to purify endogenous E32PO4^-labeled NSR1-
TAP from both wild-type NSR1-TAP and
ip6kD NSR1-TAP yeast (Fig. 4A). Lack of
IP6K resulted in an almost 60% decline in
phosphorylation of NSR1 in vivo, indicating
that this protein is physiologically phosphoryl-
ated in intact cells by endogenous IP7(Fig. 4,
B and C).
This study establishes that the inositol
pyrophosphate IP7is a physiologic phosphate
donor to a range of proteins in eukaryotic
cells. The proteins we have best characterized
as IP7targets, yeast NSR1 and SRP40 and
mammalian Nopp140 and TCOF1, are nucle-
olar proteins involved in ribosomal biogene-
sis. Additionally, IP7phosphorylation of
proteins involved in endocytosis may me-
diate roles of inositol pyrophosphates in
this process (6), consistent with the phos-
phorylation by IP7of the adaptin b3A sub-
unit (18), a regulator of vesicular trafficking
References and Notes
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18. A. Saiardi, R. Bhandari, S. H. Snyder, unpublished
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26. Materials and methods are available as supporting
material on Science online.
27. We thank A. Riccio, B. Wendland, H. R. Luo, and T. R.
Raghunand for suggestions, discussions, and helpful
comments and all the members of the Snyder lab
for creating a stimulating environment. This work
was supported by U.S. Public Health Service Grant
MH18501, Conte Center Grant MH068830-02, and
Research Scientist Award DA00074 (to S.H.S.).
Supporting Online Material
Materials and Methods
Figs. S1 to S7
References and Notes
28 July 2004; accepted 29 October 2004
Regulates SIRT1 Through a
Shino Nemoto, Maria M. Fergusson, Toren Finkel*
Nutrient availability regulates life-span in a wide range of organisms. We dem-
onstrate that in mammalian cells, acute nutrient withdrawal simultaneously
augments expression of the SIRT1 deacetylase and activates the Forkhead
transcription factor Foxo3a. Knockdown of Foxo3a expression inhibited the
starvation-induced increase in SIRT1 expression. Stimulation of SIRT1 tran-
scription by Foxo3a was mediated through two p53 binding sites present in the
SIRT1 promoter, and a nutrient-sensitive physical interaction was observed
between Foxo3a and p53. SIRT1 expression was not induced in starved p53-
deficient mice. Thus, in mammalian cells, p53, Foxo3a, and SIRT1, three
proteins separately implicated in aging, constitute a nutrient-sensing pathway.
In the yeast Saccharomyces cerevisiae and in
the nematode Caenorhabditis elegans, life-
span can be extended by increasing the
expression of the deacetylase Sir2, an enzyme
whose activity depends on the oxidized form
of nicotinamide adenine dinucleotide (NAD)
(1, 2). In these model organisms, the ability
of Sir2 to extend life may be related to its
role in gene silencing. In both the nematode
and yeast, certain simple environmental
stresses can also increase life-span. In yeast,
reducing the amount of available glucose has
this effect. The ability of glucose restriction
to increase the life-span of yeast requires
Sir2 (3). In C. elegans, activation of the
Forkhead transcription factor DAF-16 is also
associated with increased life-span (4) and its
activation depends in part on nutrient avail-
ability (5). Genetic evidence further suggests
that in worms, DAF-16 and Sir2 work
through a common pathway (2), and recent
evidence suggests that their mammalian
counterparts physically interact (6, 7).
Here, we further analyzed the interrela-
tionship of the closest mammalian orthologs
% of wild type
Fig. 4. In vivo 5b[32P]IP7phosphorylation. (A) Presence of an 80-kD phosphorylated band in the
NSR1-TAP strain. Wild-type, nsr1D, and wild-type NSR1-TAP yeast extracts were incubated with
5b[32P]IP7and processed as described in Fig. 2. (B and C) IP6K deletion reduces phosphorylation of
NSR1 by endogenous IP7. (B) Wild-type NSR1-TAP and NSR1-TAP ip6kD yeast were labeled with
[32PO4]iorthophosphate, and TAP-NSR1 was purified as described (26). Yeast homogenates (2 mg)
were subjected to NuPAGE, and the gel was silver stained and autoradiographed to demonstrate
equal levels of basal phosphorylation (left). Silver staining and autoradiogram of purified NSR1-
TAP from wild-type and ip6kD yeast (right). (C) Quantification of the relative phosphorylation of
NSR1-TAP purified from wild-type or ip6kD yeast. Data represent the mean values and SEM from
three independent experiments.
Cardiovascular Branch, National Heart, Lung, and
Blood Institute (NHLBI), Bethesda, MD 20892, USA.
*To whom correspondence should be addressed.
R E P O R T S
www.sciencemag.org SCIENCEVOL 30617 DECEMBER 2004