this brain-specific 
c-src cDNA to compare the functional activity of this 
variant form of the c-src protein, pp60'^'^'^*^"'", with 
the insert-minus form of this protein, pp60'^'*'^'^, ex- 
pressed in other cell types. The pp60'^'^'^'^"'" protein 
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