MECHANISM OF ACTION OF POLYPEPTIDE GROWTH FACTORS 
Linda J. Pike, Ph.D., Associate Investigator 
Dr. Pike is interested in the control of cell 
growth. In particular her research focuses on the 
mechanism by which epidermal growth factor 
(EGF) transmits its signal across the cell membrane. 
Binding of EGF to the extracellular domain of its 
cell surface receptor stimulates a protein tyrosine 
kinase activity located on the intracellular domain 
of the receptor. Other biological processes, includ- 
ing phosphatidylinositol (PI) turnover and receptor 
internalization, are also stimulated by ligand bind- 
ing. Although the binding of EGF stimulates numer- 
ous responses, prolonged treatment of cells with 
EGF leads to a loss of responsiveness of the cells to 
the growth factor, a process known as desensitiza- 
tion. The current goals of the laboratory are 1) to 
understand the regulation of phosphoinositide me- 
tabolism and its role in signal transduction and 2) 
to elucidate the molecular mechanism of EGF re- 
ceptor desensitization. 
I. Phosphoinositide Metabolism. 
A. Phosphatidylinositol kinase. Previous work has 
shown that EGF stimulates the activity of a PI kinase 
in A43 1 cells, a human epidermal carcinoma cell line. 
This activation is retained in membranes prepared 
from EGF-treated cells. The EGF-stimulated PI kinase 
was purified from A431 cells and shown to be a 55 
kDa monomeric protein. Because cultured cells pro- 
vide only limited amounts of material for purifica- 
tion, the PI kinase has recently been purified from 
human placenta. With this material, peptides were 
produced by cleavage with cyanogen bromide, and 
the sequence of an extremely hydrophobic peptide 
was determined. 
Polyclonal antibodies were raised to the purified 
PI kinase. The antibodies identify a 55 kDa protein 
on Western blots. These antibodies were used to 
screen a Xgtll human placental cDNA library. 
Twenty-eight putative positive cDNA clones were 
selected by this procedure. The protein sequence 
obtained from the placental enzyme was used to 
synthesize oligonucleotide probes and to rescreen 
the antibody-positive clones and the library. All pos- 
itive clones will be further characterized. Sequence 
information obtained from this cloning work will 
provide useful structural information on the PI ki- 
nase. An analysis of the PI kinase sequence will pro- 
vide information on the relationship of this enzyme 
to other known families of small-molecule kinases 
or protein kinases. 
The PI kinase is stimulated in cells treated with 
EGF. In addition, the activity of the enzyme is en- 
hanced in cells treated with the phorbol ester 12-0- 
tetradecanoylphorbol-13-acetate (TPA), a known 
activator of protein kinase C. Treatment of A431 
cell membranes with protein kinase C also leads to 
the activation of PI kinase activity, which supports a 
role for protein kinase C in the regulation of PI ki- 
nase activity. Despite the fact that both EGF and 
protein kinase C appear to activate the PI kinase, 
incubation of the purified PI kinase with either pro- 
tein kinase C or the EGF receptor kinase under 
phosphorylating conditions leads to neither phos- 
phorylation nor activation of the PI kinase. These 
data suggest that a third component is required to 
mediate the effects of EGF and protein kinase C on 
the PI kinase. Work is in progress to reconstitute ac- 
tivation of the PI kinase in vitro by adding back var- 
ious extracts from A431 cells. 
B. Phosphatidylinositol-4-monophosphate (PIP) 
phosphatase. The reactions in the pathway for the 
synthesis of the polyphosphoinositides have been 
presumed to be reversible; however, the enzymes 
responsible for the dephosphorylation of PIP and 
PIP^ have not been characterized. A PIP phospha- 
tase from A431 cells has been partially purified and 
characterized in Dr. Pike's laboratory. It appears to 
be specific for dephosphorylating PIP, as other 
phosphoinositides or inositol phosphates that have 
phosphate groups in position 4 of the inositol ring 
do not appear to function as alternate substrates or 
competitive inhibitors. Although protein phospha- 
tases do not dephosphorylate PIP, the PIP phospha- 
tase is inhibited by some common protein phos- 
phatase inhibitors, such as fluoride, zinc ions, and 
sodium orthovanadate. Physical and biochemical 
studies suggest that PIP phosphatase is a transmem- 
brane glycoprotein with a molecular weight of 
—140,000. Future work will concentrate on the pu- 
rification of the enzyme and the generation of anti- 
bodies and protein sequence necessary for cloning 
of PIP phosphatase. 
II. Desensitization of the EGF Receptor. 
Previous work in the laboratory has shown that 
treatment of A431 cells with high concentrations of 
EGF reduces the ability of the treated cells to inter- 
nalize ^^^I-EGF. This desensitization of ligand-in- 
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