INSULIN AND POLYPEPTIDE HORMONE ACTION 
Perry J. Blackshear, M.D., D.Phil., Investigator 
The mechanisms of action of insulin and 
related polypeptide hormones and growth factors 
are the major interest of Dr. Blackshear's labora- 
tory. The laboratory is investigating the interac- 
tions among the often multiple intracellular reac- 
tion pathways activated after a given agonist of 
this type binds to its cell surface receptor. The ac- 
tivation of cellular processes by insulin is of pri- 
mary concern, not only because the molecular 
mechanism of insulin action is clinically relevant 
but also because there is evidence that the tyro- 
sine kinase pathways activated by insulin serve as 
a paradigm for similar pathways activated by cer- 
tain growth factors. 
I. Insulin Regulation of Protein Biosynthesis. 
A. Induction of c-fos proto-oncogene transcription 
by insulin and related growth factors. Recent stud- 
ies in Dr. Blackshear's laboratory have focused on 
the molecular mechanisms that are responsible for 
the induction of cfos proto-oncogene transcrip- 
tion. In sensitive cells this strong response occurs 
within minutes of exposure to insulin, occurs with 
a dose-response relationship that parallels insulin 
receptor occupancy, and can be mimicked by a pro- 
tein kinase C-independent pathway activated by 
various growth factors. In the past year, Dr. Debo- 
rah J. Stumpo has demonstrated that the protein ty- 
rosine kinase activity of the insulin receptor is nec- 
essary for the insulin-stimulated effect to occur. The 
goal of these experiments is to link activation of 
this kinase to the rapid transcription of the c-fos 
gene by a series of biochemical reactions. Dr. Black- 
shear and his colleagues recently identified an ele- 
ment in the c-fos promoter that is responsible for 
the induction by insulin. Furthermore, Dr. Michael 
W Roe has identified at least three proteins from 
cellular nuclear extracts that bind to this region of 
the gene with appropriate sequence specificity. In- 
sulin stimulation of the cells results in an increase 
in the binding of one of these proteins to the spe- 
cific promoter sequence of interest; this suggests 
that insulin is modifying the protein to cause this 
apparent increase in binding. Dr. Blackshear's lab- 
oratory is currently attempting to elucidate how 
this protein is modified in response to insulin 
treatment of cells; the answers to this question 
could lead to the activated insulin receptor protein 
kinase. 
B. Insulin-stimulated mRNA translation. When 
sensitive cells are treated with insulin, numerous 
anabolic processes are activated, including general- 
ized increases in protein synthesis and inhibition of 
protein breakdown. Dr. Richard M. Levenson has 
recently shown that part of this generalized in- 
crease in protein synthesis could be due to effects 
of insulin on a crucial component of the protein 
biosynthetic apparatus, elongation factor 2 (EF-2). 
Insulin not only rapidly stimulates the translation 
of EF-2 mRNA, it also appears to alter its phosphor- 
ylation state in such a way as to activate its function 
in stimulating nascent protein elongation. Dr. 
Levenson also found that, over and above a gener- 
alized effect of insulin on protein synthesis, approx- 
imately 35 proteins are synthesized much more rap- 
idly in the presence of insulin, in most cases 
apparently by stimulation of mRNA translation. 
Joyce M. Manzella is concentrating on the mecha- 
nism of this translational activation by insulin, 
using the enzyme ornithine decarboxylase (GDC) 
as a model system. In recent studies involving the 
GDC mRNA 5 '-untranslated region sandwiched be- 
tween an exogenous promoter and a reporter gene, 
she has found that the GDC sequences dramatically 
inhibit translation of the reporter mRNA, both in 
vitro and in vivo. She has also determined that in- 
sulin can stimulate the translation of these mRNAs 
by a mechanism that involves the GDC sequences. 
She is currently attempting to determine which 
component of the GDC sequence is involved in 
both the constitutive inhibitory and insulin-stimula- 
tory effects. 
II. Molecular Characterization of a Prominent Pro- 
tein Kinase C Substrate Protein. 
Within the past year. Dr. Stumpo and Dr. Jona- 
than M. Graff have cloned, sequenced, and 
expressed cDNAs encoding a major cellular sub- 
strate for protein kinase C, which they have named 
the myristoylated alanine-rich C kinase substrate 
(MARCKS). They have determined that two regions 
of sequence identity between the chicken and bo- 
vine proteins are involved in three different behav- 
iors of the proteins: myristoylation, phosphoryla- 
tion, and calmodulin binding. Dr. Graff determined 
that a mutation that prevented amino-terminal 
myristoylation of the protein caused it to remain 
exclusively in the cytosol, yet the protein remained 
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