Page 71 - 76_03

VOL. 76 (3), 357-377, 2010 TSC1-TSC2 COMPLEX ON THE CROSSROAD OF PANCREATIC...

tivation of AMPK by glucose resulted in an enhancement of mTOR
and p70S6K phosphorylation (Figure 3B). Thus, activation of AMPK
can directly regulate mTORC1 inhibition. In a similar manner, AMPK
activation caused Raptor-Ser792 phosphorylation, which has been de-
scribed as an additional mechanism to downregulate mTORC1 signal-
ing, independently from TSC1-TSC2 complex (30). The role of the TSC
complex in energy sensing response was observed, as the phosphory-
lation of TSC2 by AMPK activation causes a mobility shift of the TSC2
protein in poliacrylamide gel electrophoresis, which is reverted by ?-
phosphatase treatment of the samples (Figure 3C).

Figure 3. Glucose and energy dependent modulation of TSC2 and mTORC1.
A) IR -/- were stimulated with glucose 5 mM for 15 min. U0126 5 µM was added 30
min prior to the stimulus. Protein extracts were submitted to immunoprecipitation
with anti-TSC2 antibody, and blotted with a phosphospecific anti TSC2-Ser664 anti-
body. Total lysates were used for Western-blot against P-ERK 1/2. B) IR +/+ cells were
stimulated with glucose 5 mM, 2-DG (2-5 mM) or AICAR 4 mM. C) IR +/+ cells were
stimulated or not with 2-DG 5 mM for 15 minutes, protein extracts were collected
and part of the samples were treated with ?-phosphatase. Samples were subjected to
SDS-PAGE and incubated with specific antibodies. Blots are representative of at least
three independent experiments.

365