SIGNAL TRANSDUCTION BY GROWTH FACTOR RECEPTORS 
Roger J. Davis, Ph.D., Assistant Investigator 
The molecular biology of transmembrane signal 
transduction by cell surface receptors for polypep- 
tide growth factors is the major focus of Dr. Davis's 
laboratory. To gain an understanding of the proper- 
ties of a typical cell surface receptor, the laboratory 
has initiated a detailed analysis of the structure- 
function relationships for the transferrin receptor. 
In addition, further studies are in progress that are 
designed to establish the molecular basis of epider- 
mal growth factor (EOF) receptor function and the 
mechanism of mitogenic signaling by this receptor. 
Signal Transduction Pathways Activated 
by the EGF Receptor 
One mechanism of signaling by the EGF receptor 
is the stimulation of the activity of protein kinases 
located within signal transduction pathways. The 
molecular characterization of these enzymes is 
therefore an important goal. Dr. Davis and his col- 
leagues previously demonstrated that the major site 
of EGF-stimulated phosphorylation of the EGF re- 
ceptor is located at Thr^^^. The growth factor- 
stimulated phosphorylation of the receptor at 
this site can be accounted for by MAP (mitogen- 
activated protein) kinases that are activated by 
phosphorylation on tyrosine and threonine resi- 
dues. 
During the past year, four human isoforms of this 
kinase family were molecularly cloned in Dr. 
Davis's laboratory: pAQ"""'\ p4l""'f"', pii""""', and 
p(53'nfl/)fe Immunofluorescence analysis using both 
confocal and three-dimensional reconstruction tech- 
niques have shown that the p63'""^* isoform is pre- 
dominantly located within the cytoplasm and is 
largely excluded from the nucleus. In contrast, the 
p4Qmapk p4l'napk ^^^mapk ^^q^q^^^ local- 
ized in both the cytoplasmic and nuclear compart- 
ments of cells. Significantly, it was found that 
growth factor treatment caused an increase in the 
nuclear accumulation of these protein kinases. 
The growth factor-stimulated nuclear transloca- 
tion of MAP kinases suggests that these enzymes may 
have a significant role in the process of signal trans- 
duction between cell surface receptors and the nu- 
clear targets of growth factor action. To identify po- 
tential nuclear substrates for the MAP kinases, Dr. 
Davis and his colleagues examined the substrate 
specificity of the purified kinases. Substrates identi- 
fied include the EGF receptor (Thr^^^), c-Myc 
(Ser**^), and c-Jun (Ser^'*^). Inspection of the pri- 
mary sequences surrounding these phosphorylation 
sites indicates the consensus sequence Pro-Leu-Ser/ 
Thr-Pro. A systematic analysis of the effects of point 
mutations within this region demonstrated that the 
consensus sequence for phosphorylation can be gen- 
eralized as Pro-Xaa„-Ser/Thr-Pro (where Xaa is an 
aliphatic or basic amino acid, and n = 1 or 2). The 
identification of this consensus sequence has great 
value, because it allows the prediction of potential 
MAP kinase phosphorylation sites, using the se- 
quence of cloned proteins. 
Regulation of c-Myc Function 
by MAP Kinases 
Dr. Davis and his colleagues previously identified 
the major site of grov^Tih factor-regulated phosphor- 
ylation of c-Myc as Ser''^. This phosphorylation site, 
which is located within the transcriptional activa- 
tion domain of c-Myc, is a substrate for phosphoryla- 
tion by MAP kinases. During the past year studies 
have been performed that are designed to establish 
the physiological significance of the phosphoryla- 
tion of c-Myc at the MAP kinase phosphorylation 
site. A GAL4 fusion protein strategy was employed to 
examine the effects of phosphorylation on tran- 
scriptional activation. It was found that the phos- 
phorylation site is required for high levels of 
transactivation of gene expression. Thus it is likely 
that the MAP kinase phosphorylation site (Ser^^) 
represents a physiologically important mechanism 
of regulation of the transcriptional activity of the 
c-Myc protein. 
Role of Multisite Phosphorylation 
of the EGF Receptor 
The EGF receptor is a 170-kDa transmembrane 
glycoprotein that is expressed at the cell surface. 
The receptor is composed of an extracellular do- 
main that binds EGF, a single transmembrane- 
spanning domain, and a cytoplasmic domain with 
intrinsic protein-tyrosine kinase activity. Binding of 
EGF to the extracellular domain of the receptor stim- 
ulates the cytoplasmic domain protein-tyrosine ki- 
nase activity and results in the autophosphorylation 
of the receptor and the phosphorylation of ex- 
ogenous substrates, e.g., phospholipase C-7 and 
GTPase-activating protein (GAP). This process of 
transmembrane signaling is regulated by multisite 
phosphorylation of the receptor at serine and threo- 
nine residues. 
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