Download SM-20, EGL-9, and the EGLN Family of Hypoxia

Mol. Cells, Vol. 16, No. 1, pp. 1-12
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KSMCB 2003
SM-20, EGL-9, and the EGLN Family of Hypoxia-inducible Factor
Prolyl Hydroxylases
Robert S. Freeman*, Daphne M. Hasbani†, Elizabeth A. Lipscomb‡, Jennifer A. Straub, and Liang Xie
Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
(Received June 23, 2003; Accepted June 24, 2003)
Key to the transduction of signals from the environment to the cell nucleus are enzymes that posttranslationally modify proteins. Modifications such as
protein phosphorylation have long been known to
regulate protein interactions, stability, and localization,
as well as enzyme activity. Recent investigations into
how cells respond to varying oxygen levels have identified a new mechanism for regulating signal transduction involving the post-translational hydroxylation of
proline. The enzymes that catalyze this reaction comprise a novel family of prolyl hydroxylases, which include a growth-factor-responsive and cell-death-related protein (SM-20) in mammals, and a protein
(EGL-9) in C. elegans important for normal egg laying.
Introduction
Cells respond to changes in their environment by activating networks of signal transduction pathways that culminate in adaptive changes in gene expression. Key to most
(if not all) signal transduction pathways are enzymes that
catalyze post-translational modifications. Over the last 25
years a nearly universal role for phosphorylation and
dephosphorylation as mechanisms for altering the function, stability, and subcellular location of signaling proteins has been firmly established (Graves and Krebs,
1999). In more recent times, the transfer of a signal from
the plasma membrane to the nucleus has been shown to
involve other types of protein modifications including
regulated proteolysis (Ebinu and Yankner, 2002), fatty acid
acylation (Resh, 1996), ubiquitination (Yang and Yu,
2003), and acetylation (Magnaghi-Jaulin et al., 1999). Recent studies on the regulation of hypoxia-inducible factor
(HIF), an important oxygen-sensing transcription factor in
animals from nematodes to man, have now identified an
additional mechanism for signal transduction involving a
not-so-new post-translational modification, hydroxylation
of proline residues. Enzyme catalyzed proline hydroxylation has long been known as a post-translational mechanism in collagen biosynthesis (Peterkofsky and Udenfriend,
1965), where it is important for stabilizing the triplehelical conformation in collagen fibers (reviewed by
Kivirikko and Pihlajaniemi, 1998). Now with the recent
discovery of a novel family of prolyl hydroxylases,
proline hydroxylation has emerged as an important component in the signaling pathway that regulates how cells
adapt to changes in oxygen (O2) levels.
When exposed to abnormally low O2 concentrations
(i.e., hypoxia), multicellular organisms respond by increasing their O2 transport capacity, glycolytic energy
production, and cell survival mechanisms. These responses are a direct consequence of HIF-dependent transcriptional transactivation of genes such as erythropoietin,
vascular endothelial growth factor (VEGF), glucose
transporter-1, and insulin-like growth factor (see Semenza,
1999, for a review of HIF biology and biochemistry).
†
Present address: Department of Anatomy and Neurobiology,
Washington University School of Medicine, St. Louis, MO
63110, USA.
‡
Present address: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
02215, USA.
Demo
* To whom correspondence should be addressed.
Tel: 1-585-273-4893; Fax: 1-585-273-2652
E-mail: [email protected]
Abbreviations: Ang II, angiotensin II; ARNT, aryl hydrocarbon
nuclear translocator; CODD, C-terminal oxygen-dependent degradation domain; CsA, cyclosporin A; EGLN, Egl-nine; EST,
expressed sequence tag; HIF, hypoxia-inducible factor; HRE,
hypoxia-inducible factor response element; MTS, mitochondria
targeting sequence; NGF, nerve growth factor; NODD, Nterminal oxygen-dependent degradation domain; pVHL, von
Hippel-Lindau tumor suppressor protein; VEGF, vascular endothelial growth factor.
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Prolyl Hydroxylases and Signal Transduction
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There are three distinct forms of HIF in mammalian cells,
two of which (HIF-1 and HIF-2) are known to be induced
by hypoxia. For HIF to be activated as a transcription
factor, one of two unique HIF α-subunits (HIF-1α or HIF2α) must enter the nucleus and bind a common HIF βsubunit (identical to ARNT, the aryl hydrocarbon nuclear
translocator). Both α- and β-subunits are members of the
basic helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) superfamily, but unlike the α-subunit, the β-subunit can
form complexes with various other bHLH/PAS domain
proteins. Hypoxia-induced activation of HIF results from
stabilization of the α-subunits, which otherwise have a
half-life of just a few minutes under normoxic conditions.
Maxwell et al. (1999) were the first to show that ubiquitination and proteasomal degradation of HIF-1α requires the
von Hippel-Lindau tumor suppressor protein (pVHL), the
substrate recognition subunit for an E3 ubiquitin ligase (see
Conaway and Conaway, 2002, for a recent review). Subsequent studies indicated that stabilization of HIF was due to
disruption of the HIF-pVHL interaction under hypoxic
conditions (Cockman et al., 2000), but the oxygen-sensing
mechanism underlying the change in affinity remained unknown. Then, two laboratories simultaneously reported that
oxygen-dependent hydroxylation of a conserved proline
residue in HIF-1α was critical for mediating its interaction
with pVHL (Ivan et al., 2001; Jaakkola et al., 2001). (Note
that Yu et al., 2001, reported similar findings a short time
later.) Because the well-known collagen prolyl hydroxylases are located in the endoplasmic reticulum, whereas
HIF is found in the cytoplasm and nucleus, the enzyme
responsible for hydroxylating HIF was believed to be novel.
Within months of the initial reports of post-translational
modification of HIF by proline hydroxylation, Epstein et al.
(2001) identified the HIF prolyl hydroxylase in C. elegans
and provided evidence for a related family of enzymes in
humans.
In this review we describe the proteins that comprise
the Egl-nine (EGLN) family of HIF prolyl hydroxylases.
We begin with rat SM-20, the first family member identified, by reviewing what was known about its function
prior to the discovery of the other EGLN proteins. The
identification of egl-9 and the three mammalian EGLN
genes is described next followed by a critical review of
their function as prolyl hydroxylases and oxygensensitive regulators of HIF. We conclude with the hypothesis that proline hydroxylation may prove to be a
general mechanism for regulating protein-protein interactions in a variety of signal transduction pathways.
SM-20 is a growth factor regulated gene in
muscle cells that can promote cell death in
neurons
Treatment of cells with growth factors and a variety of
other stimuli induces the rapid and typically transient expression of so-called ‘immediate early genes’ such as cjun and c-fos. While comparing gene expression in rat
vascular smooth muscle cells treated with or without
platelet-derived growth factor, Taubman and colleagues
(Wax et al., 1994) identified a novel gene called SM-20
that displayed characteristics of an immediate early gene.
SM-20 mRNA levels peaked 1−2 h after treatment with
serum, PDGF, angiotensin II (Ang II), isoproterenol, or
forskolin and, like other immediate early genes, its expression was superinduced when stimulated in the presence of the protein synthesis inhibitor cycloheximide.
SM-20 mRNA levels are also regulated by growth factors
in C2C12 and C25 skeletal myoblast lines, although
somewhat differently than in smooth muscle cells
(Moschella et al., 1999). In skeletal myoblasts, SM-20
expression is enhanced during differentiation initiated in
vitro by serum reduction. Upregulation of SM-20 expression in myoblasts was prevented by the addition of differentiation-blocking growth factors such as basic fibroblast
growth factor. Based on these and other results described
below, a role for SM-20 in myogenesis was postulated
(Moschella et al., 1999).
The rat SM-20 gene is predicted to encode a 355 amino
acid protein (calculated molecular mass of 39.8 kDa). At
the time it was first described, the predicted SM-20 protein lacked any resemblance to known proteins, although
a region within its C-terminus shared a high degree of
sequence identity with an uncharacterized open reading
frame in the C. elegans genome (Wax et al., 1994). Antibodies generated against SM-20 detected a 40 kDa protein
in immunoblots of rat aorta smooth muscle cells, consistent with its calculated molecular mass (Wax et al., 1996).
In immunocytochemical studies, anti-SM-20 antibodies
revealed a punctate or sometimes striated pattern in the
cytoplasm of smooth muscle cells. Similar SM-20 immunoreactivity was detected in cardiac and skeletal muscle,
in the smooth muscle cells of atherosclerotic plaques from
humans, and in the neointima in a rat aorta balloon injury
model. In addition to the relatively high levels of SM-20
antigen in muscle-rich tissues, lower levels were found in
the epithelial cells of various organs (e.g., intestine, lung,
pancreas, kidney, and skin) (Wax et al., 1996). SM-20
mRNA is also expressed in peripheral neurons and in
brain (Lipscomb et al., 1999; Wax et al., 1994). In developing mouse embryos, low levels of SM-20 antigen were
detected in proliferating myoblasts as early as embryonic
day 8.5 (Moschella et al., 1999). However, SM-20 immunoreactivity increased substantially in postmitotic myocytes present by day 10.5 and it remained elevated in
adult muscle.
Using differential display, we found that expression of
the SM-20 gene is upregulated in rat sympathetic neurons
after withdrawal of nerve growth factor (NGF) (Lipscomb
et al., 1999). Sympathetic and certain sensory neurons
Robert S. Freeman et al.
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require NGF for survival. Apoptosis initiated by NGF
withdrawal is inhibited by agents that block RNA and
protein synthesis, giving rise to the widely accepted idea
that trophic factor deprivation-induced death requires the
expression of genes that activate the apoptotic machinery.
SM-20 is one of a small number of genes (others include
c-jun, c-fos, cyclin D1, Bim, and DP5/HRK) whose mRNA
levels increase in NGF-deprived neurons (reviewed by
Freeman et al., 2003). SM-20 expression increases within
5 hours after NGF removal, peaking between 10 and 15 h.
Other apoptosis-inducing stimuli also upregulate SM-20
expression in neurons, including the phosphatidylinositol
3-kinase inhibitor LY294002 and the anti-tumor drug cytosine arabinoside (Lipscomb et al., 1999).
Over-expression of SM-20 is sufficient to promote cell
death in NGF-maintained sympathetic neurons (Lipscomb
et al., 1999; 2001). Induction of SM-20 expression in proliferating or NGF-differentiated neuronal PC12 cells
stably expressing a tetracycline-inducible SM-20 transgene also leads to significant cell death (Straub et al.,
2003). In both neurons and PC12 cells, SM-20-induced
death is blocked by broad spectrum caspase inhibitors.
Induction of SM-20 expression in PC12 cells is followed
by an increase in cytosolic cytochrome c and an unexpected increase in the total amount of cytochrome c. Precisely how SM-20 expression leads to increased cytochrome c is still under investigation, but it is not accompanied by elevated cytochrome c mRNA expression nor
an increase in several other mitochondrial proteins
(Straub et al., 2003).
We recently demonstrated that the N-terminus of SM20 contains a functional mitochondria targeting sequence
(MTS) as defined by deletion analysis and its ability to
target normally cytoplasmic proteins to mitochondria
(Lipscomb et al., 2001). Unfortunately in performing
these experiments we had to rely on over-expression of
SM-20, as the anti-SM-20 antibodies available to us at the
time were not sufficient for localizing endogenous SM-20
in cells. Nonetheless, a mitochondrial localization for at
least a portion of the endogenous protein seems likely
when images of smooth muscle cells stained with antiSM-20 antibodies (Wax et al., 1996) are compared with
those of cells over-expressing SM-20 (Lipscomb et al.,
2001).
In addition to mitochondrial-localized SM-20, we always observe some cells that contain a pool of SM-20 in
their nuclei. Nuclear-localized SM-20 has been detected
in immunofluorescence experiments, in experiments with
an SM-20-EGFP fusion protein, and in immunoblots of
subcellular fractions enriched for nuclei (Figs. 1A and
1B). The SM-20 protein in nuclear fractions migrates at
40 kDa by SDS-PAGE while the majority of the SM-20
detected in mitochondria-enriched fractions migrates at
33–34 kDa - the molecular mass difference probably due
to a cleavage event within mitochondria that removes the
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A
B
a
a
a
Fig. 1. Localization of SM20 to mitochondria and nuclei. A.
CV-1 cells were transfected with an expression vector encoding
an SM-20/GFP fusion protein. After 24 h, the cells were stained
with Mitotracker Orange and analyzed by fluorescence microscopy (magnification is 400×). The cell marked with an arrow
contains SM-20/GFP fluorescence in mitochondria and the nucleus. B. Subcellular fractions enriched for either mitochondria
or nuclei were prepared from metabolically 35S-labeled NIH3T3
cells (see Lipscomb et al., 2001, for details) 24 h after transfection with SM-20 expression vector or empty vector. SM-20 protein was immunoprecipitated with a C-terminal anti-SM-20
antibody and then analyzed by SDS-PAGE and fluorography.
Bands labeled ‘a’ and ‘b’ correspond to full-length SM-20 (40
kDa) enriched in the nuclear fraction and a truncated SM-20
protein (33–34 kDa) found only in the mitochondrial fraction.
Aliquots of each fraction were immunoblotted for cytochrome c
and proliferating cell nuclear antigen (PCNA) to indicate their
purity.
MTS (Lipscomb et al., 2001). It is feasible that nuclear
localization of some of the over-expressed SM-20 occurs
only when the mitochondrial import machinery or other
processes involved in targeting SM-20 to mitochondria
become saturated. However, much of the endogenous protein detected in muscle cells, sympathetic neurons, and
PC12 cells migrates at 40 kDa, suggesting that at least
some of the endogenous SM-20 may reside outside of
mitochondria (Lipscomb et al., 1999; Wax et al., 1996;
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Prolyl Hydroxylases and Signal Transduction
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Wolf et al., 2002). Determining where SM-20 is localized
in cells is important for at least two reasons. First, the
SM-20 orthologs in humans and mice are predicted to
encode smaller proteins that lack a classical N-terminal
MTS (discussed below). If the localization of SM-20 in
rat cells does not at least partially overlap with that of its
ortholog in humans, then the relevance of studies on rat
SM-20 to humans may be questionable. Second, a truncated form of SM-20 lacking its MTS retains the ability to
promote cell death in sympathetic neurons, despite being
diffusely localized throughout the cytoplasm and nucleus
(Lipscomb et al., 2001). Ultimately, resolving the issue of
SM-20 localization will require specific antibodies that
are sufficiently sensitive to detect endogenous SM-20
under various conditions. Moreover, the possibility that
SM-20 may move between mitochondria, nucleus, or
other intracellular sites also warrants investigation. Evidence that much of the endogenous SM-20 protein in cells
is unprocessed and that the over-expressed protein can be
found in nuclei and mitochondria suggests that endogenous SM-20 will also be found in both organelles in rat
cells.
The EGLN gene family
Recently, a family of three SM-20-like genes in the human and mouse genomes was described (Bruick and
McKnight, 2001; Epstein et al., 2001; Taylor, 2001). Unfortunately, an equal number of acronyms were coined to
describe the genes (Table 1). To avoid confusion in future
reports, we recommend the adoption of the EGLN nomenclature (as done in this review) for its evolutionary
significance, its acceptance by the Human Genome Organization (Taylor, 2001), and because it is less restrictive
with respect to biochemical function. The human and
mouse EGLN genes use conserved splice donor/acceptor
sites to generate major transcripts consisting of 5 exons.
Overall, the predicted EGLN proteins share 50% identity
over the 239 amino acids that comprise the smallest family member, EGLN3. More striking, however, is a stretch
of 109 amino acids encoded by exons 2–4 that is 75%
identical between family members. As their name indicates, EGLN genes are homologous to the egl-9 gene in C.
elegans (Darby et al., 1999), a gene required for normal
egg laying. Over the 239 amino acid region of shared homology, the protein encoded by egl-9 is 40% identical to
the mammalian EGLNs. The egl-9 gene is most similar to
mammalian EGLN1, with the proteins encoded by each
gene having two well-conserved MYND-type zinc finger
domains of unknown function near their N-termini
(Taylor, 2001). Because there is just a single EGLN family member in C. elegans (and apparently in Drosophila),
the appearance of EGLN2 and EGLN3 is likely the result
of gene duplication events in higher metazoans.
Table 1. Alternative nomenclatures for EGLN family genesa.
Egl-nine (EGLN)b
Prolyl hydroxylase HIF prolyl hydroxydomainc (PHD)
lased (HIF)
EGLN1
PHD2
HPH2
EGLN2
PHD1
HPH3
EGLN3
PHD3
HPH1
a
Names across each row stand for the same gene.
Taylor (2001).
c
Epstein et al. (2001).
d
Bruick and McKnight (2001).
b
The egl-9 locus was originally characterized over 20
years ago together with a large number of other loci in
which mutations resulted in defective egg laying (Trent et
al., 1983). Sixteen years later, Darby et al. (1999) cloned
the egl-9 gene while screening for C. elegans strains that
were resistant to a rapid and lethal paralysis induced by
Pseudomonas. The mutations in egl-9 conferring resistance to Pseudomonas toxicity are almost certainly lossof-function mutations since they prematurely terminate or
otherwise disrupt the egl-9 open reading frame. An analysis of green fluorescence protein expression in transgenic
animals containing a gfp reporter gene fused in frame
with exons 1 and 2 of egl-9 suggests that EGL-9 is expressed in various muscle cells and sensory neurons in C.
elegans. These results indicate that activation of EGL-9 or
a pathway involving it underlies the toxin-induced paralysis and death of the worm. A more recent report from this
laboratory describes the surprising finding that the toxic
substance produced by Pseudomonas is cyanide (Gallagher
and Manoil, 2001).
Within the EGLN family, the human and mouse
EGLN3 cDNAs (compiled from a large number of overlapping expressed sequence tags, or ESTs) contain open
reading frames of 239 codons that are 98 and 100% identical, respectively, to amino acids 117–355 of rat SM-20
(Taylor, 2001)1. The extremely high degree of sequence
identity between SM-20 and EGLN3 indicates that they
represent orthologous proteins in rats, mouse and humans.
Assuming the predicted EGLN3 open reading frames are
correct, then translation of the mouse and human genes
would initiate at a site downstream of the sequences en1
The SM-20 amino acid sequence used in the alignment reported by Taylor (2001) is based on the sequence originally
described by Wax et al. (1994) and has a serine at amino acid
304. In ot er SM-20 EST and cDNA sequences and in the SM20 gene itself (GenBank accession No. NW_043947) there is
a codon for phenylalanine (‘TTC’ instead of the ‘TCC’ reported
in the original cDNA) at this position. Since all other EGLN
genes including egl-9 encode phenylalanine at this position,
amino acid 304 in SM-20 is almost certainly a phenylalanine.
Robert S. Freeman et al.
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coding the MTS in SM-20 (hereafter, the term SM-20 will
be used exclusively for the rat gene). This would occur
despite significant conservation at the nucleotide level
between the 5′ ends of the EGLN3 cDNAs and the 5′ coding sequences in SM-20. In the mouse cDNA, a stop
codon is present in place of the codon for Glu83 in SM-20.
The corresponding region in human EGLN3 is not interrupted by a stop codon; instead, it lacks an upstream AUG
analogous to the start site in SM-20. Therefore, barring
alternative splicing in the 5′ exon or initiation of translation at a non-AUG codon, the first available AUG in
mouse and human EGLN3 would correspond to Met117 in
SM-20. Thus, the mouse and human EGLN3 proteins apparently lack the first 116 residues found in SM-20, although it should be noted that the endogenous proteins
have not yet been studied.
Expression of the 239-codon EGLN3 cDNA in COS-1
and U2OS osteosarcoma cells gives rise to a protein that
is distributed throughout the cytoplasm and nucleus
(Huang et al., 2002; Metzen et al., 2002), virtually identical to the distribution of an SM-20 deletion mutant lacking its MTS (Lipscomb et al., 2001). As with EGLN3, the
predicted EGLN1 and EGLN2 proteins are highly conserved across species showing 80 and 86% identity, respectively, between their human and mouse orthologs.
Cells transfected with EGLN1 or EGLN2 cDNAs (encoding proteins of 427 and 407 amino acids, respectively)
reveal EGLN1 to be mostly in the cytoplasm while
EGLN2 appears exclusively in the nucleus (Huang et al.,
2002; Metzen et al., 2002).
A handful of reports in addition to those discussed
above are consistent with a role for EGLN proteins in
controlling cell growth. For example, SM-20 has been
identified as a gene that is upregulated in response to p53
activation in temperature-sensitive-mutant rat embryo
fibroblasts (Madden et al., 1996). Colony formation assays
in p53-deficient cell lines selected for stable expression of
ectopic SM-20 resulted in reduced numbers of colonies,
suggesting that SM-20 might negatively impact cell growth
downstream of p53 function. However in PC12 cells, SM20 mRNA and protein levels are partially suppressed following treatment with Ang II, which was found to inhibit
proliferation in these cells by acting through the angiotensin AT2-receptor (Wolf et al., 2002). This was opposite
to the effect of Ang II in smooth muscle cells, where it induces immediate-early expression of SM-20 (Wax et al.,
1994) by acting through AT1 receptors. Antisense inhibition of SM-20 by itself decreased proliferation of PC12
cells while over-expression of SM-20, which resulted in
similarly decreased cell numbers in the presence or absence
of Ang II, reversed the growth inhibitory effect of Ang II
(Wolf et al., 2002). Moreover, a partial suppression of basal
apoptosis was noted in the angiotensin-treated cells, which
could also be related to inhibition of SM-20 expression
given the finding that over-expression of SM-20 can pro-
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mote cell death in PC12 cells (Straub et al., 2003).
Expression of an incomplete mouse EGLN2 cDNA
(called Falkor) was found to confer resistance to cisplatin-induced growth arrest in mouse embryo fibroblasts
(Erez et al., 2002). Because over-expression of full-length
EGLN2 reversed the effect, the authors suggested that the
truncated protein encoded by the partial cDNA might
counteract a growth inhibitory function of the endogenous
EGLN2 protein induced by cisplatin. Treatment with antisense EGLN2 oligonucleotides modestly enhanced the
growth of cells in the absence of cisplatin lending support
to a possible growth inhibitory function for EGLN2. An
opposite conclusion was reached concerning EGLN2
function in experiments with estrogen-responsive breast
cancer cells. Using SAGE, EGLN2 was identified as an
estrogen-induced gene in select estrogen-responsive breast
cancer cell lines (Seth et al., 2002). In colony formation
assays with these cells, stable expression of EGLN2 led to
an increase in colony number irrespective of estrogen,
suggesting that EGLN2 might stimulate growth in certain
tumor cells. Together these diverse studies suggest growth
regulatory functions for EGLN2 and SM-20, but whether
they act to enhance or inhibit cell growth depends on the
particular cell type and stimulus studied.
EGLN genes are expressed in a quasi tissue-specific
manner in adult humans (Fig. 2A). Two prominent EGLN1
transcripts are expressed at highest levels in skeletal muscle and heart and are also readily detected in kidney, liver,
brain, and placenta. Roughly similar levels of a single
EGLN2 mRNA are detected in heart, skeletal muscle,
brain, kidney, and liver with somewhat lower levels in
other tissues. The major EGLN3 transcript is most abundant in heart but is present at lower levels in other muscle
containing tissues and in kidney, liver, and brain. In adult
brain, EGLN3 appears to be expressed ubiquitously in all
regions (Fig. 2B). Some differences with these expression
patterns have been noted in mouse tissues; in addition,
high levels of EGLN2 were seen in mouse testis where
little if any EGLN1 or EGLN3 was detected (Lieb et al.,
2002).
EGLN proteins are novel iron- and 2-oxoglutarate-dependent dioxygenases
A major breakthrough towards understanding SM-20 and
EGLN function came when secondary structure prediction
algorithms identified the gene products of egl-9 and an
EST derived from the human EGLN3 gene as potential new
members of the iron- and 2-oxoglutarate-dependent dioxygenase superfamily (Aravind and Koonin, 2001; Epstein et
al., 2001). The defining structural features of this large and
diverse family are an α-helix followed by 7–8 β-strands
arranged in a jelly roll β-barrel topology. Situated within
the large cavity that forms the active site is a conserved 2-
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A
B
Fig. 2. Expression of EGLN genes in human tissues. A. A human multi-tissue northern blot (Clontech) was hybridized (sequentially) with 32P-labeled cDNA probes for EGLN3, EGLN2,
and EGLN1. Autoradiography was for 10 d (EGLN3), 3 d
(EGLN2), or 23 h (EGLN1). Lane 1, brain; lane 2, heart; lane 3,
skeletal muscle; lane 4, colon; lane 5, thymus; lane 6, spleen;
lane 7, kidney; lane 8, liver; lane 9, small intestine; lane 10,
placenta; lane 11, lung; lane 12, peripheral blood lymphocytes.
B. An adult human brain northern blot (Clontech) was hybridized with a 32P-labeled EGLN3 cDNA probe. Lane 1, cerebellum; lane 2, cerebral cortex; lane 3, medulla; lane 4, spinal cord;
lane 5, occipital pole; lane 6, frontal lobe; lane 7, temporal lobe;
lane 8, putamen; lane 9, amygdala; lane 10, caudate nucleus;
lane 11, corpus callosum; lane 12, hippocampus; lane 13, whole
brain; lane 14, substantia nigra; lane 15, thalamus. The two blots
(lanes 1−8 and 9−15) were hybridized and washed simultaneously and exposed to film for 15 h with an intensifying screen.
Subsequent blotting with an actin cDNA probe indicated approximately equal RNA loading across the lanes in the blots in
(A) and (B).
histidine-1-carboxylate motif (H-X-D/E…H) involved in
chelating nonheme iron. Iron(II) is critical for activating O2
and as a template for the orderly binding of reactants. In
dioxygenase-catalyzed reactions both oxygen atoms of an
O2 molecule are incorporated into substrate. The conserved
C-terminal region of EGL-9 and EGLN3 showed greatest
similarity to the catalytic domain of mammalian prolyl-4hydroxylase (Aravind and Koonin, 2001) (Fig. 3). In the
reaction catalyzed by prolyl-4-hydroxylase, one oxygen
atom is added to a peptidyl proline to form hydroxyproline
while the other is used in a coupled decarboxylation reaction that converts 2-oxoglutarate to succinate. In addition to
requirements for O2, iron(II), and 2-oxoglutarate, ascorbate
(although not stoichiometrically consumed) is necessary for
maximal prolyl hydroxylase activity (reviewed by
Kivirikko and Pihlajaniemi, 1998).
At the same time that EGL-9 and related proteins were
suggested to be novel prolyl hydroxylases, researchers
investigating the oxygen-dependent regulation of HIF
were searching for an enzyme that could catalyze hydroxylation of HIF α-subunits at a critical proline residue
(Mole et al., 2001). This proline (Pro564 in HIF-1α) lies
within the C-terminal half of a previously characterized
oxygen-dependent degradation domain, abbreviated
‘CODD’. Hydroxylation of Pro564 in HIF-1α had just
been shown to mediate its interaction with the VHL protein present in an E3 ubiquitin ligase complex, resulting
in its rapid ubiquitination and proteasomal degradation
(Ivan et al., 2001; Jaakkola et al., 2001; Yu et al., 2001)
(Fig. 4). In these studies, the hydroxyproline-dependent
association of HIF-1α with pVHL was inhibited by hypoxia and by various other agents including iron chelators,
cobalt(II), and structural analogs of 2-oxoglutarate. These
compounds had been known for some time to mimic hypoxia and more recently to stabilize HIF-1α under normoxic conditions.
Armed with HIF and pVHL homologs in C. elegans
and the realization that egl-9 might encode a novel prolyl
hydroxylase, Ratcliffe and colleagues (Epstein et al.,
2001) carried out a series of genetic and biochemical experiments that elegantly established EGL-9 as a HIF prolyl hydroxylase in C. elegans. Analysis of endogenous
HIF protein levels clearly demonstrated a defect in the
ability of C. elegans strains with inactivating mutations in
egl-9 to downregulate HIF expression under ambient O2
conditions. Inactivating mutations in the C. elegans collagen-prolyl-hydroxylase genes or in other potential HIFregulating genes did not affect the oxygen-mediated destabilization of HIF. Thus, loss of EGL-9 function specifically correlated with a failure to destabilize HIF protein in
normoxic animals. Additional experiments showed that a
recombinant EGL-9 protein (residues 359−723) could
hydroxylate the conserved proline in C. elegans HIF in an
O2, iron, and 2-oxoglutarate dependent manner. Interestingly, the four egl-9 inactivating alleles that resulted in
constitutive HIF expression in normoxia were the same
loss-of-function alleles found by Darby et al. (1999) to
confer resistance to paralysis induced by Pseudomonas.
Mammalian EGLN proteins function as HIF
prolyl hydroxylases
Along with the discovery that EGL-9 functions as a HIF
prolyl hydroxylase, Epstein et al. (2001) and Buick and
Robert S. Freeman et al.
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Fig. 3. The iron- and 2-oxoglutarate-dependent dioxygenase catalytic domain in EGLN family members. The amino acid sequence in
the catalytic domains of rat SM20, three human EGLN proteins, and C. elegans EGL-9 are aligned with the corresponding domain in
human prolyl-4-hydroxylase (P4H). The predicted secondary structure is indicated at the top of the alignment: H = α helix; E = β
strand. Asterisks identify the conserved 2-histidine-1-carboxylate motif involved in coordinating iron(II) in the active site. Vertical
bars indicate residues conserved in all six proteins.
Fig. 4. Regulation of HIF-1α stability by oxygen-sensitive proline hydroxylation. Under normal oxygen concentrations, HIF-1α is
hydroxylated at Pro402 and Pro564 by one or more HIF prolyl hydroxylases. The reaction requires O2 and iron(II) and utilizes peptidyl
proline and 2-oxoglutarate as co-substrates; ascorbate is a cofactor needed for maximal activity. Hydroxyproline, succinate, and CO2
are produced. Proline hydroxylation of HIF-1α increases its affinity for pVHL, which targets HIF-1α for ubiquitination and degradation. Under hypoxic conditions, prolyl hydroxylase activity decreases and non-hydroxylated HIF-1α is free to accumulate in the nucleus where it associates with the HIF β-subunit (ARNT) to activate transcription of target genes such as VEGF.
McKnight (2001) used in vitro pull-down assays to show
that all three EGLN proteins could enhance the pVHLbinding activity of HIF-1α or synthetic HIF-1α peptides
centered around Pro564. As expected, peptides mutated at
Pro564 and incubated with EGLN proteins failed to bind
pVHL, although this was not always the case when fulllength HIF-1α was used as the substrate (see below for a
discussion of a second proline hydroxylation site in HIF1α). Importantly, the ability of a recombinant EGLN protein (in this case EGLN2) to enhance the HIF-1α-pVHL
association showed a similar graded response to changes
in O2 tension (0–21%) as the endogenous activity present
in RCC4 renal carcinoma cell extracts (Epstein et al.,
2001). In these and other studies (Ivan et al., 2002) the
EGLN-dependent effects were shown to be inhibited by
iron chelators, CoCl2, the 2-oxoglutarate analog N-oxalylglycine, and several newly described prolyl hydroxylase
inhibitors. These compounds inhibit prolyl hydroxylases
through distinct mechanisms; however, with the possible
exception of the most recent compounds, their use is lim-
ited due to lack of specificity (Günzler and Weidmann,
1998). Mutations in the critical iron-binding residues in
the 2-histidine-1-carboxylate motif block the ability of
EGLN proteins to increase the HIF-pVHL interaction.
Purified recombinant EGLN proteins have been shown to
directly hydroxylate Pro564 in synthetic peptide substrates
confirming that the activity is intrinsic to the EGLNs
(Bruick and McKnight, 2001; Epstein et al., 2001; Ivan et
al., 2002).
HIF-dependent transcriptional transactivation assays
done in HEK293 and COS-1 cells under normoxic cell
culture conditions (i.e., 21% O2) have shown that coexpression of either EGLN1, EGLN2, or EGLN3 together
with wild-type HIF-1α significantly reduces transactivation of the HIF response elements (HREs) driving expression of the reporter genes (Bruick and McKnight, 2001;
Huang et al., 2002). The ability of over-expressed EGLN
proteins to repress HRE activation by over-expressed
HIF-1α depended on an intact iron-chelating center in the
enzyme and was inhibited when cells were cultured at O2
8
Prolyl Hydroxylases and Signal Transduction
/
< 1%2. Decreased HRE activity in normoxic cells overexpressing each of the EGLN proteins is accompanied by
a reduction in the level of ectopic HIF-1α protein (Huang
et al., 2002). This result, along with the simple observation that ectopically expressed HIF-1α can be detected in
normoxic cells (in contrast to endogenous HIF-1α), is
consistent with the notion that the endogenous prolyl hydroxylases are rate-limiting for destabilizing HIF-1α
when O2 is not limiting. Together these results are consistent with a model whereby HIF-1α levels are kept low
under normoxic conditions through the action of HIF prolyl hydroxylases but then increase dramatically during
hypoxia when these same enzymes are inhibited by inadequate O2. Oddly enough, endogenous HIF-1α protein
levels are not substantially increased by CoCl2 treatment
or even hypoxia (O2 < 1%) in cells over-expressing
EGLN1 or EGLN3 (Cioffi et al., 2003; Metzen et al.,
2002), raising the possibility that such conditions do not
completely inhibit these enzymes when over-expressed.
Consequently, experiments involving over-expression
should be interpreted with caution due to the possibility
that a portion of the over-expressed enzyme or its substrate might escape its normal regulatory controls.
Reports published in recent months raise the possibility
that HIF prolyl hydroxylases are regulated in ways other
than just changes in O2 tension. An endogenous HIF prolyl hydroxylase activity in Hep3B cells has been partially
purified using the CODD of HIF-1α as substrate and a
pVHL pull-down assay as an indirect measure of Pro564
hydroxylation (Wang et al., 2002). The prolyl hydroxylase activity was suppressed by CoCl2, iron chelators, or
low levels of O2 as expected, but the suppression was reversed by nitric oxide (NO) donors such as sodium nitroprusside. NO had previously been implicated in the inactivation of HIF but the mechanism was unknown (see
Semenza, 2001). Because treatment with NO donors led
to reduced levels of endogenous HIF-1α protein and activity under conditions that would otherwise inhibit the
prolyl hydroxylase and stabilize HIF (i.e., iron chelation
or very low O2), NO was postulated to directly activate
the prolyl hydroxylase (Wang et al., 2002). These results
suggest that NO may function in a novel mechanism for
activating prolyl hydroxylases at suboptimal O2 levels.
Like NO, the immunosuppressant cyclosporin A (CsA)
was recently shown to inhibit hypoxia-induced stabilization of endogenous HIF-1α (D’Angelo et al., 2003). Using an assay for 2-oxoglutarate-dependent dioxygenases,
2
Note that the O2 levels achieved in various studies may not be
directly comparable as it is difficult to obtain reproducibly low
O2 levels, especially < 1%, in cell culture experiments. In addition, the indicated O2 levels are not always maintained during
the preparation of extracts and in reactions. This is particularly
important since HIF-1α can undergo extensive degradation
within minutes of re-oxygenation.
a Pro564-dependent activity was identified in a ‘light mitochondria’ fraction from rat kidney that was enhanced by
treatment with CsA. Incubating recombinant CODD with
the light mitochondria fraction resulted in binding to
[35S]-labeled pVHL that too was enhanced by CsA. Mutation of Pro564 in the CODD to alanine prevented the interaction with pVHL and its augmentation by CsA, providing evidence that CsA was acting via the prolyl hydroxylase. Although the molecular nature of the prolyl
hydroxylase activity in the rat kidney extracts is unknown,
the localization of SM-20 to mitochondria makes it an
attractive candidate. The studies described above suggest
that both CsA and NO may act to suppress hypoxiainduced responses by maintaining or enhancing the activity of HIF prolyl hydroxylases under conditions that would
otherwise be inhibitory. Future experiments will obviously be aimed at identifying the prolyl hydroxylases implicated in these studies.
In addition to Pro564, a second conserved proline
(Pro402) located in the N-terminal half of the oxygendependent degradation domain (NODD) of HIF-1α has
been found to be important for the interaction with pVHL
(Masson et al., 2001). By mutating these residues either
individually or in tandem, Epstein et al. (2001) showed
that the two prolines are differentially utilized, at least in
vitro, by the EGLN proteins. All three human EGLN proteins as well as rat SM-20 could hydroxylate HIF-1α mutated at Pro402 (as assessed by its ability to bind pVHL).
In contrast, only EGLN1 and EGLN2 could hydroxylate
HIF-1α mutated at Pro564. None of the EGLN proteins
could enhance the affinity of HIF-1α for pVHL when
both prolines were mutated, suggesting that these are the
only sites hydroxylated by the EGLNs - or more precisely,
the only hydroxylated residues permissive for pVHL
binding. The presence of two distinct sites for proline
hydroxylation may help explain, at least partly, the existence of multiple EGLN prolyl hydroxylases. More than
one EGLN enzyme might also be necessary in order to
modify the different HIF α-subunits. HIF-2α, which is
also stabilized by hypoxia, contains prolines equivalent to
the two hydroxylated residues in HIF-1α. In each case,
the hydroxylated proline is contained within a Leu-X-XLeu-Ala-Pro motif (Ivan et al., 2001; Jaakkola et al.,
2001). Recently, Chan et al. (2002) described an antibody
that preferentially binds HIF-1α when it is hydroxylated
at Pro564. This exciting reagent, and future antibodies designed to recognize other hydroxyproline-containing sequences, may prove as useful for studying pathways involved in HIF regulation as phosphorylation site-specific
antibodies have in other aspects of signal transduction.
The development of this antibody is remarkable for another reason; it may indicate that similar hydroxyprolinetargeting antibodies designed with less sequence specificity towards HIF could be used in the search for novel substrates of EGLN prolyl hydroxylases.
Robert S. Freeman et al.
/
Three-dimensional structure determinations of Pro564containing peptides bound to the pVHL/E3 ligase complex reveal a single pocket in pVHL for binding hydroxyproline (Hon et al., 2002; Min et al., 2002). The
pocket is lined by five amino acids highly conserved from
C. elegans to humans, all of which are affected by missense mutations in VHL disease, an autosomal dominant
cancer syndrome that predisposes afflicted individuals to
a variety of highly vascularized tumors. Comparisons
between Pro402 and Pro564 peptides suggest that both hydroxylation sites in HIF-1α bind the same hydroxyprolinebinding pocket in pVHL (Hon et al., 2002), providing a
possible explanation for why hydroxylation at just one
site may under certain conditions be sufficient for destabilizing HIF-1α (Pereira et al., 2003).
Results from pVHL pull-down assays using HIF fragments and recombinant prolyl hydroxylases made in insect cells suggest a tentative order of reactivity with respect to hydroxylation of Pro564 - EGLN1 > EGLN3 >
EGLN2 (Huang et al., 2002). The activities of these enzymes with respect to Pro402 and, even more importantly,
their activities in vivo remain to be determined. Nonetheless, the relatively low activity of EGLN2 is interesting in
light of other experiments showing that the EGLN1 and
EGLN3 genes, but not the EGLN2 gene, are transcriptionally upregulated in various cells exposed to hypoxia
(Cioffi et al., 2003; Epstein et al., 2001; Metzen et al.,
2002). Thus, EGLN1 and EGLN3 may themselves be HIF
target genes that are upregulated in response to hypoxia.
Increased EGLN expression in cells while they are still
hypoxic could be part of a negative feedback mechanism
for the rapid hydroxylation and degradation of HIF-1α
when O2 supplies are restored.
The ability of different EGLN proteins to hydroxylate
HIF-1α in vivo is likely to be a function of their intracellular location as well as the location of HIF-1α. Since HIF1α can be found in both the cytoplasm and the nucleus, and
may even be regulated by mitochondrial function (Agani et
al., 2000), all of the EGLN proteins could be involved in
regulating HIF at distinct intracellular sites. For example,
treatment of HeLa cells with the prolyl hydroxylase inhibitor dimethyloxalylglycine blocks not only HIF-1α degradation upon reoxygenation, but also its export from the nucleus (Groulx and Lee, 2002). Thus degradation of the nuclear pool of HIF-1α in reoxygenated cells appears to require a nuclear prolyl hydroxylase, possibly EGLN2 or
EGLN3.
Evidence that EGLNs are the endogenous HIF hydroxylases also comes from experiments using novel
small molecule prolyl hydroxylase inhibitors and polypeptides derived from the hydroxylation sites in HIF-1α
(Huang et al., 2002; Ivan et al., 2002; Willam et al.,
2002). Treatment of a variety of normal and tumor cells
with a novel prolyl hydroxylase inhibitor (FG0041) at
concentrations that inhibit EGLN1 in vitro led to stabili-
9
zation of endogenous HIF-1α protein levels and increased
synthesis of VEGF, a well-characterized HIF target (Ivan
et al., 2002). In other studies, over-expression of the HIF1α NODD (containing Pro402) or CODD (containing
Pro564) was found to enhance HIF-1α protein levels,
transactivation activity, and expression of HIF target
genes (Huang et al., 2002; Willam et al., 2002). Promising results from in vivo angiogenesis assays suggest that
introduction of these HIF-1α fragments into mice can
increase blood vessel growth and VEGF and glucose
transporter-1 immunostaining, apparently as the result of
HIF activation (Willam et al., 2002). Over-expression of
these domains could block HIF-1α degradation by competitive inhibition with respect to either the prolyl hydroxylase or, maybe more likely, the HIF-binding pocket
in pVHL. Ultimately, direct proof that an EGLN protein
functions as an endogenous HIF prolyl hydroxylase will
come from experiments in which the EGLN genes are
individually, or perhaps collectively, inactivated. Nonetheless, recent demonstrations that inactivation of egl-9 in
C. elegans and the EGLN homolog in Drosophila (Bruick
and McKnight, 2001; Lavista-Llanos et al., 2002) results
in normoxic stabilization of HIF strongly suggest that at
least one member of the mammalian EGLN family serves
a similar function.
Although abundant data now implicate the EGLN proteins as HIF prolyl hydroxylases, the relative importance
of these enzymes as critical regulators of HIF stability in
vivo remains to be verified. Sensitive assays that directly
measure EGLN prolyl hydroxylase activity will be needed
in order to fully address this issue. The ability to directly
measure EGLN activity is even more critical given recent
reports that HIF’s ability to transactivate gene expression
and its stabilization by hypoxia are regulated by factors in
addition to proline hydroxylation. HIF-1α was recently
shown to undergo an additional hydroxylation event, this
time at an asparagine residue (Asn803) (Lando et al., 2002).
Like proline hydroxylation, hydroxylation of asparagine
is catalyzed by an iron- and 2-oxoglutarate-dependent
dioxygenase, but in this case an asparaginyl hydroxylase
that was originally identified as ‘Factor Inhibiting HIF-1’,
or FIH-1 (Lando et al., 2002; Mahon et al., 2001). This
reaction, which is also sensitive to O2, regulates the interaction between HIF-1α and the nuclear transcriptional coactivator CBP/p300, thus influencing HIF transcription
factor activity. A third post-translational mechanism for
regulating HIF-1α, also discovered within the last year,
involves acetylation at a lysine residue in the oxygendependent degradation domain (Jeong et al., 2002). Acetylation of HIF-1α by a cytosolic enzyme called ARD1
enhanced its interaction with pVHL resulting in increased
ubiquitination and ultimately decreased HIF stability. Mutation of the target lysine (Lys532) increased the stability
of the over-expressed HIF-1α under normoxic conditions.
Determining the relative contributions of proline hy-
10
Prolyl Hydroxylases and Signal Transduction
/
droxylation, asparagine hydroxylation, and lysine acetylation for the overall activity of HIF will undoubtedly take
some time to resolve and may well depend on cell type,
O2 levels, and other factors that influence HIF function.
Conclusion
HIF function is clearly important for a proper physiologic
response to changes in O2 tension through its ability to
regulate genes involved in glycolysis, angiogenesis,
erythropoiesis, and cell survival (Maxwell and Ratcliffe,
2002). Moreover, it is highly likely that one or more
EGLN proteins will have a critical role in regulating HIF
function in oxygen-sensitive tissues, and perhaps in response to other stimuli under non-hypoxic conditions.
But do all three EGLN proteins function exclusively as
HIF prolyl hydroxylases? Clearly O2 plays an important
role in regulating EGLN function but other factors
(growth factors, neurotrophic factors, CsA, NO) also appear to modulate the expression or activity of EGLN proteins. Unpublished data from our laboratory showing that
prolyl hydroxylase inhibitors can protect neurons from
cell death under normoxic conditions support a role for
EGLN proteins in neuronal apoptosis that may or may not
involve HIF. A recent report that the large subunit (Rpb1)
of RNA polymerase II can bind pVHL in a hydroxyproline-dependent manner hints that additional substrates
for EGLN prolyl hydroxylation exist and will soon be
identified (Kuznetsova et al., 2003). Even in C. elegans
which has only one EGLN gene, the observation that one
of four egl-9 mutants defective in HIF regulation and resistant to Pseudomonas toxicity does not appear to have
an egg-laying defect (Darby et al., 1999), suggests that at
least one egl-9-associated phenotype need not involve the
HIF pathway. Data implicating EGLN proteins in such
disparate processes as O2 sensing, neuronal cell death,
cell proliferation, and muscle cell differentiation in
mammals, as well as egg laying and toxin-induced paralysis in C. elegans, point to the realization that prolyl hydroxylation as a signal transduction mechanism will soon
extend well beyond the interaction between HIF and
pVHL.
Acknowledgments We thank current and past members of the
Freeman laboratory for their help in formulating the ideas expressed in this review. R. S. Freeman acknowledges support
from the National Institutes of Health grants NS34400 and
NS42224.
References
Agani, F. H., Pichiule, P., Chavez, J. C., and LaManna, J. C.
(2000) The role of mitochondria in the regulation of hypoxiainducible factor 1 expression during hypoxia. J. Biol. Chem.
275, 35863−35867.
Aravind, L. and Koonin, E. V. (2001) The DNA-repair protein
AlkB, EGL-9, and leprecan define new families of 2oxoglutarate- and iron-dependent dioxygenases. Genome
Biol. 2, 7.1−7.8.
Bruick, R. K. and McKnight, S. L. (2001) A conserved family
of prolyl-4-hydroxylases that modify HIF. Science 294,
1337−1340.
Chan, D. A., Sutphin, P. D., Denko, N. C., and Giaccia, A. J.
(2002) Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1α. J. Biol. Chem. 277, 40112−
40117.
Cioffi, C. L., Liu, X. Q., Kosinski, P. A., Garay, M., and Bowen,
B. R. (2003) Differential regulation of HIF-1α prolyl-4hydroxylase genes by hypoxia in human cardiovascular cells.
Biochem. Biophys Res. Commun. 303, 947−953.
Cockman, M. E., Masson, N., Mole, D. R., Jaakkola, P., Chang,
G. W., Clifford, S. C., Maher, E. R., Pugh, C. W., Ratcliffe, P.
J., and Maxwell, P. H. (2000) Hypoxia inducible factor-α
binding and ubiquitylation by the von Hippel-Lindau tumor
suppressor protein. J. Biol. Chem. 275, 25733−25741.
Conaway, R. C. and Conaway, J. W. (2002) The Von HippelLindau tumor suppressor complex and regulation of hypoxiainducible transcription. Adv. Cancer Res. 85, 1−12.
D’Angelo, G., Duplan, E., Vigne, P., and Frelin, C. (2003) Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1α Pro-564 hydroxylation. J. Biol.
Chem. 278, 15406−15411.
Darby, C., Cosma, C. L., Thomas, J. H., and Manoil, C. (1999)
Lethal paralysis of Caenorhabditis elegans by Pseudomonas
aeruginosa. Proc. Natl. Acad. Sci. USA 96, 15202−15207.
Ebinu, J. O. and Yankner, B. A. (2002) A RIP tide in neuronal
signal transduction. Neuron 34, 499−502.
Epstein, A. C., Gleadle, J. M., McNeill, L. A., Hewitson, K. S.,
O’Rourke, J., Mole, D. R., Mukherji, M., Metzen, E., Wilson,
M. I., Dhanda, A., Tian, Y. M., Masson, N., Hamilton, D. L.,
Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh,
C. W., Schofield, C. J., and Ratcliffe, P. J. (2001) C. elegans
EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107,
43−54.
Erez, N., Milyavsky, M., Goldfinger, N., Peles, E., Gudkov, A.
V., and Rotter, V. (2002) Falkor, a novel cell growth regulator
isolated by a functional genetic screen. Oncogene 21, 6713−
6721.
Freeman, R. S., Burch, R. L., Crowder, R. J., Lomb, D. J.,
Schoell, M. C., Straub, J. A., and Xie, L. (2003) NGF
deprivation-induced gene expression: after ten years, where
do we stand? Prog. Brain Res. (in press).
Gallagher, L. A. and Manoil, C. (2001) Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J. Bacteriol. 183, 6207−6214.
Graves, J. D. and Krebs, E. G. (1999) Protein phosphorylation
and signal transduction. Pharmacol. Ther. 82, 111−121.
Groulx, I. and Lee, S. (2002) Oxygen-dependent ubiquitination
and degradation of hypoxia-inducible factor requires nuclearcytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein. Mol. Cell. Biol. 22, 5319−5336.
Robert S. Freeman et al.
11
/
Günzler, V. and Weidmann, K. (1998) Prolyl 4-hydroxylase
inhibitors; in Prolyl hydroxylase, Protein Disulfide Isomerase,
and Other Structurally Related Proteins, Guzman, N. A.
(ed.), pp. 65−95, Marcel Dekker, New York.
Hon, W. C., Wilson, M. I., Harlos, K., Claridge, T. D., Schofield,
C. J., Pugh, C. W., Maxwell, P. H., Ratcliffe, P. J., Stuart, D.
I., and Jones, E. Y. (2002) Structural basis for the recognition
of hydroxyproline in HIF-1α by pVHL. Nature 417, 975−978.
Huang, J., Zhao, Q., Mooney, S. M., and Lee, F. S. (2002) Sequence determinants in hypoxia-inducible factor-1α for hydroxylation by the prolyl hydroxylases PHD1, PHD2, and
PHD3. J. Biol. Chem. 277, 39792−39800.
Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M.,
Salic, A., Asara, J. M., Lane, W. S., and Kaelin, W. G. Jr.
(2001) HIFα targeted for VHL-mediated destruction by
proline hydroxylation: implications for O2 sensing. Science
292, 464−468.
Ivan, M., Haberberger, T., Gervasi, D. C., Michelson, K. S.,
Gunzler, V., Kondo, K., Yang, H., Sorokina, I., Conaway, R.
C., Conaway, J. W., and Kaelin, W. G. Jr. (2002) Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor.
Proc. Natl. Acad. Sci. USA 99, 13459−13464.
Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J.,
Gaskell, S. J., Kriegsheim, A., Hebestreit, H. F., Mukherji,
M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and
Ratcliffe, P. J. (2001) Targeting of HIF-α to the von HippelLindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468−472.
Jeong, J. W., Bae, M. K., Ahn, M. Y., Kim, S. H., Sohn, T. K.,
Bae, M. H., Yoo, M. A., Song, E. J., Lee, K. J., and Kim, K.
W. (2002) Regulation and destabilization of HIF-1α by
ARD1-mediated acetylation. Cell 111, 709−720.
Kivirikko, K. I. and Pihlajaniemi, T. (1998) Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4hydroxylases; in Advances in Enzymology & Related Areas
of Molecular Biology, Purich, D. L. (ed.), Vol. 72, pp. 325−
398, Wiley, New York.
Kuznetsova, A. V., Meller, J., Schnell, P. O., Nash, J. A.,
Ignacak, M. L., Sanchez, Y., Conaway, J. W., Conaway, R. C.,
and Czyzyk-Krzeska, M. F. (2003) von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it
for ubiquitination. Proc. Natl. Acad. Sci. USA 100, 2706−
2711.
Lando, D., Peet, D. J., Whelan, D. A., Gorman, J. J., and Whitelaw, M. L. (2002) Asparagine hydroxylation of the HIF
transactivation domain a hypoxic switch. Science 295, 858−
861.
Lavista-Llanos, S., Centanin, L., Irisarri, M., Russo, D. M.,
Gleadle, J. M., Bocca, S. N., Muzzopappa, M., Ratcliffe, P. J.,
and Wappner, P. (2002) Control of the hypoxic response in
Drosophila melanogaster by the basic helix-loop-helix PAS
protein similar. Mol. Cell. Biol. 22, 6842−6853.
Lieb, M. E., Menzies, K., Moschella, M. C., Ni, R., and Taubman, M. B. (2002) Mammalian EGLN genes have distinct
patterns of mRNA expression and regulation. Biochem. Cell
Biol. 80, 421−426.
Lipscomb, E. A., Sarmiere, P. D., Crowder, R. J., and Freeman,
R. S. (1999) Expression of the SM-20 gene promotes death
in nerve growth factor-dependent sympathetic neurons. J.
Neurochem. 73, 429−432.
Lipscomb, E. A., Sarmiere, P. D., and Freeman, R. S. (2001)
SM-20 is a novel mitochondrial protein that causes caspasedependent cell death in nerve growth factor-dependent neurons. J. Biol. Chem. 276, 5085−5092.
Madden, S. L., Galella, E. A., Riley, D., Bertelsen, A. H., and
Beaudry, G. A. (1996) Induction of cell growth regulatory
genes by p53. Cancer Res. 56, 5384−5390.
Magnaghi-Jaulin, L., Ait-Si-Ali, S., and Harel-Bellan, A. (1999)
Histone acetylation in signal transduction by growth
regulatory signals. Semin. Cell Dev. Biol. 10, 197−203.
Mahon, P. C., Hirota, K., and Semenza, G. L. (2001) FIH-1: a
novel protein that interacts with HIF-1α and VHL to mediate
repression of HIF-1 transcriptional activity. Genes Dev. 15,
2675−2686.
Masson, N., Willam, C., Maxwell, P. H., Pugh, C. W., and
Ratcliffe, P. J. (2001) Independent function of two destruction domains in hypoxia-inducible factor-α chains activated
by prolyl hydroxylation. EMBO J. 20, 5197−5206.
Maxwell, P. H. and Ratcliffe, P. J. (2002) Oxygen sensors and
angiogenesis. Semin. Cell Dev. Biol. 13, 29−37.
Maxwell, P. H., Wiesener, M. S., Chang, G. W., Clifford, S. C.,
Vaux, E. C., Cockman, M. E., Wykoff, C. C., Pugh, C. W.,
Maher, E. R., and Ratcliffe, P. J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399, 271−275.
Metzen, E., Berchner-Pfannschmidt, U., Stengel, P., Marxsen, J.
H., Stolze, I., Klinger, M., Huang, W. Q., Wotzlaw, C., Hellwig-Burgel, T., Jelkmann, W., Acker, H., and Fandrey, J.
(2002) Intracellular localisation of human HIF-1α hydroxylases: implications for oxygen sensing. J. Cell Sci. 116, 1319−
1326.
Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G. Jr., and
Pavletich, N. P. (2002) Structure of an HIF-1α/pVHL complex: hydroxyproline recognition in signaling. Science 296,
1886−1889.
Mole, D. R., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J.
(2001) Regulation of HIF by the von Hippel-Lindau tumour
suppressor: implications for cellular oxygen sensing. IUBMB
Life 52, 43−47.
Moschella, M. C., Menzies, K., Tsao, L., Lieb, M. A., Kohtz, J.
D., Kohtz, D. S., and Taubman, M. B. (1999) SM-20 is a
novel growth factor-responsive gene regulated during skeletal muscle development and differentiation. Gene Exp. 8, 59−
66.
Pereira, T., Zheng, X. W., Ruast, J. L., Tanimoto, K., and Poellinger, L. (2003) Identification of residues critical for regulation of protein stability and the transactivation function of
the hypoxia-inducible factor-1α by the von Hippel-Lindau
tumor suppressor gene product. J. Biol. Chem. 278, 6816−
6823.
Peterkofsky, B. and Udenfriend, S. (1965) Enzymatic hydroxylation of proline in microsomal polypeptide leading to formation of collagen. Proc. Natl. Acad. Sci. USA 53, 335−342.
Resh, M. D. (1996) Regulation of cellular signalling by fatty
acid acylation and prenylation of signal transduction proteins.
Cell Signal. 8, 403−412.
Semenza, G. L. (1999) Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu. Rev. Cell Dev. Biol.
12
Prolyl Hydroxylases and Signal Transduction
/
15, 551−578.
Semenza, G. L. (2001) HIF-1 and mechanisms of hypoxia sensing. Curr. Opin. Cell Biol. 13, 167−171.
Seth, P., Krop, I., Porter, D., and Polyak, K. (2002) Novel estrogen and tamoxifen induced genes identified by SAGE (serial
analysis of gene expression). Oncogene 21, 836−843.
Straub, J. A., Lipscomb, E. A., Yoshida, E. S., and Freeman, R.
S. (2003) Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c
in the cytosol, and caspase-dependent cell death. J. Neurochem. 85, 318−328.
Taylor, M. S. (2001) Characterization and comparative analysis
of the EGLN gene family. Gene 275, 125−132.
Trent, C., Tsuing, N., and Horvitz, H. R. (1983) Egg-laying
defective mutants of the nematode Caenorhabditis elegans.
Genetics 104, 619−647.
Wang, F., Sekine, H., Kikuchi, Y., Takasaki, C., Miura, C.,
Heiwa, O., Shuin, T., Fujii-Kuriyama, Y., and Sogawa, K.
(2002) HIF-1α prolyl hydroxylase: molecular target of nitric
oxide in the hypoxic signal transduction pathway. Biochem.
Biophys. Res. Commun. 295, 657−662.
Wax, S. D., Rosenfield, C. L., and Taubman, M. B. (1994) Iden-
tification of a novel growth factor-responsive gene in vascular smooth muscle cells. J. Biol. Chem. 269, 13041−13047.
Wax, S. D., Tsao, L., Lieb, M. E., Fallon, J. T., and Taubman, M.
B. (1996) SM-20 is a novel 40-kd protein whose expression
in the arterial wall is restricted to smooth muscle. Lab. Invest.
74, 797−808.
Willam, C., Masson, N., Tian, Y. M., Mahmood, S. A., Wilson,
M. I., Bicknell, R., Eckardt, K. U., Maxwell, P. H., Ratcliffe,
P. J., and Pugh, C. W. (2002) Peptide blockade of HIF-α degradation modulates cellular metabolism and angiogenesis.
Proc. Natl. Acad. Sci. USA 99, 10423−10428.
Wolf, G., Harendza, S., Schroeder, R., Wenzel, U., Zahner, G.,
Butzmann, U., Freeman, R. S., and Stahl, R. A. (2002) Angiotensin II’s antiproliferative effects mediated through AT2receptors depend on down-regulation of SM-20. Lab. Invest.
82, 1305−1317.
Yang, Y. and Yu, X. (2003) Regulation of apoptosis: the ubiquitous way. FASEB J. 17, 790−799.
Yu, F., White, S. B., Zhao, Q., and Lee, F. S. (2001) HIF-1α
binding to VHL is regulated by stimulus-sensitive proline
hydroxylation. Proc. Natl. Acad. Sci. USA 98, 9630−9635.