Mechanism of Action of Polypeptide Growth Factors 
Linda J. Pike, Ph.D. — Associate Investigator 
Dr. Pike is also Associate Professor in the Department of Biochemistry and Molecular Biophysics at Wash- 
ington University School of Medicine. She received her B.S. degree in chemistry from the University of Del- 
aware and her Ph.D. degree in biochemistry from Duke University, where she studied with Robert Lefko- 
witz. Her postdoctoral training was done in the laboratory of Edwin Krebs at the University of Washington. 
A number of low-molecular-weight polypep- 
tides have been shown to regulate cell 
growth. These growth factors bind to specific re- 
ceptors on the surface of cells. Through a com- 
plex series of reactions, the binding of the growth 
factor to its receptor stimulates the cell to grow 
and divide. Until recently, little was known of the 
mechanism by which growth factors induce cell 
division. It is now recognized, however, that the 
receptors not only bind the appropriate growth 
factor but also have an enzymatic activity. 
The receptors catalyze the transfer of a phos- 
phate group from adenosine triphosphate to tyro- 
sine residues in selected protein substrates. The 
enzyme effecting this activity is called a tyrosine 
protein kinase. Typically, the phosphorylation of 
a protein by a kinase leads to changes in the activ- 
ity of the protein. Although much is known about 
the groMTih factor receptor kinases, the substrates 
for these enzymes have not been identified. 
My laboratory is involved in studies of the 
mechanism by which the binding of epidermal 
grov^Tih factor (EGF) to the outside of the cell 
elicits a biological response inside. This is re- 
ferred to as signal transduction. The model sys- 
tem used in our studies employs the A43 1 line of 
human epidermal carcinoma cells, which are 
highly responsive to EGF. 
Phosphatidylinositol Metabolism 
One of the earliest responses of A431 cells to 
EGF is an increase in the metabolism of a particu- 
lar phospholipid, phosphatidylinositol. This im- 
portant lipid serves as a precursor for the genera- 
tion of two intracellular compounds that activate 
various enzymes and thereby mediate the effects 
of EGF within the cell. One of the enzymes in- 
volved in phosphatidylinositol metabolism is 
phosphatidylinositol kinase. Since this enzyme is 
stimulated by EGF, it represents a potential sub- 
strate for phosphorylation by the EGF receptor 
tyrosine protein kinase. 
We have purified the phosphatidylinositol ki- 
nase from both A431 cells and human placenta. 
This 55-kDa enzyme is active as a monomer — 
that is, as a single polypeptide chain. It phos- 
phorylates phosphatidylinositol on the 4 position 
of the inositol ring and hence is distinct from an- 
other phosphatidylinositol kinase that phosphor- 
ylates the ring on the 3 position. Preliminary data 
suggest that in membranes and whole cells this 
activity is regulated via phosphorylation, and a 
protein kinase has been identified that phosphor- 
ylates and activates the phosphatidylinositol ki- 
nase in vitro. Work is under way to characterize 
this event. 
Another enzyme involved in phosphatidylino- 
sitol metabolism is a phosphatidylinositol mono- 
phosphate phosphatase. This enzyme catalyzes 
the reverse of the reaction catalyzed by the phos- 
phatidylinositol kinase — that is, it removes the 
phosphate from the 4 position of the inositol ring 
on phosphatidylinositol monophosphate. Al- 
though this enzyme has not been studied previ- 
ously, its position in the metabolic pathway of 
phosphatidylinositol suggests it may be impor- 
tant in the overall regulation of the pathway. We 
have identified its activity in A43 1 cells and have 
characterized it with respect to its kinetic proper- 
ties, substrate specificity, and response to various 
inhibitors. We have purified the enzyme to a high 
degree and have shown that it is a 140,000-Da 
glycoprotein. Unlike other enzymes in this path- 
way, this enzyme appears to have an extracellular 
domain. The possibility that the activity of this 
enzyme is regulated through the binding of an 
extracellular mediator is being investigated. 
It has recently been postulated that the inositol 
phospholipids may function in the regulation of 
cell shape via interactions with proteins that con- 
trol the polymerization of actin. Using EGF and 
A43 1 cells, we have shown that there is no corre- 
lation between EGF-induced changes in cell 
shape and changes in the levels of the classical 
phosphoinositides, phosphatidylinositol 4-phos- 
phate and phosphatidylinositol 4,5-bisphos- 
phate. Further experiments are in progress to de- 
termine whether EGF-stimulated changes in cell 
shape can be attributed to the generation of novel 
inositol phospholipids or is due to the ability of 
the EGF receptor to catalyze the phosphorylation 
of cytoskeletal proteins. 
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