Molecular Mechanisms of Insulin and Polypeptide 
Hormone Action 
Perry J. Blackshear, M.D., D.Phil. — Investigator 
Dr. Blackshear is also Professor of Medicine and Assistant Professor of Biochemistry at Duke University. 
He received his D.Phil, degree in biochemistry at Trinity College, Oxford University, and his M.D. degree 
from Harvard Medical School. Before moving to Duke University, Dr. Blackshear was Assistant Professor 
of Medicine at Harvard Medical School. Dr. Blackshear has received the Young Investigator Award for 
Clinical Research from the American Federation for Clinical Research. 
OUR laboratory has been studying the molecu- 
lar mechanisms of action of insulin and 
other polypeptide growth factors. The goal of 
these studies is to understand the biochemical 
steps between the binding of insulin to its recep- 
tors on the surface of its target cells and the ulti- 
mate stimulation of changes in enzyme activity, 
protein synthesis, and gene expression. An under- 
standing of the molecular details of insulin's ac- 
tions may lead to the biochemical characteriza- 
tion of the insulin resistance seen in certain 
common disorders, such as obesity and type II 
diabetes, and possibly to novel therapies for these 
disorders. 
We are studying the intracellular actions of in- 
sulin and related polypeptide growth factors at 
several levels. One of the first effects of insulin 
on cells is the activation of an enzyme activity 
intrinsic to those receptors, a protein kinase activ- 
ity that transfers a phosphate group from the cel- 
lular energy source ATP to a specific amino acid, 
tyrosine, in target proteins. Later effects of insu- 
lin include the activation of other cellular pro- 
tein kinases that catalyze the phosphorylation of 
various cellular proteins on serine and threonine 
residues. In the past year we identified one of 
these insulin-activated kinases as the Raf-1 pro- 
tein kinase, a proto-oncogene product that is re- 
lated to the transforming oncogene of a murine 
sarcoma virus. The activation of this protein ki- 
nase was accompanied by its phosphorylation on 
serine and threonine residues. In our laboratory 
Ruey-Min Lee recently identified two insulin- 
activated protein kinases that in turn phosphory- 
late the Raf-1 protein; these kinases are activated 
within two minutes of cellular exposure to insu- 
lin and may represent one of the missing links 
between the tyrosine kinase activity of the insulin 
receptor and the serine kinase activity of the 
Raf-1 protein kinase. 
Another well-known action of insulin is the 
stimulation of protein synthesis in its target cells. 
The synthesis of many proteins is increased as 
part of insulin's general anabolic effect. How- 
ever, insulin also stimulates the synthesis of sev- 
eral proteins to a much greater extent than the 
overall average of cellular proteins; the synthesis 
of many of these proteins appears to be stimu- 
lated at the level of messenger RNA translation. 
Joyce Manzella is currently studying the transla- 
tion of the mRNA for the enzyme ornithine decar- 
boxylase as an example of insulin-stimulated 
mRNA translation. She recently found a direct 
correlation between the ability of insulin to stim- 
ulate mRNA translation and the extent of pre- 
dicted intrachain folding or secondary structure 
for that mRNA. In searching for a mechanism for 
this effect, she found that insulin stimulated the 
phosphorylation — and presumably the activa- 
tion — of two translation initiation factors whose 
actions involve unwinding or "melting" those 
secondary mRNA structures. These studies have 
led her to propose a general model for insulin- 
stimulated mRNA translation based on the insulin- 
stimulated unwinding of mRNA secondary 
structures. 
Insulin also stimulates the transcription of 
many genes shortly after its addition to sensitive 
target cells. One such gene, the c-fos proto-onco- 
gene, has been studied in detail. The transcrip- 
tion of this gene is stimulated by insulin within 
approximately 5-10 min of cellular exposure. 
Recent studies by Rajesh Malik and Michael Roe 
have identified two cellular proteins that appear 
to be modified by insulin and growth factor treat- 
ment, so that their binding to the regulatory re- 
gion of the c-fos gene is increased. They are 
currently attempting to purify and clone these 
proteins, with the goal of determining how insu- 
lin stimulates their interaction with the c-fos 
gene promoter. Wi-Sheung Lai has recently 
cloned another gene whose rapid transcription is 
stimulated by insulin, the tris-tetraproline (TTP) 
gene; similar studies of its regulatory elements 
are under way. The protein encoded by this rap- 
idly turned on gene appears to belong to a novel 
class of zinc finger-containing proteins that are 
probably DNA-binding proteins; efforts to deter- 
mine the DNA targets of this protein are ongoing. 
Other protein kinases are involved in the intra- 
cellular responses to a variety of polypeptide 
hormones and growth factors other than insulin. 
For example, some of these growth factors also 
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