FORUM REVIEW ARTICLE
DAF-16=Forkhead Box O Transcription Factor:
Many Paths to a Single Fork(head) in the Road
Kelvin Yen, Sri Devi Narasimhan, and Heidi A. Tissenbaum
The Caenorhabditis elegans Forkhead box O transcription factor (FOXO) homolog DAF-16 functions as a central
mediator of multiple biological processes such as longevity, development, fat storage, stress resistance, and
reproduction. In C. elegans, similar to other systems, DAF-16 functions as the downstream target of a conserved,
well-characterized insulin=insulin-like growth factor (IGF)-1 signaling pathway. This cascade is comprised of an
insulin=IGF-1 receptor, which signals through a conserved PI 3-kinase=AKT pathway that ultimately down-
regulates DAF-16=FOXO activity. Importantly, studies have shown that multiple pathways intersect with the
insulin=IGF-1 signaling pathway and impinge on DAF-16 for their regulation. Therefore, in C. elegans, the single
FOXO family member, DAF-16, integrates signals from several pathways and then regulates its many down-
stream target genes. Antioxid. Redox Signal. 14, 623–634.
systems for biological discovery. C. elegans are 1-mm-long,
by feeding them lawns of Escherichia coli on standard agar
plates (102) (Fig. 1). Their transparency allows for ease of
observation, especially when using fluorescent reporters to
observe specific tissues (17). Adult worms contain only 959
cells, and the positions of cells as well as the number of cells
are constant, which gives an incredibly rich resource for
studying individual cell fate (118). In addition, C. elegans is
amenable to genetic manipulations with forward and reverse
genetic approaches being applied to study multiple aspects of
cellular function (102, 124).
C. elegans has been a powerful tool to study the molecular
biology of aging over the past two decades. They have a con-
sistent mean lifespan of *2 weeks, and single-gene manipula-
over 100% of control (27, 54). These genes have homologs in
higher organisms, with many of them belonging to conserved
molecular pathways that regulate energy metabolism and de-
velopment (8, 81). Of these aging genes, the most important
identification has arguably been that of the single Forkhead box
O transcription factor (FOXO) homolog DAF-16 (66, 85).
In C. elegans, DAF-16 functions as a central regulator of
multiple biologicalprocesses, includinglongevity, fatstorage,
stress response, development, and reproduction (8, 77). DAF-
ince its isolation *30 years ago, the nematode Cae-
norhabditis elegans has been one of the most useful model
16 is the downstream target of a conserved, well-character-
ized insulin=insulin-like growth factor (IGF)-1 signaling (IIS)
pathway. Besides IIS, a number of additional signalling cas-
cades have been identified that can regulate DAF-16. Here we
will discuss the pathways that feed into DAF-16, the levels of
regulation of DAF-16 activity and its many transcriptional
targets, and how these numerous signals are coupled to ulti-
mately control multiple biological functions.
A FOXO in the Context of a Whole Organism
One benefit of working with C. elegans has been the ability
to assess the role of a protein on an organismal level. As such,
single-gene manipulations can be directly measured as a
phenotypic consequence in a worm. The multiple biological
processes that are regulated by DAF-16 can be tested in the
laboratory using simple well-defined assays. Modulation of
DAF-16 signaling results in changes in organismal lifespan
that can be measured directly with a lifespan assay (104). In
addition, the role of DAF-16 in regulating the response to
stress can assayed by monitoring worm survival under con-
ditions of heat or oxidative stress (43, 68). Changes in fat
storage are qualitatively assessed using Oil Red O and Sudan
Black staining and quantitatively assessed using gas chro-
motography, mass spectrophotometry, and coherent anti-
Stokes Raman spectroscopy (56, 58, 101, 115). An additional
phenotype associated with changes in DAF-16 signaling is
changes in pathogen resistance; this can be tested by exposing
worms to a pathogen and measuring their survival (30).
Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester,
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 4, 2011
ª Mary Ann Liebert, Inc.
Further, expression of DAF-16 in distinct tissues in the worm
can influence specific physiological processes, such as lon-
gevity (65, 122). Therefore, C. elegans provides a unique op-
portunity to analyze the multiple functions of a single-FOXO
gene in a relatively simple, genetically tractable organism.
Identification of daf-16
In a favorable growth environment, the life cycle of C. ele-
gans begins from an egg and develops through four larval
stages (each with a molt; abbreviated L1–L4) to a final molt as
an adult hermaphrodite (Fig. 2) (94). Each adult hermapro-
hodite can produce *300 self-progeny under favorable
growth conditions. In an unfavorable growth environment,
primarily determined by the levels of a secreted pheromone,
worms can enter an alternative larval three stage and form a
dauer larva (36, 46). A dauer larva is a developmentally ar-
rested, nonfeeding stage that is well suited for long-term
survival (25). Studies have shown that C. elegans continuously
secrete a dauer pheromone that is a complex mixture of dif-
ferent ascarosides (21). In conjunction with other enviro-
mental factors such as temperature and food availability, this
pheromone acts as a critical modulator of dauer formation
and will molt into a reproductive hermaphrodite when con-
ditions are favorable (25, 46). During the early stages of C.
elegans research, genetic screens were performed to identify
genes that modulate dauer formation (2, 95). Two classes of
mutations were identified in this screen: dauer constitutive
(daf-c) and dauer defective (daf-d). Many of the genes (daf) that
were isolated in this screen were later shown to encode
homologs of the well-studied insulin=IGF-1 signaling (IIS)
pathway (56, 76). daf-16 was isolated in these screens because
daf-16 mutants have a dauer defective phenotype (2).
16 encodes a FOXO transcription factor (66, 85). Despite the
presence of several different FOXO proteins in mammals,
DAF-16 is thesingle-FOXO homolog in C. elegans. Subsequent
studies showed that there are several alternatively spliced
forms of daf-16—daf-16a1 (R13H8.b), daf-16a2 (R13H8.1c), and
daf-16b (R13H8.1a)—that have distinct tissue expression pat-
terns (59, 66, 67, 85). Functional assays have shown that daf-
16a is more important for lifespan, whereas daf-16b is more
important for dauer formation (41, 59, 67).
gram of Caenorhabditis ele-
gans labeled with important
features. (A) Differential in-
terference contrast image of
an adult worm. (B) Schematic
diagram of the major features
found in adult worms.
A picture and dia-
vironment, the worm life cycle proceeds from an egg to
successive larval stages designated as L1–L4 before becom-
ing an adult. In an unfavorable growth environment, pri-
marily determined by a continuously secreted pheromone,
with temperature and food conditions also modulating this
process, worms can proceed to an alternative developmental
stage from the L1 or L2 stage to become stress-resistant
Life cycle of C. elegans. In a favorable growth en-
624YEN ET AL.
DAF-16 is a member of the FOXO family, which includes
AFX (FOXO4), FKHR (FOXO1), and FKHR-L1 (FOXO3a).
FOXO family of transcription factors bind DNA as monomers
at consensus binding sites (TTG=ATTTAC) (28). Microarray
analysis in C. elegans suggested the existence of a second po-
tential binding site for DAF-16, although this has not been
tested biochemically (79). Thus far, many of the in vivo inferred
molecular and biochemical characteristics of DAF-16 are de-
duced from its similarity with mammalian FOXOs, due to the
lack of a C. elegans cell culture system similar to Drosophila S2
cells or mammalian tissue culture systems.
DAF-16 activity is regulated by several different kinases,
including AKT-1, AKT-2, the serum, and glucocortoid kinase
SGK-1, Jun kinase 1 (JNK-1), and CST-1 (worm homolog of
phosphorylate DAF-16 (discussed below). It is presumed that
similar to mammalian FOXOs, the phosphorylation status of
DAF-16, aided by nuclear transporter proteins, is responsible
for determining its sub-cellular localization (109, 112).
Structurally, DAF-16 contains an evolutionarily conserved
forkhead box DNA binding domain adjacent to a nuclear lo-
calization signal and two possible 14-3-3 protein binding sites
[reviewed in (11, 48, 84)]. Near this region there are many
conserved phosphorylation and acetylation sites for proteins
such as CST-1, AKT, AMP kinase (AMPK), and transcription
factor cAMP response element-binding protein (CBP), the
roles of which will be discussed subsequently (Fig. 3). In
mammals and in C. elegans, these sites affect DNA binding
and localization of FOXO proteins (11, 48, 59, 67).
Pathways Upstream of DAF-16=FOXO
DAF-16=FOXO is a central regulator of many biological
processes. To regulate these processes, DAF-16 receives sig-
nals from several upstream signaling pathways, including
direct phosphorylation by multiple independent kinases and
interaction with additional proteins. As a consequence of
these interactions, DAF-16 either remains in the cytoplasm or
translocates to the nucleus, where it affects hundreds of direct
and indirect target genes. We focus on the multiple pathways
that regulate DAF-16 activity below.
Insulin=IGF-1-like signaling pathway
Initial genetic studies identified that a loss-of-function
mutations in several genes, including daf-2 (worm homolog of
the insulin=IGF receptor gene) and age-1 (20, 37, 54, 57, 105).
These studies also showed that in addition to dauer devel-
opment, the daf-16, daf-2, and age-1 genes could modulate
longevity, stress resistance, and reproduction. Later molecu-
larcloning studies revealed thatthese genes formed part of an
IIS pathway where daf-2 is an ortholog that is equally similar
to the mammalian insulin and IGF-1 receptors (56), age-1 en-
codedfor thecatalyticsubunitofPI3-kinase (76),anddaf-16,a
FOXO that was downstream of and negatively regulated by
daf-2 and age-1 (Fig. 4) (66, 85).
Further genetic and molecular genetic studies defined
conserved components of a pathway homologous to mam-
malian insulin=IGF-1 signaling, consisting of a PI 3-kinase
signaling cascade downstream of daf-2. Activation of the C.
elegans PI 3-kinase AGE-1 results in the conversion of phos-
phatidylinositol (3–5) bisphosphate to phosphatidylinositol
(3–5) trisphosphate (116). In mammals, phosphatidylinositol
bisphosphate and=or phosphatidylinositol trisphosphate re-
cruit the downstream kinases PDK1 and AKT-1 to the plasma
membrane, where PDK1 activates AKT-1 by phosphorylation
(4, 99). The homologs of these kinases in C. elegans correspond
to PDK-1, AKT-1, and AKT-2. Signaling through PDK-1 also
activates SGK-1 in a conserved manner (14, 42).
Biochemical studies in C. elegans have shown that AKT-1,
AKT-2, and SGK-1 can phosphorylate DAF-16 (42, 107). This
molecular interaction is conserved in mammals, as AKT and
SGK1 can phosphorylate FOXO proteins (12, 14). Phosphor-
ylation of DAF-16 results in its sequestration in the cytosol by
virtue of its association with 14-3-3 proteins (10, 63). In con-
trast, under low signaling conditions or when a mutation is
presumed to be less phosphorylated and then translocates
microarrays and a chromatin immunoprecipitation (ChIP)
have shown that upon entering the nucleus, DAF-16 binds to
and transactivates=represses numerous target genes involved
3-3 binding motifs surrounding a DNA binding domain and
nuclear localization signal. Phosphorylation sites are marked
with a P with single-bordered white circles denoting AKT-1=2
phosphorylation sites, double-outlined circles indicating CST-1
sites, gray indicating AMP kinase sites, and dual-colored circles
denoting a site recognized by multiple kinases. cAMP re-
sponse element-binding protein-1 acetylation sites are denoted
by white triangles marked with A. The DAF-16B isoform is
similar in structure except for the absence of the first CST-1
site. BD, binding domain; BM, binding motifs; DAF-16, worm
homolog of Forkhead box O transcription factor protein; NLS,
nuclear localization signal.
Diagram of the DAF-16A isoform. There are two 14-
cies. Black labels indicate worm proteins, gray is used for fly
proteins, and light gray is for mammalian proteins. This figure
is modified from reference (3).
Insulin=IGF signaling is conserved between spe-
CENTRAL ROLE FOR C. ELEGANS FOXO/DAF-16 625
in lifespan regulation, stress response, dauer formation, and
metabolism (Fig. 6) (60, 73, 79, 87). Consistent with this,
studies have shown that RNAi or reduction of function mu-
tations of daf-2, age-1, aap-1, pdk-1, sgk-1, or akt-1=2 will result
in changes in all or some of these phenotypes such as lifespan
phenotype. Mutations in daf-2 and age-1 have been also re-
ported to show increased fat storage (20, 41, 42, 57, 92, 123).
The well-studied JNK signaling cascade has also been
shown to modulate lifespan through a direct interaction with
DAF-16 (88). The JNK family is a subgroup of the mitogen-
activated protein kinase superfamily and is associated with
regulation of critical biological processes, including develop-
ment, apoptosis, and cell survival (117). In C. elegans, the JNK
pathway is activated by several different stresses such as heat
stress and oxidative stress (88, 120). Previous studies using
mammalian cell culture had connected insulin signaling with
components of the JNK signaling pathway through interac-
1 protein kinase (55). In worms and flies, JNK overexpression
extends lifespan and increases stress resistance, and this
lifespan extension is dependent on DAF-16 (88, 113). In C.
elegans and mammalian cell culture, JNK physically interacts
with and phosphorylates DAF-16 at sites different from the
AKT phosphorylation sites (23, 88). In C. elegans, this phos-
phorylation results in enhanced nuclear translocation of
DAF-16 (88). Therefore, the JNK pathway represents an ad-
ditional input into DAF-16 under conditions of stress.
Proteins Interacting with DAF-16
AKT-1 is reported to phosphorylate DAF-16 on four dis-
tinct sites, and three of these are conserved in mammalian
FOXO (48, 59, 67, 112). Studies using DAF-16::green fluor-
escent protein (GFP) constructs where the four AKT-1 phos-
phorylation sites on DAF-16 were mutated and then added
back to complement a daf-16 mutant worm have been used to
look for restoration of mutant phenotypes such as altered
lifespan and=or dauer formation. In one study, absence of
AKT phosphorylation was sufficient to cause dauer arrest
(59). This implies that DAF-16 is the major target of AKT. In
contrast, another study found that although absence of AKT
phosphorylation facilitates DAF-16 entry into the nucleus,
nuclear localization was not sufficient to induce either dauer
formation or lifespan extension (67), suggesting a role for un-
identified proteins that activate DAF-16 (67). The differences
pictures. (A) Under high insulin=IGF signaling, the protein is
located in the cytosol (arrow). (B) Under low insulin=IGF
signaling, DAF-16::green fluorescent protein is located in the
nucleus as indicated by the gray arrows. White arrow indicates
the pharynx of the worm.
DAF-16::green fluorescent protein localization
cascade in two different states. In
a fed state, under high insulin=IGF-
1 signaling, DAF-16 is located in
the cytosol, whereas under stress or
starvation, DAF-16 is located in the
6. Insulin=IGF signaling
626YEN ET AL.
between the two studies could have arisen because different
transgenic worms were used (59). The study from the Kenyon
lab used an integrated genomic construct tagged with GFP,
whereas the Ruvkun lab used an extrachromosomal cDNA
construct. Additionally, the expression levels of the DAF-16
transgenes could have been substantially different.
The C. elegans serum-glucocoriticoid kinase SGK-1, which
functions at the level of AKT-1=2 (42), forms a protein com-
plex containing AKT-1=AKT-2=SGK-1 and transduces the PI
3-kinase signals via PDK-1 to control the localization and ac-
tivation of DAF-16 by direct phosphorylation. Although,
biochemically, AKT-1=AKT-2=SGK-1 have been shown to
form a protein complex, tissue expression patterns of these
kinases show little overlap. AKT-1::GFP and AKT-2::GFP are
expressed in the head and tail neurons, pharynx, and sper-
mathecae; SGK-1::GFP is primarily in the intestine (80, 91).
Recently, using mammalian cell culture under conditions
of oxidative stress, it was shown that STK4 (serine=threonine
kinase 4, formerly known as MST1) phosphorylates FOXO3A
at a conserved site in the forkhead domain (61). This phos-
phorylation results in disruption of the interactions between
FOXO3A and the 14-3-3 proteins (described below), thus
in C. elegans, as STK4 can robustly phosphorylate C. elegans
DAF-16 at the conserved STK4 site. Consistent with this idea,
overexpression of the C. elegans Stk4 (serine=threonine kinase
4 gene formerly known as Mst1) ortholog, named cst-1 (worm
homolog of mammalian Stk4=Stk3 gene), promotes longevity
in a daf-16-dependent manner (61).
In contrast to the inactivating phosphorylations by AKT-
1=AKT-2=SGK-1, AMPK activates DAF-16 by direct phos-
phorylation (Fig. 7) (38). AMPK phosphorylates DAF-16 on at
least six different sites (Fig. 3) (38). AMPK also indirectly
regulates DAF-16 activity through its inhibitory effects on
target of rapamycin (TOR) signaling (Fig. 7). TOR is a highly
conserved kinase that integrates many nutritional signals (40,
81). In C. elegans, the TORC2 complex activates SGK-1 and
inhibition by AMPK should likely results in the activation of
DAF-16 signaling (51, 101). Therefore, AMPK can both di-
rectly and indirectly activate DAF-16 signaling.
The translocation of mammalian FOXOs between the nu-
cleus and the cytoplasm is regulated in part through interac-
tion of the 14-3-3 family of proteins. Under active signaling
conditions, AKT=SGK phosphorylates FOXO, resulting in an
increased binding affinity to the 14-3-3 proteins. This in-
creased binding affinity causes the release of the FOXO pro-
tein from the DNA and relocalization to the cytosol (12, 16).
After translocation to the cytosol, the bound 14-3-3 prevents
re-entry of FOXO into the nucleus by masking the nuclear
localization signal (13, 15, 48). Therefore, 14-3-3 proteins in-
teract with FOXO and provide additional regulation for the
Similar to mammalian FOXO signaling, regulation of DAF-
16 also includes association with 14-3-3 proteins. Recent work
using worm extracts suggest that C. elegans 14-3-3 proteins
(PAR-5 and FTT-2) interact with DAF-16 to regulate its nu-
clear=cytoplasmic distribution (10, 63, 114). In addition, C.
elegans 14-3-3 proteins have been shown to modulate lifespan
through the IIS pathway, as overexpression of either par-5 or
ftt-2 extends lifespan dependent on daf-16 (114). This result is
somewhat counterintuitive as overexpression of the 14-3-3
proteins should result in more retention of DAF-16 in the
cytosol and thereby a decrease in DAF-16 activity and a
shortening of lifespan. One possible reason this is not the case
may be linked to the 14-3-3 proteins interacting with silent
information regulator (SIR2), discussed below. In addition,
recent studies have shown that the 14-3-3 proteins can also
modulate lifespan in a DAF-16-independent manner (6).
Therefore, taken together, these observations imply that C.
elegans DAF-16 shares many of the similarities to mammalian
FOXO transription factors, including the important interac-
tion with the 14-3-3 proteins.
Originally identified in S. cerevisiae as a gene important for
gene silencing, silent information regulator 2 (sir2) (97) has
emerged as an important lifespan regulator for yeast, worms,
and flies (52, 98, 103). In C. elegans, overexpression of sir-2.1
(C. elegans SIR2 ortholog) results in lifespan extension. This
extension is completely dependent on daf-16 (103). Epistasis
and dauer formation analysis ofworms overexpressing sir-2.1
also suggest that sir-2.1 functions in the IIS pathway (103).
both directly activates DAF-16 by direct phosphorylation
and indirectly activates DAF-16 by inhibiting TOR complex
2. Worm protein names are on top and mammalian names
are on the bottom where applicable. AMPK, AMP kinase;
TOR, target of rapamycin.
AMPK influence on DAF-16 signaling. AMPK
CENTRAL ROLE FOR C. ELEGANS FOXO/DAF-16 627
Genetic, molecular, and biochemical studies showed that the
14-3-3 proteins mediated the direct interaction between SIR-
2.1 and DAF-16 (10, 114). Therefore, the 14-3-3 proteins
modulate the nuclear=cytoplasmic translocation of DAF-16
and mediate interactions with additional partners.
Cofactors for DAF-16
A number of additional proteins have been identified that
modulate DAF-16 function. These proteins are listed as co-
factors since they have not been shown to directly interact
with DAF-16 in whole worms.
(suppressor of MEK null gene), was identified (121). Studies
of SMK-1 may help to address how DAF-16 acheives its
specificity. Genetic, molecular, and physiological analysis of
of MEK1 mutants in Dictyostellium discoideum. In C. elegans,
SMK-1 is required for DAF-16-dependent regulation of
lifespan (121). SMK-1 does not affect dauer formation or
regulation of lifespan by the reproductive tissues, two other
functions associated with DAF-16 (121). Transcription and
physiological studies show that smk-1 (suppressor of MEK
null gene) is required for oxidative and UV stress responses
and innate immunity, but is not necessary for the thermal
stress function of DAF-16 (121.) Therefore, SMK-1 possesses
all of the requirements for an IIS-mediated-longevity cofactor
of DAF-16, although direct biochemical data are needed to
confirm this function.
Recently, a potential cofactor of DAF-16, SMK-1
Heat shock factor-1.
heat stress induces a set of specialized molecular chaperones
regulated at the transcriptional level by a specialized tran-
scription factor,heat shock factor1 (HSF-1),which can bindto
promoters of proteins containing a heat shock element, con-
sisting of inverted repeats of the sequence nGAAn, where ‘‘n’’
can be an arbitrary nucleotide (24, 83). This process, whereby
exposure to heat stress increases transcription of a subset of
genes, is termed the ‘‘heat shock response.’’
In C. elegans, a genome-wide RNAi screen for mutants that
data on DAF-16 and HSF-1 have suggested a model where
these two proteins function together to promote longevity.
Importantly, overexpression of hsf-1 results in lifespan
extension, which is dependent on daf-16 and on a subset of
DAF-16 target genes (45, 75). Taken together, one possible
explanation is that HSF-1 and DAF-16 may interact directly
or through an intermediate protein to regulate a subset of
DAF-16 target genes specific for the heat shock and lifespan
responses. Future biochemical studies should be able to
Across phylogeny, the exposure to
have been identified that either positively or negatively reg-
ulate DAF-16 activity. The C. elegans host-cell factor homolog
host cell factor 1 (HCF-1) interacts with and inhibits DAF-16
transcriptional activity in the nucleus (62). Consistent with
this, knockdown of hcf-1by RNAiresults in increased lifespan
(62). In addition, the Wnt signalling pathway has been found
to intersect with IIS at the level of DAF-16=FOXO, where the
A number of additional cofactors
required for the transcription of oxidative stress-related genes
(22). These studies highlight the remarkable conservation
from nematodes to mammals in the oxidative stress response.
Taken together, cofactors provide another means for DAF-16
to transduce upstream signals into specific outputs.
Tissue Input into DAF-16
Worms continuously sense their environment to determine
if growth conditions are favorable for reproduction and, in
response, develop into reproductive adults or form dauer
larvae. Environmental stimuli are sensed through pairs of
ciliated sensory neurons that are located in the head (amphid
neurons) and tail (phasmid neurons) (96). Mutating certain
genes involved in the structure of the sensory cilia or ablating
the sensory neurons with a laser can have effects on lifespan
and dauer formation, as well as on sensory perception. These
genes in the sensory neurons are generally called che (abnor-
mal chemotaxis gene) or osm (osmotic avoidance abnormal
gene), and mutating either of them results in defects in sen-
sory perception, where worms do not respond properly to
changes in food or other sensory stimuli. These genes are
genetically positioned upstream of daf-16 (Fig. 8) (3, 5). Con-
sistent with the important role of sensory neurons, neuronal
activity ofdaf-16ismoreimportantfor dauerformation (lesser
for lifespan), whereas intestinal expression is critical for
lifespan regulation (65, 67).
In C. elegans, the gonadal primordium consists of four cells
(Z1, Z2, Z3, and Z4) (49). The Z1 and Z4 cell lineage gives rise
risetothegermcells.Laserablationof thegermline precursor
cells (Z2, Z3) results in an increase in lifespan of up to 60%
(44). However, removal of the entire reproductive system
(germ line plus somatic gonad; ablation of Z1, Z2, Z3, and
Z4) has no effect on lifespan. Importantly, both ablation of
the germline precursor cells and removal of the entire re-
productive system result in sterility. Therefore, since only
ablation of the germline percursor cells in lifespan exten-
sion, the lifespan effect is a result of a specific signal from
the reproductive tissue rather than a nonspecific effect of
sterility. Germ line ablation in a daf-16 mutant background
has no effect on lifespan, suggesting that the active signal
from the reproductive signal requires DAF-16 (Fig. 8) (44).
that are required for the increased longevity in worms lacking
a germline (44). The gene kri-1 was identified as a positive
regulator of the extended lifespan observed in germline-
deficient animals and DAF-16 nuclear localization (44). Reg-
ulation of DAF-16 through KRI-1 is independent of the IIS
pathway (44). The gene tcer-1, a predicted elongation factor,
has also been demonstrated to be required for lifespan ex-
tension by germline ablation (33). When germ cells of worms
are removed, TCER-1 is suggested to function with DAF-16 to
express a set of genes that result in the increased longevity in
affect the lifespan of long-lived IIS mutants, suggesting an
independent input into DAF-16 activity (33). Further studies
628YEN ET AL.
are required to determine which molecular components of the
pathway link signals from the germline to modulation of
DAF-16 activity and how these signals are transduced to
regulate lifespan in the context of a whole organism.
Further Regulation of DAF-16 Activity
Besides interacting with several cofactors and being phos-
phorylated by multiple upstream kinases, DAF-16 is also
regulated by acetylation, proteasomal degradation, and pos-
sibly dephosphorylation similar to mammalian FOXO (109).
We briefly discuss each of these below.
Acetylation of lysine residues is an important posttransla-
tional modification that can regulate transcription factor ac-
tivity. The transcription factor CBP can act as a histone acetyl-
transferase, and has been found to physically interact with
DAF-16 and FOXO (82). Studies in mice have shown that
CBP-mediated acetylation of histones enhances FOXO1 tran-
scriptional activity, though direct acetylation of lysine resi-
in vitro studies using acetylated FOXO1 demonstrate a de-
creased DNA binding affinity (11, 84). In C. elegans, cbp-1
RNAi reduces the lifespan of long-lived daf-2 mutants, but
does not further decrease the lifespan of daf-16 mutants (126).
dependent histone deacetylase SIR-2.1 modulates lifespan in a
DAF-16-dependent manner (103). Mammalian SIRT1 can bind
and deacetylate FOXO proteins, thereby activating them, sug-
gesting both histone and histone-independent functions for the
sirtuins (18, 48). The counteracting effects of CBP and SIR2 on
FOXO1 acetylation are another way by which FOXO activity is
Protein degradation is a dynamic and regulated process
that is important to maintain cellular proteostasis. Mamma-
lian FOXO proteins undergo proteasomal degradation under
conditions of active IIS (47, 93, 110). Consistent with this, the
P13-kinase inhibitor LY294002 inhibits FOXO degradation in
mammals (72). In C. elegans, the RLE-1 E3 ubiquitin ligase
alters DAF-16 protein levels by modulating its ubiquitination
and degradation (64). In addition, the SKP1-CUL1-F-Box E3
ligase complex has been identified as a positive regulator of
DAF-16, as knockdown of the components of this complex by
RNAi decreases the lifespan of daf-2 mutants as well as the
transcriptional activity of DAF-16 (34). This is consistent with
findings in mammals that the SKP1=CUL1=F-Box E3 ligase
protein complex regulates FOXO proteasomal degradation in
an AKT-dependent manner (47, 48).
Phosphorylation–dephosphorylation cycles are one of the
most robust regulators of protein function. DAF-16 is phos-
phorylated by multiple upstream kinases. Phosphorylation
and negative regulation by the AKT and SGK kinases un-
doubtedly provides the most potent regulation of DAF-16
surprising that no DAF-16 phosphatases have been identi-
fied that can counterbalance kinase activity. However, a
number of phosphatases that act on pathways upstream of
DAF-16 can modulate its localization and activity. Among
these, the mammalian phosphatase and tensin homolog is
a lipid phosphatase that antagonizes IIS at the level of PI
3-kinase (69). In C. elegans, mutation or RNAi of the phos-
phatase and tensin homolog daf-18 (worm homolog of the
Pten gene) decreases daf-2 lifespan, and results in more
cytosolic and inactive DAF-16 (74, 86, 91). In addition, the
protein phosphatase 2A (PP2A)-B56 phosphatase holoen-
zyme positively regulates DAF-16=FOXO nuclear localiza-
tion and transcriptional activity by modulating AKT
dephosphorylation (80, 91, 108). The PP2A catalytic subunit
can interact with FOXO1 and dephosphorylate FOXO3a
(100, 125). However, without its regulatory subunit, which
confers substrate specificity, the PP2A catalytic subunit
is fairly undiscriminatory in its dephosphorylation of
serine=threonine residues (111).
Additional studies are required to test whether specific
regulatory subunits of PP2A do indeed direct the PP2A-
B56 phosphatase holoenzyme to dephosphorylate DAF-
16=FOXO. In addition, because the kinases and cofactors that
associate with DAF-16=FOXO depend upon the upstream
signals, it is likely that the associated phosphatase(s) also in-
teracts and dephosphorylates specific residues in a stimulus-
of DAF-16 signaling. Sen-
germline both inhibit DAF-
16 signaling, and ablation
of either can extend lifespan
CENTRAL ROLE FOR C. ELEGANS FOXO/DAF-16629
Signals Downstream of DAF-16
Modulating levels of DAF-16 have been shown to result in
multiple biological changes, including changes in lifespan,
development, stress resistance, reproduction, and metabo-
lism. DAF-16 targets have been identified by many different
approaches and include superoxide dismutase (sod-3) (43),
transmembrane tyrosine kinase (old-1) (78), metallothioneine
(mtl-1) (9), SCP-like extracellular protein (scl-1) (89), raptor
(daf-15) (50), and small heat shock proteins (45). cDNA mi-
croarrays (73, 79) have identified a number of genes whose
expression level depends on DAF-16. Lee et al. used a com-
bination of bioinformatics and molecular studies to identify
additional targets (60). Together, these studies identified
genes that could be linked to lifespan regulation since they
included antioxidant genes (such as superoxide dismutase,
metallothioneine, catalase, and glutathione S-transferase),
small heat shock protein genes, metabolic genes (such as
apolipoprotein genes, glyoxylate-cycle genes, genes in-
volved in amino acid turnover), and antibacterial genes.
These results generally agree with the concept that an in-
crease in cellular defense results in an extended lifespan
Direct targets of DAF-16 have been identified by a ChIP-
based cloning strategy. ChIP fundamentally relies on the
physical interaction of a transcription factor and its target
promoter. The ChIP studies showed, for the first time, that
DAF-16 directly binds to previously known (including sod-3)
and numerous novel target promoters in C. elegans (87). In
addition, the large number of direct target genes identified
suggests that there is a complex regulation downstream of
DAF-16=FOXO: Implications in Disease
DAF-16=FOXO is at the heart of regulating multiple bio-
logical processes in response to numerous upstream signals,
be kept in tight check. In mammals, control of FOXO activity
maintains the balance between anabolic and catabolic path-
ways as well as cell cycle arrest versus progression (90, 110).
Hyperactivation of FOXO, while beneficial to cells during
starvation or oxidative stress, can have pleotropic effects
during normal fed conditions, depending on the tissue type.
Overexpression of FOXO in skeletal muscle has been associ-
ated with reduced mucle mass and dysregulated glucose
metabolism (53), whereas constituitively nuclear FOXO in the
liver decreased insulin sensitivity and increased liver tri-
glyceride levels (39, 71).
Mutations in the FOXOs have been identified in a number
of human cancers. Expression of dominant-negative FOXO
can accelerate lymphoma formation, consistent with the role
for FOXOs as tumor suppressors (7, 19). In addition, muta-
tions in FOXOs can result in depletion of hematopoietic stem
cell pools (7, 106). Lastly, a role for FOXOs in regulating
lifespan wasfirst established inC. elegans,andconservation of
this function has been observed across phylogeny, with a
number of studies identifying a correlation between naturally
occurring polymorphisms in FOXOs and increased longevity
in humans (26, 119).
For more on the role of DAF-16=FOXOs in disease, we refer
the reader to a number of reviews that discuss this topic in
greater detail (35, 39, 70, 110).
The single-FOXO homolog DAF-16 is an important
downstream effector of the IIS pathway. Recent studies have
revealed that DAF-16 is at the crossroads of several path-
ways and transduces upstream signals to specify distinct
biological processes. Work from several laboratories over
the last two decades has identified proteins that modulate
many aspects of DAF-16 function, including its localization,
stability,andtranscriptional targets. Importantly, all of these
pathways and proteins have a conserved function in regu-
lating mammalian FOXOs. However, there are several
questions that still remain unexplored. How does a single
transcription factor respond to both activatory and inhibi-
tory signals to regulate various cellular outputs? What are
the cofactors that activate or repress DAF-16-mediated
transcription? The lifespan-regulating function of DAF-16 is
likely to be attributed to the combinatorial function of its
several target genes, including antioxidant, immunity, and
stress-related genes. It is unclear what the spatial and tem-
poral aspects of their regulation are, and whether all or a
subset of them are responsible for the role of DAF-16 in
enhancing longevity. Exploring some of these questions
using the powerful genetic tools of C. elegans may have im-
plications for our understanding of not only FOXO biology
but also age-associated diseases such as cancer and diabetes.
H.A.T. is a William Randolph Hearst Young Investigator.
This publication was made possible by an endowment from
NIA (1R01AG025891 and 1R01AG031237) and the Ellison
1. Aguirre V, Uchida T, Yenush L, Davis R, and White MF.
The c-Jun NH2-terminal kinase promotes insulin resistance
during association with insulin receptor substrate-1 and
phosphorylation of Ser307. J Biol Chem 275: 9047–9054, 2000.
2. Albert PS, Brown SJ, and Riddle DL. Sensory control of
dauer larva formation in Caenorhabditis elegans. J Comp
Neurol 198: 435–451, 1981.
3. Alcedo J and Kenyon C. Regulation of C. elegans Longevity
by specific gustatory and olfactory neurons. Neuron 41: 45–
4. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney
PR, Reese CB, and Cohen P. Characterization of a 3-
phosphoinositide-dependent protein kinase which phos-
phorylates and activates protein kinase Balpha. Curr Biol 7:
5. Apfeld J and Kenyon C. Regulation of lifespan by sensory
perception in Caenorhabditis elegans. Nature 402: 804–809, 1999.
6. Araiz C, Chateau MT, and Galas S. 14-3-3 regulates life
span by both DAF-16-dependent and -independent mech-
anisms in Caenorhabditis elegans. Exp Gerontol 43: 505–519,
7. Arden KC. FoxOs in tumor suppression and stem cell
maintenance. Cell 128: 235–237, 2007.
8. Barbieri M, Bonafe M, Franceschi C, and Paolisso G. In-
sulin=IGF-I-signaling pathway: an evolutionarily con-
served mechanism of longevity from yeast to humans. Am J
Physiol Endocrinol Metab 285: E1064–E1071, 2003.
630YEN ET AL.
9. Barsyte D, Lovejoy DA, and Lithgow GJ. Longevity and
heavy metal resistance in daf-2 and age-1 long-lived mu-
tants of Caenorhabditis elegans. FASEB J 15: 627–634, 2001.
10. Berdichevsky A, Viswanathan M, Horvitz HR, and Guar-
ente L. C. elegans SIR-2.1 interacts with 14-3-3 proteins to
activate DAF-16 and extend life span. Cell 125: 1165–1177,
11. Brent MM, Anand R, and Marmorstein R. Structural basis
for DNA recognition by FoxO1 and its regulation by post-
translational modification. Structure 16: 1407–1416, 2008.
12. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS,
Anderson MJ, Arden KC, Blenis J, and Greenberg ME. Akt
promotes cell survival by phosphorylating and inhibiting a
Forkhead transcription factor. Cell 96: 857–868, 1999.
13. Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni
JV, Dalal SN, DeCaprio JA, Greenberg ME, and Yaffe MB.
14-3-3 transits to the nucleus and participates in dynamic
nucleocytoplasmic transport. J Cell Biol 156: 817–828, 2002.
14. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, and
Greenberg ME. Protein kinase SGK mediates survival sig-
nals by phosphorylating the forkhead transcription factor
FKHRL1 (FOXO3a). Mol Cell Biol 21: 952–965, 2001.
15. Burgering BM and Kops GJ. Cell cycle and death control:
long live Forkheads. Trends Biochem Sci 27: 352–360, 2002.
16. Cahill CM, Tzivion G, Nasrin N, Ogg S, Dore J, Ruvkun G,
and Alexander-Bridges M. Phosphatidylinositol 3-kinase
signaling inhibits DAF-16 DNA binding and function via
14-3-3-dependent and 14-3-3-independent pathways. J Biol
Chem 276: 13402–13410, 2001.
17. Chalfie M, Tu Y, Euskirchen G, Ward WW, and Prasher
DC. Green Fluorescent protein as a marker for gene ex-
pression. Science 263: 802–805, 1994.
18. Daitoku H, Hatta M, Matsuzaki H, Aratani S, Ohshima T,
Miyagishi M, Nakajima T, and Fukamizu A. Silent infor-
mation regulator 2 potentiates Foxo1-mediated transcrip-
tion through its deacetylase activity. Proc Natl Acad Sci USA
101: 10042–10047, 2004.
19. Dansen TB and Burgering BM. Unravelling the tumor-
suppressive functions of FOXO proteins. Trends Cell Biol 18:
20. Dorman JB, Albinder B, Shroyer T, and Kenyon C. The age-
1 and daf-2 genes function in a common pathway to control
the lifespan of Caenorhabditis elegans. Genetics 141: 1399–
21. Edison AS. Caenorhabditis elegans pheromones regulate
multiple complex behaviors. Curr Opin Neurobiol 19: 378–
22. Essers MA, de Vries-Smits LM, Barker N, Polderman PE,
Burgering BM, and Korswagen HC. Functional interaction
between beta-catenin and FOXO in oxidative stress sig-
naling. Science 308: 1181–1184, 2005.
23. Essers MA, Weijzen S, de Vries-Smits AM, Saarloos I, de
Ruiter ND, Bos JL, and Burgering BM. FOXO transcription
factor activation by oxidative stress mediated by the small
GTPase Ral and JNK. EMBO J 23: 4802–4812, 2004.
24. Feder ME and Hofmann GE. Heat-shock proteins, molec-
ular chaperones, and the stress response: evolutionary and
ecological physiology. Annu Rev Physiol 61: 243–282, 1999.
25. Fielenbach N and Antebi A. C. elegans dauer formation
and the molecular basis of plasticity. Genes Dev 22: 2149–
26. Flachsbart F, Caliebe A, Kleindorp R, Blanche H, von Eller-
Eberstein H, Nikolaus S, Schreiber S, and Nebel A.
Association of FOXO3A variation with human longevity
confirmed in German centenarians. Proc Natl Acad Sci USA
106: 2700–2705, 2009.
27. Friedman DB and Johnson TE. A mutation in the age-1 gene
in Caenorhabditis elegans lengthens life and reduces her-
maphrodite fertility. Genetics 118: 75–86, 1988.
28. Furuyama T, Nakazawa T, Nakano I, and Mori N. Identi-
fication of the differential distribution patterns of mRNAs
and consensus binding sequences for mouse DAF-16 ho-
mologues. Biochem J 349: 629–634, 2000.
29. Garigan D, Hsu AL, Fraser AG, Kamath RS, Ahringer J,
and Kenyon C. Genetic analysis of tissue aging in Cae-
norhabditis elegans: a role for heat-shock factor and bacterial
proliferation. Genetics 161: 1101–1112, 2002.
30. Garsin DA, Villanueva JM, Begun J, Kim DH, Sifri CD,
Calderwood SB, Ruvkun G, and Ausubel FM. Long-lived
C. elegans daf-2 mutants are resistant to bacterial patho-
gens. Science 300: 1921, 2003.
31. Gems D. Longevity and ageing in parasitic and free-living
nematodes. Biogerontology 1: 289–307, 2000.
32. Gems D and McElwee JJ. Broad spectrum detoxification:
the major longevity assurance process regulated by insu-
lin=IGF-1 signaling? Mech Ageing Dev 126: 381–387, 2005.
33. Ghazi A, Henis-Korenblit S, and Kenyon C. A transcription
elongation factor that links signals from the reproductive
system to lifespan extension in Caenorhabditis elegans. PLoS
Genet 5: e1000639, 2009.
34. Ghazi A, Henis-Korenblit S, and Kenyon C. Regulation of
Caenorhabditis elegans lifespan by a proteasomal E3 ligase
complex. Proc Natl Acad Sci USA 104: 5947–5952, 2007.
35. Glauser DA and Schlegel W. The emerging role of FOXO
transcription factors in pancreatic beta cells. J Endocrinol
193: 195–207, 2007.
36. Golden JW and Riddle DL. A pheromone influences larval
development in the nematode Caenorhabditis elegans. Science
218: 578–580, 1982.
37. Gottlieb S and Ruvkun G. daf-2, daf-16, and daf-23: Ge-
netically interacting genes controlling dauer formation in C.
elegans. Genetics 137: 107–120, 1994.
38. Greer EL, Dowlatshahi D, Banko MR, Villen J, Hoang K,
Blanchard D, Gygi SP, and Brunet A. An AMPK-FOXO
pathway mediates longevity induced by a novel method
of dietary restriction in C. elegans. Curr Biol 17: 1646–1656, 2007.
39. Gross DN, van den Heuvel AP, and Birnbaum MJ. The role
of FoxO in the regulation of metabolism. Oncogene 27:
40. Guertin DA and Sabatini DM. Defining the role of mTOR in
cancer. Cancer Cell 12: 9–22, 2007.
41. Henderson ST and Johnson TE. daf-16 integrates develop-
mental and environmental inputs to mediate aging in
the nematode Caenorhabditis elegans. Curr Biol 11: 1975–
42. Hertweck M, Gobel C, and Baumeister R. C. elegans SGK-1
is the critical component in the Akt=PKB kinase complex to
control stress response and life span. Dev Cell 6: 577–588, 2004.
43. Honda Y and Honda S. The daf-2 gene network for longevity
regulates oxidative stress resistance and Mn-superoxide
dismutase gene expression in Caenorhabditis elegans. FASEB J
13: 1385–1393, 1999.
44. Hsin H and Kenyon C. Signals from the reproductive sys-
tem regulate the lifespan of C. elegans. Nature 399: 362–366,
45. Hsu AL, Murphy CT, and Kenyon C. Regulation of aging
and age-related disease by DAF-16 and heat-shock factor.
Science 300: 1142–1145, 2003.
CENTRAL ROLE FOR C. ELEGANS FOXO/DAF-16631
46. Hu PJ. Dauer. WormBook 1–19, 2007.
47. Huang H, Regan KM, Wang F, Wang D, Smith DI, van
Deursen JM, and Tindall DJ. Skp2 inhibits FOXO1 in tumor
suppression through ubiquitin-mediated degradation. Proc
Natl Acad Sci USA 102: 1649–1654, 2005.
48. Huang H and Tindall DJ. Dynamic FoxO transcription
factors. J Cell Sci 120: 2479–2487, 2007.
49. Hubbard EJ and Greenstein D. The Caenorhabditis elegans
gonad: a test tube for cell and developmental biology. Dev
Dyn 218: 2–22, 2000.
50. Jia K, Chen D, and Riddle DL. The TOR pathway interacts
with the insulin signaling pathway to regulate C. elegans
larval development, metabolism and life span. Development
131: 3897–3906, 2004.
51. Jones KT, Greer ER, Pearce D, and Ashrafi K. Rictor=TORC2
regulates Caenorhabditis elegans fat storage, body size, and
development through sgk-1. PLoS Biol 7: e60, 2009.
52. Kaeberlein M, McVey M, and Guarente L. The SIR2=3=4
complex and SIR2 alone promote longevity in Sacchar-
omyces cerevisiae by two different mechanisms. Genes Dev
13: 2570–2580, 1999.
53. Kamei Y, Miura S, Suzuki M, Kai Y, Mizukami J, Taniguchi
T, Mochida K, Hata T, Matsuda J, Aburatani H, Nishino I,
and Ezaki O. Skeletal muscle FOXO1 (FKHR) transgenic
mice have less skeletal muscle mass, down-regulated Type
I (slow twitch=red muscle) fiber genes, and impaired gly-
cemic control. J Biol Chem 279: 41114–41123, 2004.
54. Kenyon C, Chang J, Gensch E, Rudner A, and Tabtiang R.
A C. elegans mutant that lives twice as long as wild type.
Nature 366: 461–464, 1993.
55. Kim AH, Sasaki T, and Chao MV. JNK-interacting protein 1
56. Kimura KD, Tissenbaum HA, Liu Y, and Ruvkun G. daf-2,
an insulin receptor-like gene that regulates longevity and
diapause in Caenorhabditis elegans. Science 277: 942–946, 1997.
57. Larsen PL, Albert PS, and Riddle DL. Genes that regulate
both development and longevity in Caenorhabditis elegans.
Genetics 139: 1567–1583, 1995.
58. Le TT, Duren HM, Slipchenko MN, Hu CD, and Cheng JX.
Label-free quantitative analysis of lipid metabolism in liv-
ing Caenorhabditis elegans. J Lipid Res 51: 672–677.
59. Lee RY, Hench J, and Ruvkun G. Regulation of C. elegans
DAF-16 and its human ortholog FKHRL1 by the daf-2 in-
sulin-like signaling pathway. Curr Biol 11: 1950–1957, 2001.
60. Lee SS, Kennedy S, Tolonen AC, and Ruvkun G. DAF-16
target genes that control C. elegans life-span and metabo-
lism. Science 300: 644–647, 2003.
61. Lehtinen MK, Yuan Z, Boag PR, Yang Y, Villen J, Becker
EB, DiBacco S, de la Iglesia N, Gygi S, Blackwell TK, and
Bonni A. A conserved MST-FOXO signaling pathway me-
diates oxidative-stress responses and extends life span. Cell
125: 987–1001, 2006.
62. Li J, Ebata A, Dong Y, Rizki G, Iwata T, and Lee SS. Cae-
norhabditis elegans HCF-1 functions in longevity mainte-
nance as a DAF-16 regulator. PLoS Biol 6: e233, 2008.
63. Li J, Tewari M, Vidal M, and Lee SS. The 14-3-3 protein
FTT-2 regulates DAF-16 in Caenorhabditis elegans. Dev Biol
301: 82–91, 2007.
64. Li W, Gao B, Lee SM, Bennett K, and Fang D. RLE-1, an E3
ubiquitin ligase, regulates C. elegans aging by catalyzing
DAF-16 polyubiquitination. Dev Cell 12: 235–246, 2007.
65. Libina N, Berman JR, and Kenyon C. Tissue-specific ac-
tivities of C. elegans DAF-16 in the regulation of lifespan.
Cell 115: 489–502, 2003.
66. Lin K, Dorman JB, Rodan A, and Kenyon C. daf-16: An
HNF-3=forkhead family member that can function to
double the life-span of Caenorhabditis elegans. Science 278:
67. Lin K, Hsin H, Libina N, and Kenyon C. Regulation of the
Caenorhabditis elegans longevity protein DAF-16 by insulin=
IGF-1 and germline signaling. Nat Genet 28: 139–145, 2001.
68. Lithgow GJ, White TM, Melov S, and Johnson TE. Ther-
motolerance and extended life-span conferred by single-
gene mutations and induced by thermal stress. Proc Natl
Acad Sci USA 92: 7540–7544, 1995.
69. Maehama T and Dixon JE. The tumor suppressor,
PTEN=MMAC1, dephosphorylates the lipid second mes-
senger, phosphatidylinositol 3,4,5-trisphosphate. J Biol
Chem 273: 13375–13378, 1998.
70. Maiese K, Chong ZZ, and Shang YC. OutFOXOing disease
and disability: the therapeutic potential of targeting FoxO
proteins. Trends Mol Med 14: 219–227, 2008.
71. Matsumoto M, Han S, Kitamura T, and Accili D. Dual role
of transcription factor FoxO1 in controlling hepatic insulin
sensitivity and lipid metabolism. J Clin Invest 116: 2464–
72. Matsuzaki H, Ichino A, Hayashi T, Yamamoto T, and
Kikkawa U. Regulation of intracellular localization and
transcriptional activity of FOXO4 by protein kinase B
through phosphorylation at the motif sites conserved
among the FOXO family. J Biochem 138: 485–491, 2005.
73. McElwee J, Bubb K, and Thomas JH. Transcriptional out-
puts of the Caenorhabditis elegans forkhead protein DAF-16.
Aging Cell 2: 111–121, 2003.
74. Mihaylova VT, Borland CZ, Manjarrez L, Stern MJ, and
Sun H. The PTEN tumor suppressor homolog in Cae-
norhabditis elegans regulates longevity and dauer formation
in an insulin receptor-like signaling pathway. Proc Natl
Acad Sci USA 96: 7427–7432, 1999.
75. Morley JF and Morimoto RI. Regulation of longevity in C.
elegans by heat shock factor and molecular chaperones. Mol
Biol Cell, 2003.
76. Morris JZ, Tissenbaum HA, and Ruvkun G. A phosphatidy-
diapause in Caenorhabditis elegans. Nature 382: 536–539, 1996.
77. Mukhopadhyay A, Oh SW, and Tissenbaum HA. Worming
pathways to and from DAF-16=FOXO. Exp Gerontol 41:
78. Murakami S and Johnson TE. The OLD-1 positive regulator
of longevity and stress resistance is under DAF-16 regula-
tion in Caenorhabditis elegans. Curr Biol 11: 1517–1523, 2001.
79. Murphy CT, McCarroll SA, Bargmann CI, Fraser A,
Kamath RS, Ahringer J, Li H, and Kenyon C. Genes that act
downstream of DAF-16 to influence the lifespan of Cae-
norhabditis elegans. Nature 424: 277–283, 2003.
80. Narasimhan SD, Mukhopadhyay A, and Tissenbaum HA.
InAKTivation of insulin=IGF-1 signaling by dephosphory-
lation. Cell Cycle 8: 3878–3884, 2009.
81. Narasimhan SD, Yen K, and Tissenbaum HA. Converging
82. NasrinN,OggS,CahillCM,BiggsW,Nui S,DoreJ, Calvo D,
Shi Y, Ruvkun G, and Alexander-Bridges MC. DAF-16 re-
cruits the CREB-binding protein coactivator complex to the
insulin-like growth factor binding protein 1 promoter in
HepG2 cells. Proc Natl Acad Sci USA 97: 10412–10417, 2000.
83. Nollen EA and Morimoto RI. Chaperoning signaling
pathways: molecular chaperones as stress-sensing ‘‘heat
shock’’ proteins. J Cell Sci 115: 2809–2816, 2002.
632YEN ET AL.
84. Obsil T and Obsilova V. Structure=function relationships
underlying regulation of FOXO transcription factors. On-
cogene 27: 2263–2275, 2008.
85. Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissen-
baum HA, and Ruvkun G. The Fork head transcription
factor DAF-16 transduces insulin-like metabolic and lon-
gevity signals in C. elegans. Nature 389: 994–999, 1997.
86. Ogg S and Ruvkun G. The C. elegans PTEN homolog, DAF-
18, acts in the insulin receptor-like metabolic signaling
pathway. Mol Cell 2: 887–893, 1998.
87. Oh SW, Mukhopadhyay A, Dixit BL, Raha T, Green MR,
and Tissenbaum HA. Identification of direct DAF-16 targets
controlling longevity, metabolism and diapause by chro-
matin immunoprecipitation. Nat Genet 38: 251–257, 2006.
88. Oh SW, Mukhopadhyay A, Svrzikapa N, Jiang F, Davis RJ,
and Tissenbaum HA. JNK regulates lifespan in Cae-
norhabditis elegans by modulating nuclear translocation of
forkhead transcription factor=DAF-16. Proc Natl Acad Sci
USA 102: 4494–4499, 2005.
89. Ookuma S, Fukuda M, and Nishida E. Identification of a
DAF-16 transcriptional target gene, scl-1, that regulates
longevity and stress resistance in Caenorhabditis elegans.
Curr Biol 13: 427–431, 2003.
90. Osaki M, Oshimura M, et al. PI3K-Akt pathway: its functions
and alterations in human cancer. Apoptosis 9: 667–676, 2004.
91. Padmanabhan S, Mukhopadhyay A, Narasimhan SD, Tesz
G, Czech MP, and Tissenbaum HA. A PP2A regulatory
subunit regulates C. elegans insulin=IGF-1 signaling by
modulating AKT-1 phosphorylation. Cell 136: 939–951, 2009.
92. Paradis S, Ailion M, Toker A, Thomas JH, and Ruvkun G.
A PDK1 homolog is necessary and sufficient to transduce
AGE-1 PI3 kinase signals that regulate diapause in Cae-
norhabditis elegans. Genes Dev 13: 1438–1452, 1999.
93. Plas DR and Thompson CB. Akt activation promotes deg-
radation of tuberin and FOXO3a via the proteasome. J Biol
Chem 278: 12361–12366, 2003.
94. Riddle D, Blumenthal T, Meyer B, and Priess J. C. Elegans II.
Cold Spring Harbor, NY: Cold Spring Harbor Press, 1997.
95. Riddle DL. A genetic pathway for dauer larva formation in
Caenorhabditis elegans. Stadler Symposia, University of Mis-
souri, Columbia, 1977, pp. 101–120.
96. Riddle DL and Albert PS. Genetic and environmental reg-
ulation of dauer larva development. In: C. elegans II, edited
by Riddle DL, Blumenthal T, Meyer BJ, and Priess JR. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press,
1997, pp. 739–768.
97. Rine J and Herskowitz I. Four genes responsible for a po-
sition effect on expression from HML and HMR in Sac-
charomyces cerevisiae. Genetics 116: 9–22, 1987.
98. Rogina B and Helfand SL. Sir2 mediates longevity in the fly
through a pathway related to calorie restriction. Proc Natl
Acad Sci USA 101: 15998–16003, 2004.
99. Scheid MP, Marignani PA, and Woodgett JR. Multiple
phosphoinositide 3-kinase-dependent steps in activation of
protein kinase B. Mol Cell Biol 22: 6247–6260, 2002.
100. SinghA,Ye M,BucurO,ZhuS,TanyaSantosM,Rabinovitz I,
Wei W, Gao D, Hahn WC, and Khosravi-Far R. Protein
phosphatase 2A reactivates FOXO3a through a dynamic in-
101. Soukas AA, Kane EA, Carr CE, Melo JA, and Ruvkun G.
Rictor=TORC2 regulates fat metabolism, feeding, growth,
and life span in Caenorhabditis elegans. Genes Dev 23: 496–
102. Stiernagle T. Maintenance of C. elegans. WormBook 1–11, 2006.
103. Tissenbaum HA and Guarente L. Increased dosage of a sir-
2 gene extends lifespan in Caenorhabditis elegans. Nature 410:
104. Tissenbaum HA and Guarente L. Model organisms as a
guide to mammalian aging. Dev Cell 2: 9–19, 2002.
105. Tissenbaum HA and Ruvkun G. An insulin-like signaling
pathway affects both longevity and reproduction in Cae-
norhabditis elegans. Genetics 148: 703–717, 1998.
106. Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH,
Cullen DE, McDowell EP, Lazo-Kallanian S, Williams IR,
Sears C, Armstrong SA, Passegue E, DePinho RA, and
Gilliland DG. FoxOs are critical mediators of hematopoietic
stem cell resistance to physiologic oxidative stress. Cell 128:
107. Tullet JM, Hertweck M, An JH, Baker J, Hwang JY, Liu S,
Oliveira RP, Baumeister R, and Blackwell TK. Direct inhi-
bition of the longevity-promoting factor SKN-1 by insulin-
like signaling in C. elegans. Cell 132: 1025–1038, 2008.
108. Ugi S, Imamura T, Maegawa H, Egawa K, Yoshizaki T, Shi
K, Obata T, Ebina Y, Kashiwagi A, and Olefsky JM. Protein
phosphatase 2A negatively regulates insulin’s metabolic
signaling pathway by inhibiting Akt (protein kinase B) ac-
tivity in 3T3-L1 adipocytes. Mol Cell Biol 24: 8778–8789, 2004.
109. Van Der Heide LP, Hoekman MF, and Smidt MP. The ins
and outs of FoxO shuttling: mechanisms of FoxO translo-
cation and transcriptional regulation. Biochem J 380: 297–309,
110. van der Horst A and Burgering BM. Stressing the role of
FoxO proteins in lifespan and disease. Nat Rev Mol Cell Biol
8: 440–450, 2007.
111. Virshup DM and Shenolikar S. From promiscuity to pre-
cision: protein phosphatases get a makeover. Mol Cell 33:
112. Vogt PK, Jiang H, and Aoki M. Triple layer control: phos-
phorylation, acetylation and ubiquitination of FOXO pro-
teins. Cell Cycle 4: 908–913, 2005.
113. Wang MC, Bohmann D, and Jasper H. JNK extends life
span and limits growth by antagonizing cellular and
organism-wide responses to insulin signaling. Cell 121:
114. Wang Y, Oh SW, Deplancke B, Luo J, Walhout AJ, and
Tissenbaum HA. C. elegans 14-3-3 proteins regulate life
span and interact with SIR-2.1 and DAF-16=FOXO. Mech
Ageing Dev 127: 741–747, 2006.
115. Watts JL. Fat synthesis and adiposity regulation in Cae-
norhabditis elegans. Trends Endocrinol Metab 20: 58–65, 2009.
116. Weinkove D, Halstead JR, Gems D, and Divecha N.
3-kinase-dependent translocation of DAF-16=FOXO to the
cytoplasm. BMC Biol 4: 1, 2006.
117. Weston CR and Davis RJ. The JNK signal transduction
pathway. Curr Opin Cell Biol 19: 142–149, 2007.
118. White JG, Southgate E, Thomson JN, and Brenner S. The
structure of the nervous system of the nematode Cae-
119. Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K,
Masaki KH, Willcox DC, Rodriguez B, and Curb JD.
FOXO3A genotype is strongly associated with human lon-
gevity. Proc Natl Acad Sci USA 105: 13987–13992, 2008.
120. Wolf M, Nunes F, Henkel A, Heinick A, and Paul RJ. The
MAP kinase JNK-1 of Caenorhabditis elegans: location, acti-
vation, and influences over temperature-dependent insulin-
like signaling, stress responses, and fitness. J Cell Physiol
214: 721–729, 2008.
CENTRAL ROLE FOR C. ELEGANS FOXO/DAF-16 633
121. Wolff S, Ma H, Burch D, Maciel GA, Hunter T, and Dillin
A. SMK-1, an essential regulator of DAF-16-mediated lon-
gevity. Cell 124: 1039–1053, 2006.
122. Wolkow CA, Kimura KD, Lee MS, and Ruvkun G. Reg-
ulation of C. elegans life-span by insulinlike signaling in the
nervous system. Science 290: 147–150, 2000.
123. Wolkow CA, Munoz MJ, Riddle DL, and Ruvkun G.
Insulin receptor substrate and p55 orthologous adap-
tor proteins function in the Caenorhabditis elegans daf-2=
insulin-like signaling pathway. J Biol Chem 277: 49591–
124. Wood WB. The nematode Caenorhabditis elegans. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratory Press,
125. Yan L, Lavin VA, Moser LR, Cui Q, Kanies C, and Yang E.
PP2A regulates the pro-apoptotic activity of FOXO1. J Biol
Chem 283: 7411–7420, 2008.
126. Zhang M, Poplawski M, Yen K, Cheng H, Bloss E, Zhu X,
Patel H, and Mobbs CV. Role of CBP and SATB-1 in aging,
dietary restriction, and insulin-like signaling. PLoS Biol 7:
Address correspondence to:
Dr. Heidi A. Tissenbaum
Program in Gene Function and Expression
Program in Molecular Medicine
University of Massachusetts Medical School
Aaron Lazare Research Building Suite 621
364 Plantation St.
Worcester, MA 01605
Date of first submission to ARS Central, July 19, 2010; date of
acceptance, August 1, 2010.
CBP¼cAMP response element-binding protein
che¼abnormal chemotaxis gene
cst-1¼worm homolog of mammalian Stk4=Stk3
daf-2¼worm homolog of the insulin=IGF
DAF-16¼worm homolog of FOXO protein
daf-18¼worm homolog of the Pten gene
FOXO¼Forkhead box O transcription factor
HCF-1¼host cell factor 1
HSF-1¼heat shock factor 1
IGF¼insulin-like growth factor
JNK-1¼jun kinase 1
osm¼osmotic avoidance abnormal gene
PP2A¼protein phosphatase 2A
SGK-1¼serum and glucocorticoid inducible
sir¼silent information regulator
smk-1¼suppressor of MEK null gene
Stk4¼serine=threonine kinase 4 gene formerly
known as Mst1
TOR¼target of rapamycin
634 YEN ET AL.