MECHANISM OF INSULIN ACTION 
Joseph Avruch, M.D., Investigator 
The overall goals of Dr. Avruch's work are 1) to 
identify the biochemical steps by which the insulin 
receptor tyrosine reorients cell function and 2) to 
develop methods to determine, in intact cells, the 
function of specific tyrosine and serine/threonine 
protein kinases. 
I. Targets of the Insulin Receptor Tyrosine Protein 
Kinases. 
An array of independently generated, high-affin- 
ity polyclonal and monoclonal antibodies against 
phosphotyrosine detect a common set of insulin- 
stimulated tyrosine P-containing polypeptides in a 
variety of cells. The role of these endogenous re- 
ceptor kinase substrates as signaling intermediates 
in insulin's biologic actions remains invalidated. 
Several such proteins have been purified in the last 
year to essential homogeneity, and an analysis of 
the primary structure has been initiated, in an ef- 
fort to establish the functional identity of these 
polypeptides. The major candidates are 180 kDa 
polypeptides and a doublet of —65 kDa. 
II. Insulin/Growth Factor-Stimulated Serine/Threo- 
nine Protein Kinases. 
Earlier work from Dr. Avruch's laboratory had 
shown that one of the earliest responses to insulin 
was a stimulation of serine/threonine-specific phos- 
phorylation of a subset of intracellular proteins. 
The view rapidly emerged that this phenomenon 
reflected the activation of one or more serine/threo- 
nine-specific protein kinases, as an intermediate sig- 
naling reaction consequent to receptor activation. 
The first insulin-stimulated serine/threonine phos- 
phoprotein identified was the lipogenic ATP-citrate 
lyase. Subsequent work identified such other tar- 
gets as 40 S ribosomal protein S6, acetyl-CoA car- 
boxylase, and the insulin receptor itself Recently 
an S6 protein kinase from livers of cycloheximide- 
treated rats was purified. Hepatic S6 phosphoryla- 
tion increases markedly after cycloheximide admin- 
istration in vivo. Concomitant with this is an 
increase in the activity of a protein kinase that is 
very active on S6; the chromatographic properties 
of this protein kinase are indistinguishable from the 
S6 kinase activated in regenerating rat liver or insu- 
lin-treated hepatoma cells. After purification the en- 
2yme is visualized as a 70 kDa polypeptide that 
phosphorylates, in addition to S6, glycogen syn- 
thase, a number of nonhistone nuclear proteins, 
and ATP-citrate lyase; the last substrate is phosphor- 
ylated at sites identical to those phosphorylated in 
response to insulin in vivo. The activity of the puri- 
fied S6 kinase is abolished by treatment with the 
serine/threonine phosphatase-2A. 
Antisera raised to the 70 kDa S6 kinase polypep- 
tide immunoprecipitate an insulin-stimulated S6 
protein kinase activity from insulin-treated 
hepatoma cells. When hepatoma cells are labeled 
with ^^P, these immunoprecipitates exhibit a 70 
kDa ^^P-peptide; the immunoprecipitation of ^^P- 
peptide is specifically inhibited by preincubation of 
the antisera with the purified S6 kinase. The ^^P 
content of this 70 kDa S6 kinase is increased ap- 
proximately threefold by insulin treatment of the 
cells. 
The 70 kDa rat liver S6 kinase is not cross-re- 
active with antisera raised to S6 kinase II puri- 
fied from Xenopus oocytes or to a related recombi- 
nant Xenopus S6 kinase. These latter antisera do 
immunoprecipitate a distinct 85 kDa *^P-polypep- 
tide from rat liver, which also demonstrates S6 ki- 
nase activity in the immunoprecipitate. This 85 kDa 
^^P-polypeptide is the rat liver homologue of 
Xenopus S6 kinase II. Low-stringency hybridization 
of rat liver cDNA libraries with the Xenopus cDNA 
has been used to obtain several cDNAs correspond- 
ing to this 85 kDa rat liver S6 kinase. Comparison 
of the deduced amino acid sequence for the rat 
liver 85 kDa S6 kinase to the sequence of peptide 
segments obtained from the purified 70 kDa S6 ki- 
nase reveals regions of considerable identity 
(—50%) within the protein kinase homology do- 
main. 
In summary, there appears to be a family of struc- 
turally and immunochemically distinct S6 protein 
kinases that is regulated by insulin and growth fac- 
tors via phosphorylation on serine/threonine resi- 
dues. The interrelations of these enzymes, the iden- 
tity of the important substrates, and the protein 
kinases responsible for their activation are un- 
known. 
Dr. Avruch and his colleagues have purified a sec- 
ond insulin-stimulated serine/threonine-specific 
protein kinase that phosphorylates microtubule-as- 
sociated protein-2 (MAP-2). Thus activity of this en- 
zyme is also abolished by serine/threonine dephos- 
phorylation. The native substrates for MAP-2 kinase 
Continued 
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