MOLECULAR MECHANISMS OF INSULIN AND POLYPEPTIDE 
GROWTH FACTOR ACTION 
Perry J. Blackshear, M.D., D.Phil., Investigator 
Dr. Blackshear's laboratory is interested in the mo- 
lecular mechanisms of action of insulin and other 
polypeptide hormones and growth factors. In the 
past several years, these studies have focused on two 
major areas: insulin's regulation of protein biosyn- 
thesis and the involvement of protein kinase C sub- 
strates in agonist-signaling pathways involving this 
family of protein kinases. 
The studies of insulin-stimulated protein biosyn- 
thesis involve one example of stimulated mRNA 
translation and two examples of rapid stimulated 
gene transcription. Studies by Dr. Joyce Manzella in 
the laboratory showed that insulin could rapidly 
stimulate the biosynthesis of the enzyme ornithine 
decarboxylase (GDC), primarily at the level of 
mRNA translation. She further showed that the insu- 
lin stimulation of GDC biosynthesis appeared to in- 
volve the unwinding or melting of mRNA secondary 
structure, permitting translation to proceed more 
rapidly. This effect was particularly striking in the 
case of the GDC mRNA, since the extreme 5' portion 
of the long GDC mRNA leader sequence con- 
tains a large stem-loop structure of pronounced neg- 
ative free energy. The mechanism by which insulin 
stimulated this mRNA melting activity appeared to 
involve the rapid stimulated phosphorylation of ini- 
tiation factors eIF4B and 4F, both of which are in- 
volved in either cap binding or unwinding of mRNA 
secondary structure. In this way, insulin and other 
growth factors can preferentially stimulate the 
translation of mRNAs with intense secondary struc- 
ture in their 5'-untranslated region. 
Dr. Blackshear and his colleagues have also con- 
tinued to work on the rapid induction by insulin of 
certain early response genes, including the c-fos 
proto-oncogene and a gene encoding a zinc finger 
protein of no known function. In previous studies 
with the c-fos promoter, the major insulin response 
element was localized to a promoter segment 
known as the serum response element (SRE) . More 
recent studies by Dr. Rajesh Malik showed that insu- 
lin and other growth factors stimulated formation of 
two different ternary complexes involving the SRE 
within 2 min of hormonal stimulation. Current stud- 
ies in the laboratory involve purification of the pro- 
teins involved in these complexes, their molecular 
cloning, and the elucidation of the covalent modifi- 
cation that leads to their increased binding to the 
c-fos SRE. 
The other major area under study in the laboratory 
involves the stimulated phosphorylation by protein 
kinase C (PKC) of a small family of substrate pro- 
teins related to the myristoylated alanine-rich C- 
kinase substrate (MARCKS) protein. Gne recent 
study involved an exploration of the factors respon- 
sible for the binding of this myristoylprotein to cel- 
lular membranes. Dr. Daniel George in the labora- 
tory found that the myristoylated protein bound to 
cell membranes much more rapidly than did a non- 
myristoylated mutant protein. However, this mem- 
brane association did not appear to involve binding 
to a specific membrane "receptor" protein, as has 
been shown for another myristoylprotein, but in- 
stead seems to involve hydrophobic interactions be- 
tween the fatty acid moiety and the membrane 
lipids. 
Gther studies are attempting to prove that the 
high-affinity binding of MARCKS to calmodulin, 
which occurs readily in vitro, also occurs in vivo at 
normal ambient calcium concentrations. Dr. Robert 
D. Hinrichsen and Dr. Blackshear showed that 
MARCKS calmodulin-binding peptides, when in- 
jected into Paramecium, could elicit a behavioral 
response in these organisms that is typical of de- 
creased calmodulin concentrations, suggesting 
strongly that the MARCKS peptide was interacting 
with calmodulin and preventing the normal behav- 
ioral response. Such a response was not seen with 
mutant peptides that had lower affinity for calmo- 
dulin and could be reversed by PKC-stimulated 
phosphorylation of the peptide, as occurs in cell- 
free systems. These studies demonstrated that 
MARCKS calmodulin-binding peptides can actually 
bind calmodulin at ambient cellular calcium con- 
centrations and that this binding can be reversed by 
PKC-dependent phosphorylation of MARCKS pep- 
tide and proteins. 
Another group of studies involved the elucida- 
tion of the structure and function of the MRP pro- 
tein, a recently discovered MARCKS homologue. 
This protein is homologous to the MARCKS protein 
in the three regions in which the MARCKS proteins 
from different animal species are conserved: the ex- 
treme amino-terminus myristoylation consensus se- 
quence, the site of splicing of the single intron, and 
the basic phosphorylation site/calmodulin-binding 
domain. Studies by George Verghese in the labora- 
tory identified this protein as a myristoylprotein 
both in vivo and in vitro; as a high-affinity phos- 
phorylation substrate for PKC; and as a high-affinity 
calmodulin-binding protein, with disruption of the 
complex occurring after PKC-dependent phosphor- 
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