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1.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
2.
3.
Jacamo R Sinnett-Smith J Rey O Waldron RT Rozengurt E 《The Journal of biological chemistry》2008,283(19):12877-12887
Protein kinase D (PKD) is a serine/threonine protein kinase rapidly
activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C
(PKC)-dependent pathway. Recently, PKD has been implicated in the regulation
of long term cellular activities, but little is known about the mechanism(s)
of sustained PKD activation. Here, we show that cell treatment with the
preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid
(1–5-min) PKD activation induced by bombesin stimulation, but this
inhibition was greatly diminished at later times of bombesin stimulation
(e.g. 45 min). These results imply that GPCR-induced PKD activation
is mediated by early PKC-dependent and late PKC-independent mechanisms.
Western blot analysis with site-specific antibodies that detect the
phosphorylated state of the activation loop residues Ser744 and
Ser748 revealed striking PKC-independent phosphorylation of
Ser748 as well as Ser744 phosphorylation that remained
predominantly but not completely PKC-dependent at later times of bombesin or
vasopressin stimulation (20–90 min). To determine the mechanisms
involved, we examined activation loop phosphorylation in a set of PKD mutants,
including kinase-deficient, constitutively activated, and PKD forms in which
the activation loop residues were substituted for alanine. Our results show
that PKC-dependent phosphorylation of the activation loop Ser744
and Ser748 is the primary mechanism involved in early phase PKD
activation, whereas PKD autophosphorylation on Ser748 is a major
mechanism contributing to the late phase of PKD activation occurring in cells
stimulated by GPCR agonists. The present studies identify a novel mechanism
induced by GPCR activation that leads to late, PKC-independent PKD
activation.A rapid increase in the synthesis of lipid-derived second messengers with
subsequent activation of protein phosphorylation cascades has emerged as a
fundamental signal transduction mechanism triggered by multiple extracellular
stimuli, including hormones, neurotransmitters, chemokines, and growth factors
(1). Many of these agonists
bind to G protein-coupled receptors
(GPCRs),4 activate
heterotrimeric G proteins and stimulate isoforms of the phospholipase C
family, including β, γ, δ, and ε (reviewed in Refs.
1 and
2). Activated phospholipase Cs
catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce
the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG).
Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular
stores (3,
4) whereas DAG directly
activates the classic (α, β, and γ) and novel (δ,
ε, η, and θ) isoforms of PKC
(5–7).
Although it is increasingly recognized that each PKC isozyme has specific
functions in vivo
(5–8),
the mechanisms by which PKC-mediated signals are propagated to critical
downstream targets remain incompletely defined.PKD, also known initially as PKCμ
(9,
10), and two recently
identified serine protein kinases termed PKD2
(11) and PKCν/PKD3
(12,
13), which are similar in
overall structure and primary amino acid sequence to PKD
(14), constitute a new protein
kinase family within the Ca2+/calmodulin-dependent protein kinase
group (15) and separate from
the previously identified PKCs
(14). Salient features of PKD
structure include an N-terminal regulatory region containing a tandem repeat
of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain)
that confers high affinity binding to phorbol esters and DAG
(9,
16,
17), followed by a pleckstrin
homology (PH) domain that negatively regulates catalytic activity
(18,
19). The C-terminal region of
the PKDs contains its catalytic domain, which is distantly related to
Ca2+-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity
maintained by the N-terminal domain, which represses the catalytic activity of
the enzyme by autoinhibition. Consistent with this model, deletions or single
amino acid substitutions in the PH domain result in constitutive kinase
activity
(18–20).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(21). In response to cellular
stimuli, PKD is converted from a low activity form into a persistently active
form that is retained during isolation from cells, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(21,
22). PKD activation has been
demonstrated in response to engagement of specific GPCRs either by regulatory
peptides
(23–30)
or lysophosphatidic acid (27,
31,
32); signaling through
Gq, G12, Gi, and Rho
(27,
31–34);
activation of receptor tyrosine kinases, such as the platelet-derived growth
factor receptor (23,
35,
36); cross-linking of B-cell
receptor and T-cell receptor in B and T lymphocytes, respectively
(37–40);
and oxidative stress
(41–44).Throughout these studies, multiple lines of evidence indicated that PKC
activity is necessary for rapid PKD activation within intact cells. For
example, rapid PKD activation was selectively and potently blocked by cell
treatment with preferential PKC inhibitors (e.g. GF 109203X or
Gö 6983) that do not directly inhibit PKD catalytic activity
(21,
22), implying that PKD
activation in intact cells is mediated, directly or indirectly, through PKCs.
In line with this conclusion, cotransfection of PKD with active mutant forms
of “novel” PKCs (PKCs δ, ε, η, and θ)
resulted in robust PKD activation in the absence of cell stimulation
(21,
44–46).
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
in response to multiple GPCR agonists in a broad range of cell types,
including normal and cancer cells (reviewed in Ref.
14). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as the activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation (reviewed in Ref.
14). Collectively, these
findings demonstrated the existence of rapidly activated PKC-PKD protein
kinase cascade(s) and raised the possibility that some PKC-dependent
biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular
activities and processes, including signal transduction
(30,
47–49),
chromatin modification (50),
Golgi organization and function
(51,
52), c-Jun function
(47,
53,
54), NFκB-mediated gene
expression (43,
55,
56), and cell survival,
migration, and differentiation and DNA synthesis and proliferation (reviewed
in Ref. 14). Thus, mounting
evidence indicates that PKD has a remarkable diversity of both its signal
generation and distribution and its potential for complex regulatory
interactions with multiple downstream pathways, leading to multiple responses,
including long term cellular events. Despite increasing recognition of its
importance, very little is known about the mechanism(s) of sustained PKD
activation as opposed to the well documented rapid, PKC-dependent PKD
activation.The results presented here demonstrate that prolonged GPCR-induced PKD
activation is mediated by sequential PKC-dependent and PKC-independent phases
of regulation. We report here, for the first time, that PKD
autophosphorylation on Ser748 is a major mechanism contributing to
the late phase of PKD activation occurring in cells stimulated by GPCR
agonists. The present studies expand previous models of PKD regulation by
identifying a novel mechanism induced by GPCR activation that leads to late,
PKC-independent PKD activation. 相似文献
4.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
5.
Quang-Kim Tran Jared Leonard D. J. Black Owen W. Nadeau Igor G. Boulatnikov Anthony Persechini 《The Journal of biological chemistry》2009,284(18):11892-11899
We have investigated the possible biochemical basis for enhancements in NO
production in endothelial cells that have been correlated with agonist- or
shear stress-evoked phosphorylation at Ser-1179. We have found that a
phosphomimetic substitution at Ser-1179 doubles maximal synthase activity,
partially disinhibits cytochrome c reductase activity, and lowers the
EC50(Ca2+) values for calmodulin binding and enzyme
activation from the control values of 182 ± 2 and 422 ± 22
nm to 116 ± 2 and 300 ± 10 nm. These are
similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q.
K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry
47, 7557–7566). Although combining substitutions at Ser-617 and Ser-1179
has no additional effect on maximal synthase activity, cooperativity between
the two substitutions completely disinhibits reductase activity and further
reduces the EC50(Ca2+) values for calmodulin binding and
enzyme activation to 77 ± 2 and 130 ± 5 nm. We have
confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179
and phosphomimetic substitutions at these positions have similar functional
effects. Changes in the biochemical properties of eNOS produced by combined
phosphorylation at Ser-617 and Ser-1179 are predicted to substantially
increase synthase activity in cells at a typical basal free Ca2+
concentration of 50–100 nm.The nitric-oxide synthases catalyze formation of NO and
l-citrulline from l-arginine and O2, with
NADPH as the electron donor
(1). The role of NO generated
by endothelial nitricoxide synthase
(eNOS)2 in the
regulation of smooth muscle tone is well established and was the first of
several physiological roles for this small molecule that have so far been
identified (2). The
nitric-oxide synthases are homodimers of 130–160-kDa subunits. Each
subunit contains a reductase and oxygenase domain
(1). A significant difference
between the reductase domains in eNOS and nNOS and the homologous P450
reductases is the presence of inserts in these synthase isoforms that appear
to maintain them in their inactive states
(3,
4). A calmodulin (CaM)-binding
domain is located in the linker that connects the reductase and oxygenase
domains, and the endothelial and neuronal synthases both require
Ca2+ and exogenous CaM for activity
(5,
6). When CaM is bound, it
somehow counteracts the effects of the autoinhibitory insert(s) in the
reductase. The high resolution structure for the complex between
(Ca2+)4-CaM and the isolated CaM-binding domain from
eNOS indicates that the C-ter and N-ter lobes of CaM, which each contain a
pair of Ca2+-binding sites, enfold the domain, as has been observed
in several other such CaM-peptide complexes
(7). Consistent with this
structure, investigations of CaM-dependent activation of the neuronal synthase
suggest that both CaM lobes must participate
(8,
9).Bovine eNOS can be phosphorylated in endothelial cells at Ser-116, Thr-497,
Ser-617, Ser-635, and Ser-1179
(10–12).
There are equivalent phosphorylation sites in the human enzyme
(10–12).
Phosphorylation of the bovine enzyme at Thr-497, which is located in the
CaM-binding domain, blocks CaM binding and enzyme activation
(7,
11,
13,
14). Ser-116 can be basally
phosphorylated in cells (10,
11,
13,
15), and dephosphorylation of
this site has been correlated with increased NO production
(13,
15). However, it has also been
reported that a phosphomimetic substitution at this position has no effect on
enzyme activity measured in vitro
(13). Ser-1179 is
phosphorylated in response to a variety of stimuli, and this has been reliably
correlated with enhanced NO production in cells
(10,
11). Indeed, NO production is
elevated in transgenic endothelium expressing an eNOS mutant containing an
S1179D substitution, but not in tissue expressing an S1179A mutant
(16). Shear stress or insulin
treatment is correlated with Akt-catalyzed phosphorylation of Ser-1179 in
endothelial cells, and this is correlated with increased NO production in the
absence of extracellular Ca2+
(17–19).
Akt-catalyzed phosphorylation or an S1179D substitution has also been
correlated with increased synthase activity in cell extracts at low
intracellular free [Ca2+]
(17). Increased NO production
has also been observed in cells expressing an eNOS mutant containing an S617D
substitution, and physiological stimuli such as shear-stress, bradykinin,
VEGF, and ATP appear to stimulate Akt-catalyzed phosphorylation of Ser-617 and
Ser-1179 (12,
13,
20). Although S617D eNOS has
been reported to have the same maximum activity in vitro as the wild
type enzyme (20), in our hands
an S617D substitution increases the maximal CaM-dependent synthase activity of
purified mutant enzyme ∼2-fold, partially disinhibits reductase activity,
and reduces the EC50(Ca2+) values for CaM binding and
enzyme activation (21).In this report, we describe the effects of a phosphomimetic Asp
substitution at Ser-1179 in eNOS on the Ca2+ dependence of CaM
binding and CaM-dependent activation of reductase and synthase activities. We
also describe the effects on these properties of combining this substitution
with one at Ser-617. Finally, we demonstrate that Akt-catalyzed
phosphorylation and Asp substitutions at Ser-617 and Ser-1179 have similar
functional effects. Our results suggest that phosphorylation of eNOS at
Ser-617 and Ser-1179 can substantially increase synthase activity in cells at
a typical basal free Ca2+ concentration of 50–100
nm, while single phosphorylations at these sites produce smaller
activity increases, and can do so only at higher free Ca2+
concentrations. 相似文献
6.
A key set of reactions for the initiation of new DNA strands during herpes
simplex virus-1 replication consists of the primase-catalyzed synthesis of
short RNA primers followed by polymerase-catalyzed DNA synthesis
(i.e. primase-coupled polymerase activity). Herpes primase
(UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long.
However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at
least 8 nucleotides long. Surprisingly, coupled activity was remarkably
inefficient, even considering only those primers at least 8 nucleotides long,
and herpes polymerase typically elongated <2% of the primase-synthesized
primers. Of those primers elongated, only 4–26% of the primers were
passed directly from the primase to the polymerase (UL30-UL42) without
dissociating into solution. Comparing RNA primer-templates and DNA
primer-templates of identical sequence showed that herpes polymerase greatly
preferred to elongate the DNA primer by 650–26,000-fold, thus accounting
for the extremely low efficiency with which herpes polymerase elongated
primase-synthesized primers. Curiously, one of the DNA polymerases of the host
cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended
herpes primase-synthesized RNA primers much more efficiently than the viral
polymerase, raising the possibility that the viral polymerase may not be the
only one involved in herpes DNA replication.Herpes simplex virus 1
(HSV-1)2 encodes seven
proteins essential for replicating its double-stranded DNA genome; five of
these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products)
and the heterodimeric polymerase (UL30-UL42 gene products)
(1,
2). The helicase-primase
unwinds the DNA at the replication fork and generates single-stranded DNA for
both leading and lagging strand synthesis. Primase synthesizes short RNA
primers on the lagging strand that the polymerase presumably elongates using
dNTPs (i.e. primase-coupled polymerase activity). These two protein
complexes are thought to replicate the viral genome on both the leading and
lagging strands (1,
2).Previous studies have focused on the helicase-primase and polymerase
separately. The helicase-primase contains three subunits, UL5, UL52, and UL8
in a 1:1:1 ratio
(3–5).
The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like
motifs, yet the minimal active complex that demonstrates either helicase or
primase activities contains both UL5 and UL52
(6,
7). Although the UL8 subunit
has no known catalytic activity, several functions have been proposed,
including enhancing helicase and primase activities, enhancing primer
synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA
strands, and facilitating formation of the replisome
(8–12).
Although primase will synthesize short
(2–3
nucleotides long) primers on a variety of template sequences, synthesis of
longer primers up to 13 nucleotides long requires the template sequence,
3′-deoxyguanidine-pyrimidine-pyrimidine-5′
(13). Primase initiates
synthesis at the first pyrimidine via the polymerization of two purine NTPs
(13). Even after initiation at
this sequence, however, the vast majority of products are only 2–3
nucleotides long (13,
14).The herpes polymerase consists of the UL30 subunit, which has polymerase
and 3′ → 5′ exonuclease activities
(1,
2), and the UL42 subunit, which
serves as a processivity factor
(15–17).
Unlike most processivity factors that encircle the DNA, the UL42 protein binds
double-stranded DNA and thus directly tethers the polymerase to the DNA
(18). Using pre-existing DNA
primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42)
incorporates dNTPs at a rate of 150 s–1, a rate much faster
than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01
s–1) (19,
20).We examined primase-coupled polymerase activity by the herpes primase and
polymerase complexes. Although herpes primase synthesizes RNA primers
2–13 nucleotides long, the polymerase only effectively elongates those
at least 8 nucleotides long. Surprisingly, the polymerase elongated only a
small fraction of the primase-synthesized primers (<1–2%), likely
because of the polymerase elongating RNA primer-templates much less
efficiently than DNA primer-templates. In contrast, human DNA polymerase
α (pol α) elongated the herpes primase-synthesized primers very
efficiently. The biological significance of these data is discussed. 相似文献
7.
Motoki Takaku Shinichi Machida Noriko Hosoya Shugo Nakayama Yoshimasa Takizawa Isao Sakane Takehiko Shibata Kiyoshi Miyagawa Hitoshi Kurumizaka 《The Journal of biological chemistry》2009,284(21):14326-14336
The RAD51 protein is a central player in homologous recombinational repair.
The RAD51B protein is one of five RAD51 paralogs that function in the
homologous recombinational repair pathway in higher eukaryotes. In the present
study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested
to be involved in actin-remodeling processes, unexpectedly binds to the RAD51
and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and
strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged
DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone
promotes single-stranded DNA annealing, and the recombination activities of
the EVL protein are further enhanced by the RAD51B protein. The expression of
the EVL protein is not ubiquitous, but it is significantly expressed in breast
cancer-derived MCF7 cells. These results suggest that the EVL protein is a
novel recombination factor that may be required for repairing specific DNA
lesions, and that may cause tumor malignancy by its inappropriate
expression.Chromosomal DNA double strand breaks
(DSBs)2 are potential
inducers of chromosomal aberrations and tumorigenesis, and they are accurately
repaired by the homologous recombinational repair (HRR) pathway, without base
substitutions, deletions, and insertions
(1–3).
In the HRR pathway (4,
5), single-stranded DNA (ssDNA)
tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue
of the bacterial RecA protein, binds to the ssDNA tail and forms a helical
nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact
double-stranded DNA (dsDNA) to form a three-component complex, containing
ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the
RAD51 protein promotes recombination reactions, such as homologous pairing and
strand exchange
(6–9).The RAD51 protein requires auxiliary proteins to promote the homologous
pairing and strand exchange reactions efficiently in cells
(10–12).
In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the
RAD51 protein
(13–17)
and stimulate the RAD51-mediated homologous pairing and/or strand exchange
reactions in vitro
(18–21).
The human RAD51AP1 protein, which directly binds to the RAD51 protein
(22), was also found to
stimulate RAD51-mediated homologous pairing in vitro
(23,
24). The BRCA2 protein
contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs
(25–33),
and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated
RAD51-mediated strand exchange
(34,
35). Most of these
RAD51-interacting factors are known to be required for efficient RAD51
assembly onto DSB sites in cells treated with ionizing radiation
(10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which
share about 20–30% amino acid sequence similarity with the RAD51 protein
(36–38).
RAD51B-deficient cells are hypersensitive to DSB-inducing agents,
such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the
RAD51B protein is involved in the HRR pathway
(39–44).
Genetic experiments revealed that RAD51B-deficient cells exhibited
impaired RAD51 assembly onto DSB sites
(39,
44), suggesting that the
RAD51B protein functions in the early stage of the HRR pathway. Biochemical
experiments also suggested that the RAD51B protein participates in the early
to late stages of the HRR pathway
(45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein
binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein
is known to be involved in cytoplasmic actin remodeling
(48) and is also overexpressed
in breast cancer (49). Like
the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51
foci formation after DSB induction, suggesting that the EVL protein enhances
RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound
to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange.
The EVL protein also promoted the annealing of complementary strands. These
recombination reactions that were stimulated or promoted by the EVL protein
were further enhanced by the RAD51B protein. These results strongly suggested
that the EVL protein is a novel factor that activates RAD51-mediated
recombination reactions, probably with the RAD51B protein. We anticipate that,
in addition to its involvement in cytoplasmic actin dynamics, the EVL protein
may be required in homologous recombination for repairing specific DNA
lesions, and it may cause tumor malignancy by inappropriate recombination
enhanced by EVL overexpression in certain types of tumor cells. 相似文献
8.
9.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
10.
11.
12.
A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate
Receptor-mediated
Endocytosis 总被引:1,自引:0,他引:1
Rongbao Zhao Sang Hee Min Yanhua Wang Estela Campanella Philip S. Low I. David Goldman 《The Journal of biological chemistry》2009,284(7):4267-4274
Recently, this laboratory identified a proton-coupled folate transporter
(PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate
absorption and transport into the central nervous system because there are
loss-of-function mutations in this gene in the autosomal recessive disorder,
hereditary folate malabsorption. The current study addresses the role PCFT
might play in another transport pathway, folate receptor (FR)-mediated
endocytosis. FRα cDNA was transfected into novel PCFT+ and
PCFT– HeLa sublines. FRα was shown to bind and trap
folates in vesicles but with minimal export into the cytosol in
PCFT– cells. Cotransfection of FRα and PCFT resulted in
enhanced folate transport into cytosol as compared with transfection of
FRα alone. Probenecid did not inhibit folate binding to FR, but
inhibited PCFT-mediated transport at endosomal pH, and blocked
FRα-mediated transport into the cytosol. FRα and PCFT co-localized
to the endosomal compartment. These observations (i) indicate that PCFT plays
a role in FRα-mediated endocytosis by serving as a route of export of
folates from acidified endosomes and (ii) provide a functional role for PCFT
in tissues in which it is expressed, such as the choroid plexus, where the
extracellular milieu is at neutral pH.Loss of function mutations of the proton-coupled folate transporter
(PCFT),2 which
functions optimally at low pH, are the molecular basis for the autosomal
recessive disorder, hereditary folate malabsorption (HFM)
(1–4).
Infants present with this disorder several months after birth with marked
folate deficiency anemia, hypogammaglobulinemia with immune deficiency and
infections, neurological deficits, and often seizures
(5). PCFT is highly expressed
at the apical brush-border membrane of the duodenum and proximal jejunum
(6–9)
where the pH at the microclimate of the surface of this epithelium is low (pH
5.8–6.0), and folates are absorbed
(1,
7,
10,
11). Hence, the failure to
absorb folates in the absence of this transporter in HFM is expected. However,
PCFT expression, and its associated folate transport activity at low pH, is
observed in many tissues where the transport interface is presumed to be at pH
7.4 (12). Of particular
interest is the mechanism by which PCFT mediates transport of folates into the
central nervous system (CNS) where this transporter is expressed in brain and
choroid plexus (1,
7,
13). Transport into the CNS is
impaired in patients with HFM who have very low cerebrospinal fluid (CSF)
folate levels and marked reversal of the blood:CSF folate gradient which is
normally 2–3:1 (5).Folates are also transported into cells by a receptor-mediated process.
Folate receptor-α (FRα) is anchored to cell membranes via a
glycosylphosphatidylinositol domain. Uptake begins with folate binding to
receptor at the cell surface followed by invagination of the membrane and the
formation of endosomes that traffic along microtubules to a perinuclear
compartment before returning to the plasma membrane
(14–16).
During transit in the cytoplasm, endosomes acidify to a pH of
∼6.0–6.5 (17),
folate is released from the receptor and exported from the intact endosome
into the cytoplasm. This putative exporter was shown to require a
trans-endosomal pH gradient
(18–20).The current report addresses the hypothesis that PCFT is an endosomal
folate exporter and thereby plays a role in FRα-mediated endocytosis
(1,
2,
21,
22), that the ubiquitous
expression of PCFT in mammalian tissues may be related to this function, and
that loss of this function may be a basis for the low CSF folate levels in
HFM. The experimental approach utilized a series of HeLa sublines, developed
in this laboratory, in which constitutive expression of FRα is
negligible. HeLa R5 cells lack reduced folate carrier (RFC) function due to a
genomic deletion of this gene
(23). A derivative of R5
cells, HeLa R1-11 cells lack, in addition, PCFT expression, while an R1-11
revertant re-expresses PCFT
(24). The impact of PCFT on
FRα-mediated endocytosis, achieved by transfection of the receptor into
these cell lines, was assessed under conditions in which there was negligible
PCFT-mediated transport directly across the plasma membrane into cells. 相似文献
13.
Rebecca M. Dixon Jack R. Mellor Jonathan G. Hanley 《The Journal of biological chemistry》2009,284(21):14230-14235
Oxygen and glucose deprivation (OGD) induces delayed cell death in
hippocampal CA1 neurons via Ca2+/Zn2+-permeable,
GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents
in hippocampal neurons show marked inward rectification and increased
sensitivity to channel blockers selective for GluR2-lacking AMPARs. This
occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression
and a rapid internalization of GluR2-containing AMPARs during the OGD insult,
which are replaced by GluR2-lacking receptors. The mechanisms that underlie
this rapid change in subunit composition are unknown. Here, we demonstrate
that this trafficking event shares features in common with events that mediate
long term depression and long term potentiation and is initiated by the
activation of N-methyl-d-aspartic acid receptors. Using
biochemical and electrophysiological approaches, we show that peptides that
interfere with PICK1 PDZ domain interactions block the OGD-induced switch in
subunit composition, implicating PICK1 in restricting GluR2 from synapses
during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at
synapses during OGD as a result of PICK1 PDZ interactions are involved in
OGD-induced delayed cell death. This work demonstrates that PICK1 plays a
crucial role in the response to OGD that results in altered synaptic
transmission and neuronal death and has implications for our understanding of
the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation
(OGD)3 associated with
transient global ischemia induces delayed cell death, particularly in
hippocampal CA1 pyramidal cells
(1–3),
a phenomenon that involves Ca2+/Zn2+-permeable,
GluR2-lacking AMPARs (4).
AMPARs are heteromeric complexes of subunits GluR1–4
(5), and most AMPARs in the
hippocampus contain GluR2, which renders them calcium-impermeable and results
in a marked inward rectification in their current-voltage relationship
(6–8).
Ischemia induces a delayed down-regulation of GluR2 mRNA and protein
expression (4,
9–11),
resulting in enhanced AMPAR-mediated Ca2+ and Zn2+
influx into CA1 neurons (10,
12). In these neurons,
AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification
1–2 days following ischemia and increased sensitivity to 1-naphthyl
acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs
(13–16).
Blockade of these channels at 9–40 h following ischemia is
neuroprotective, indicating a crucial role for Ca2+-permeable
AMPARs in ischemic cell death
(16).In addition to delayed changes in AMPAR subunit composition as a result of
altered mRNA expression, it was recently reported that
Ca2+-permable, GluR2-lacking AMPARs are targeted to synaptic sites
via membrane trafficking at much earlier times during OGD
(17). This subunit
rearrangement involves endocytosis of AMPARs containing GluR2 complexed with
GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3
(17). However, the molecular
mechanisms behind this trafficking event are unknown, and furthermore, it is
not known whether these trafficking-mediated changes in AMPAR subunit
composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial
involvement in long term depression (LTD) and long term potentiation (LTP),
activity-dependent forms of synaptic plasticity thought to underlie learning
and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching
events (brought about by trafficking that involves endo/exocytosis) are
central to the necessary changes in synaptic receptor complement
(7,
18–20).
It is possible that similar mechanisms regulate AMPAR trafficking during
OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that
binds, via the PDZ domain, to a number of membrane proteins including AMPAR
subunits GluR2/3. This interaction is required for AMPAR internalization from
the synaptic plasma membrane in response to Ca2+ influx via NMDAR
activation in hippocampal neurons
(21–23).
This process is the major mechanism that underlies the reduction in synaptic
strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged
as a mechanism that regulates the GluR2 content of synaptic receptors, which
in turn determines their Ca2+ permeability
(7,
20). This is likely to be of
profound importance in both plasticity and pathological mechanisms.
Importantly, PICK1 overexpression has been shown to induce a shift in synaptic
AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly
rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1
(24). This suggests that PICK1
may mediate the rapid switch in subunit composition occurring during OGD
(17). Here, we demonstrate
that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1
PDZ interactions, and importantly, that this early trafficking event that
occurs during OGD contributes to the signaling that results in delayed
neuronal death. 相似文献
14.
Dong Han Hamid Y. Qureshi Yifan Lu Hemant K. Paudel 《The Journal of biological chemistry》2009,284(20):13422-13433
In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked
to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms
paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is
phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa
bands on SDS-gel, and does not promote microtubule assembly. Upon
dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on
SDS-gels in a manner similar to tau that is isolated from normal brain and
promotes microtubule assembly. The site(s) that inhibits microtubule
assembly-promoting activity when phosphorylated in the diseased brain is not
known. In this study, when tau was phosphorylated by Cdk5 in vitro,
its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a
time-dependent manner. This mobility shift correlated with phosphorylation at
Ser202, and Ser202 phosphorylation inhibited tau
microtubule-assembly promoting activity. When several tau point mutants were
analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17,
but not nonspecific mutations S214A and S262A, promoted Ser202
phosphorylation and mobility shift to a ∼68-kDa band. Furthermore,
Ser202 phosphorylation inhibited the microtubule assembly-promoting
activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17
missense mutations, by promoting phosphorylation at Ser202, inhibit
the microtubule assembly-promoting activity of tau in vitro,
suggesting that Ser202 phosphorylation plays a major role in the
development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles
(NFTs)4 and senile
plaques are the two characteristic neuropathological lesions found in the
brains of patients suffering from Alzheimer disease (AD). The major fibrous
component of NFTs are paired helical filaments (PHFs) (for reviews see Refs.
1–3).
Initially, PHFs were found to be composed of a protein component referred to
as “A68” (4).
Biochemical analysis reveled that A68 is identical to the
microtubule-associated protein, tau
(4,
5). Some characteristic
features of tau isolated from PHFs (PHF-tau) are that it is abnormally
hyperphosphorylated (phosphorylated on more sites than the normal brain tau),
does not bind to microtubules, and does not promote microtubule assembly
in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind
to and promote microtubule assembly
(6,
7). Tau hyperphosphorylation is
suggested to cause microtubule instability and PHF formation, leading to NFT
pathology in the brain
(1–3).PHF-tau is phosphorylated on at least 21 proline-directed and
non-proline-directed sites (8,
9). The individual contribution
of these sites in converting tau to PHFs is not entirely clear. However, some
sites are only partially phosphorylated in PHFs
(8), whereas phosphorylation on
specific sites inhibits the microtubule assembly-promoting activity of tau
(6,
10). These observations
suggest that phosphorylation on a few sites may be responsible and sufficient
for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on
SDS-gel due to the presence of six isoforms that are phosphorylated to
different extents (2). PHF-tau
isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68
kDa-bands on an SDS-gel (4,
5,
11). Studies have shown that
∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau
band as an ∼69-kDa band in these studies) are present only in brain areas
affected by NFT degeneration
(12,
13). Their amount is
correlated with the NFT densities at the affected brain regions. Moreover, the
increase in the amount of ∼64- and 68-kDa band tau in the brain correlated
with a decline in the intellectual status of the patient. The ∼64- and
68-kDa tau bands were suggested to be the pathological marker of AD
(12,
13). Biochemical analyses
determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which
upon dephosphorylation, migrated as normal tau on SDS-gel
(4,
5,
11). Tau sites involved in the
tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role
in AD pathology (12,
13). It is not known whether
phosphorylation at all 21 PHF-sites is required for the tau mobility shift in
AD. However, in vitro the tau mobility shift on SDS-gel is sensitive
to phosphorylation only on some sites
(6,
14). It is therefore possible
that in the AD brain, phosphorylation on some sites also causes a tau mobility
shift. Identification of such sites will significantly enhance our knowledge
of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients
suffering from a group of neurodegenerative disorders collectively called
tauopathies (2,
11). These disorders include
frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17),
corticobasal degeneration, progressive supranuclear palsy, and Pick disease.
Each PHF-tau isolated from autopsied brains of patients suffering from various
tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands
on SDS-gel, and is incapable of binding to microtubules. Upon
dephosphorylation, the above referenced PHF-tau migrates as a normal tau on
SDS-gel, binds to microtubules, and promotes microtubule assembly
(2,
11). These observations
suggest that the mechanisms of NFT pathology in various tauopathies may be
similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may
be an indicator of the disease. The tau gene is mutated in familial FTDP-17,
and these mutations accelerate NFT pathology in the brain
(15–18).
Understanding how FTDP-17 mutations promote tau phosphorylation can provide a
better understanding of how NFT pathology develops in AD and various
tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and
P301L tau mutations reduce tau phosphorylation
(19,
20). In COS cells, although
G272V, P301L, and V337M mutations do not show any significant affect, the
R406W mutation caused a reduction in tau phosphorylation
(21,
22). When expressed in SH-SY5Y
cells subsequently differentiated into neurons, the R406W, P301L, and V337M
mutations reduce tau phosphorylation
(23). In contrast, in
hippocampal neurons, R406W increases tau phosphorylation
(24). When phosphorylated by
recombinant GSK3β in vitro, the P301L and V337M mutations do not
have any effect, and the R406W mutation inhibits phosphorylation
(25). However, when incubated
with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations
stimulate tau phosphorylation
(26). The mechanism by which
FTDP-17 mutations promote tau phosphorylation leading to development of NFT
pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that
phosphorylates tau in the brain
(27,
28). In this study, to
determine how FTDP-17 missense mutations affect tau phosphorylation, we
phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by
Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility
shift to ∼64- and 68 kDa-bands. Although the mobility shift to a
∼64-kDa band is achieved by phosphorylation at Ser396/404 or
Ser202, the mobility shift to a 68-kDa band occurs only in response
to phosphorylation at Ser202. We show that in
vitro, FTDP-17 missense mutations, by promoting phosphorylation at
Ser202, enhance the mobility shift to ∼64- and 68-kDa bands and
inhibit the microtubule assembly-promoting activity of tau. Our data suggest
that Ser202 phosphorylation is the major event leading to NFT
pathology in AD and related tauopathies. 相似文献
15.
16.
Cheuk-Lun Lee Poh-Choo Pang William S. B. Yeung Bérangère Tissot Maria Panico Terence T. H. Lao Ivan K. Chu Kai-Fai Lee Man-Kin Chung Kevin K. W. Lam Riitta Koistinen Hannu Koistinen Markku Sepp?l? Howard R. Morris Anne Dell Philip C. N. Chiu 《The Journal of biological chemistry》2009,284(22):15084-15096
Glycodelin is a human glycoprotein with four reported glycoforms, namely
glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S
(GdS). These glycoforms have the same protein core and appear to differ in
their N-glycosylation. The glycosylation of GdA is completely
different from that of GdS. GdA inhibits proliferation and induces cell death
of T cells. However, the glycosylation and immunomodulating activities of GdF
and GdC are not known. This study aimed to use ultra-high sensitivity mass
spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the
relationship between the immunological activity and glycosylation pattern
among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were
shown to contain an enormous diversity of bi-, tri-, and tetra-antennary
complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or
GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels
of fucose and sialic acid substitution. Interestingly, they all carried a
family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing
glycans, which were not identified in the earlier study because of less
sensitive methodologies used. Among the three glycodelins, GdA is the most
heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda
antennae. With the exception of the Sda epitope, the GdC N-glycome
appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase
activity, which may be responsible for transforming GdA/GdF to GdC, was
detected in cumulus cells. Both GdA and GdF inhibited the proliferation,
induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and
peripheral blood mononuclear cells. In contrast, no immunosuppressive effect
was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino
acid residues (1) with two
sites of N-linked glycosylation. There are four reported glycodelin
isoforms, namely glycodelin-A (amniotic fluid isoform,
GdA),4 glycodelin-F
(follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S
(seminal plasma, GdS)
(2–5).
Among the four glycodelin isoforms, only the N-glycan structures of
GdA and GdS have been previously determined. This was achieved using fast atom
bombardment mass spectrometry
(6,
7). The glycan structures of
GdA and GdS are completely different. In GdA, the Asn-28 site carries high
mannose, hybrid, and complex-type structures, whereas the second Asn-63 site
is exclusively occupied by complex-type glycans
(6). The major non-reducing
epitopes characterized in the complex-type glycans are
Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc),
NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc),
NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc),
Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and
GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood
group substance Lewis-x) (6).
Many of these oligosaccharides are rare in other human glycoproteins. GdS
glycans are unusually fucose-rich, and the major complex type glycan
structures are bi-antennary glycans with Lewis-x and Lewis-y antennae.
Glycosylation of GdS is highly site-specific. Asn-28 contains only high
mannose structures, whereas Asn-63 contains only complex type glycans. More
than 80% of the complex glycans have 3–5 fucose residues/glycan, and
none of the glycans is sialylated, which is unusual for a secreted human
glycoprotein (7). The glycan
structures of GdF and GdC are not known, although they differ in
lectin-binding properties and isoelectric point from the other two glycodelin
isoforms (5).Glycans are involved in various intracellular, intercellular, and
cell-matrix recognition events
(8,
9). Glycosylation determines
the biological activities of the glycodelin isoforms
(2,
10). For example, both GdA and
GdF inhibit the spermatozoa-zona pellucida binding
(11) via fucosyltransferase-5
(12), but only the latter
inhibits progesterone-induced acrosome reaction, thus preventing a premature
acrosome reaction of the spermatozoa. There is evidence that cumulus cells can
convert exogenous GdA and -F to GdC, the physicochemical properties of which
suggest that it is differently glycosylated compared with GdA/F
(5). Moreover, GdC stimulated
spermatozoa-zona pellucida binding in a dose-dependent manner, and it
effectively displaced sperm-bound GdA and -F
(4,
5). GdS suppresses capacitation
probably via its inhibitory activity on cholesterol efflux from spermatozoa
(13).Except for the effects on fertilization, GdA is involved in fetomaternal
defense. This glycodelin isoform suppresses proliferation and induces
apoptosis of T cells (2) and
inhibits natural killer cell
(14) and B-cell
(15) activities. Glycosylation
is involved in the binding of GdA to receptors on T cells
(16). The sialic acid of GdA
contributes to the apoptotic activity in T cells
(17,
18) and binding to CD45, a
potential GdA receptor (16).
The importance of glycosylation in glycodelin is further shown by the absence
of immunosuppressive activities in GdS with different glycosylation
(18). The immunomodulating
activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different
glycodelins to exhibit their binding activities and biological effects
(13,
19,
20). The present study aims to
identify the effect of all four glycodelin isoforms on lymphocyte viability,
cell death, and interleukin-2 (IL-2) secretion and to correlate these
bioactivities with their glycosylation patterns determined by mass
spectrometry. 相似文献
17.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
18.
19.
Denise A. Berti Cain Morano Lilian C. Russo Leandro M. Castro Fernanda M. Cunha Xin Zhang Juan Sironi Cl��cio F. Klitzke Emer S. Ferro Lloyd D. Fricker 《The Journal of biological chemistry》2009,284(21):14105-14116
Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme
that has been proposed to metabolize peptides within cells, thereby affecting
antigen presentation and G protein-coupled receptor signal transduction.
However, only a small number of intracellular substrates of EP24.15 have been
reported previously. Here we have identified over 100 peptides in human
embryonic kidney 293 (HEK293) cells that are derived from intracellular
proteins; many but not all of these peptides are substrates or products of
EP24.15. First, cellular peptides were extracted from HEK293 cells and
incubated in vitro with purified EP24.15. Then the peptides were
labeled with isotopic tags and analyzed by mass spectrometry to obtain
quantitative data on the extent of cleavage. A related series of experiments
tested the effect of overexpression of EP24.15 on the cellular levels of
peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10
of the cellular peptides were incubated with purified EP24.15 in
vitro, and the cleavage was monitored by high pressure liquid
chromatography and mass spectrometry. Many of the EP24.15 substrates
identified by these approaches are 9–11 amino acids in length,
supporting the proposal that EP24.15 can function in the degradation of
peptides that could be used for antigen presentation. However, EP24.15 also
converts some peptides into products that are 8–10 amino acids, thus
contributing to the formation of peptides for antigen presentation. In
addition, the intracellular peptides described here are potential candidates
to regulate protein interactions within cells.Intracellular protein turnover is a crucial step for cell functioning, and
if this process is impaired, the elevated levels of aged proteins usually lead
to the formation of intracellular insoluble aggregates that can cause severe
pathologies (1). In mammalian
cells, most proteins destined for degradation are initially tagged with a
polyubiquitin chain in an energy-dependent process and then digested to small
peptides by the 26 S proteasome, a large proteolytic complex involved in the
regulation of cell division, gene expression, and other key processes
(2,
3). In eukaryotes, 30–90%
of newly synthesized proteins may be degraded by proteasomes within minutes of
synthesis (3,
4). In addition to proteasomes,
other extralysosomal proteolytic systems have been reported
(5,
6). The proteasome cleaves
proteins into peptides that are typically 2–20 amino acids in length
(7). In most cases, these
peptides are thought to be rapidly hydrolyzed into amino acids by
aminopeptidases
(8–10).
However, some intracellular peptides escape complete degradation and are
imported into the endoplasmic reticulum where they associate with major
histocompatibility complex class I
(MHC-I)3 molecules and
traffic to the cell surface for presentation to the immune system
(10–12).
Additionally, based on the fact that free peptides added to the intracellular
milieu can regulate cellular functions mediated by protein interactions such
as gene regulation, metabolism, cell signaling, and protein targeting
(13,
14), intracellular peptides
generated by proteasomes that escape degradation have been suggested to play a
role in regulating protein interactions
(15). Indeed, oligopeptides
isolated from rat brain tissue using the catalytically inactive EP24.15 (EC
3.4.24.15) were introduced into Chinese hamster ovarian-S and HEK293 cells and
were found capable of altering G protein-coupled receptor signal transduction
(16). Moreover, EP24.15
overexpression itself changed both angiotensin II and isoproterenol signal
transduction, suggesting a physiological function for its intracellular
substrates/products (16).EP24.15 is a zinc-dependent peptidase of the metallopeptidase M3 family
that contains the HEXXH motif
(17). This enzyme was first
described as a neuropeptide-degrading enzyme present in the soluble fraction
of brain homogenates (18).
Whereas EP24.15 can be secreted
(19,
20), its predominant location
in the cytosol and nucleus suggests that the primary function of this enzyme
is not the extracellular degradation of neuropeptides and hormones
(21,
22). EP24.15 was shown in
vivo to participate in antigen presentation through MHC-I
(23–25)
and in vitro to bind
(26) or degrade
(27) some MHC-I associated
peptides. EP24.15 has also been shown in vitro to degrade peptides
containing 5–17 amino acids produced after proteasome digestion of
β-casein (28). EP24.15
shows substrate size restriction to peptides containing from 5 to 17 amino
acids because of its catalytic center that is located in a deep channel
(29). Despite the size
restriction, EP24.15 has a broad substrate specificity
(30), probably because a
significant portion of the enzyme-binding site is lined with potentially
flexible loops that allow reorganization of the active site following
substrate binding (29).
Recently, it has also been suggested that certain substrates may be cleaved by
an open form of EP24.15 (31).
This characteristic is supported by the ability of EP24.15 to accommodate
different amino acid residues at subsites S4 to S3′, which even includes
the uncommon post-proline cleavage
(30). Such biochemical and
structural features make EP24.15 a versatile enzyme to degrade structurally
unrelated oligopeptides.Previously, brain peptides that bound to catalytically inactive EP24.15
were isolated and identified using mass spectrometry
(22). The majority of peptides
captured by the inactive enzyme were intracellular protein fragments that
efficiently interacted with EP24.15; the smallest peptide isolated in these
assays contained 5 and the largest 17 amino acids
(15,
16,
22,
32), which is within the size
range previously reported for natural and synthetic substrates of EP24.15
(18,
30,
33,
34). Interestingly, the
peptides released by the proteasome are in the same size range of EP24.15
competitive inhibitors/substrates
(7,
35,
36). Taken altogether, these
data suggest that in the intracellular environment EP24.15 could further
cleave proteasome-generated peptides unrelated to MHC-I antigen presentation
(15).Although the mutated inactive enzyme “capture” assay was
successful in identifying several cellular protein fragments that were
substrates for EP24.15, it also found some interacting peptides that were not
substrates. In this study, we used several approaches to directly screen for
cellular peptides that were cleaved by EP24.15. The first approach involved
the extraction of cellular peptides from the HEK293 cell line, incubation
in vitro with purified EP24.15, labeling with isotopic tags, and
analysis by mass spectrometry to obtain quantitative data on the extent of
cleavage. The second approach examined the effect of EP24.15 overexpression on
the cellular levels of peptides in the HEK293 cell line. The third set of
experiments tested synthetic peptides with purified EP24.15 in vitro,
and examined cleavage by high pressure liquid chromatography and mass
spectrometry. Collectively, these studies have identified a large number of
intracellular peptides, including those that likely represent the endogenous
substrates and products of EP24.15, and this original information contributes
to a better understanding of the function of this enzyme in vivo. 相似文献
20.
Xavier Hanoulle Aurélie Badillo Jean-Michel Wieruszeski Dries Verdegem Isabelle Landrieu Ralf Bartenschlager Fran?ois Penin Guy Lippens 《The Journal of biological chemistry》2009,284(20):13589-13601
We report here a biochemical and structural characterization of domain 2 of
the nonstructural 5A protein (NS5A) from the JFH1 Hepatitis C virus strain and
its interactions with cyclophilins A and B (CypA and CypB). Gel filtration
chromatography, circular dichroism spectroscopy, and finally NMR spectroscopy
all indicate the natively unfolded nature of this NS5A-D2 domain. Because
mutations in this domain have been linked to cyclosporin A resistance, we used
NMR spectroscopy to investigate potential interactions between NS5A-D2 and
cellular CypA and CypB. We observed a direct molecular interaction between
NS5A-D2 and both cyclophilins. The interaction surface on the cyclophilins
corresponds to their active site, whereas on NS5A-D2, it proved to be
distributed over the many proline residues of the domain. NMR heteronuclear
exchange spectroscopy yielded direct evidence that many proline residues in
NS5A-D2 form a valid substrate for the enzymatic peptidyl-prolyl
cis/trans isomerase (PPIase) activity of CypA and CypB.Hepatitis C virus
(HCV)4 is a small,
positive strand, RNA-enveloped virus belonging to the Flaviviridae family and
the genus Hepacivirus. With 120–180 million chronically
infected individuals worldwide, hepatitis C virus infection represents a major
cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma
(1). The HCV viral genome
(∼9.6 kb) codes for a unique polyprotein of ∼3000 amino acids
(recently reviewed in Refs.
2–4).
Following processing via viral and cellular proteases, this polyprotein gives
rise to at least 10 viral proteins, divided into structural (core, E1, and E2
envelope glycoproteins) and nonstructural proteins (p7, NS2, NS3, NS4A, NS4B,
NS5A, NS5B). Nonstructural proteins are involved in polyprotein processing and
viral replication. The set composed of NS3, NS4A, NS4B, NS5A, and NS5B
constitutes the minimal protein component required for viral replication
(5).Cyclophilins are cellular proteins that have been identified first as
CsA-binding proteins (6). As
FK506-binding proteins (FKBP) and parvulins, cyclophilins are peptidyl-prolyl
cis/trans isomerases (PPIase) that catalyze the
cis/trans isomerization of the peptide linkage preceding a proline
(6,
7). Several subtypes of
cyclophilins are present in mammalian cells
(8). They share a high sequence
homology and a well conserved three-dimensional structure but display
significant differences in their primary cellular localization and in
abundance (9). CypA, the most
abundant of the cyclophilins, is primarily cytoplasmic, whereas CypB is
directed to the endoplasmic reticulum lumen or the secretory pathway. CypD, on
the other hand, is the mitochondrial cyclophilin. Cyclophilins are involved in
numerous physiological processes such as protein folding, immune response, and
apoptosis and also in the replication cycle of viruses including vaccinia
virus, vesicular stomatitis virus, severe acute respiratory syndrome
(SARS)-coronavirus, and human immunodeficiency virus (HIV) (for review see
Ref. 10). For HIV, CypA has
been shown to interact with the capsid domain of the HIV Gag precursor
polyprotein (11). CypA thereby
competes with capsid domain/TRIM5 interaction, resulting in a loss of the
antiviral protective effect of the cellular restriction factor TRIM5α
(12,
13). Moreover, it has been
shown that CypA catalyzes the cis/trans isomerization of
Gly221-Pro222 in the capsid domain and that it has
functional consequences for HIV replication efficiency
(14–16).
For HCV, Watashi et al.
(17) have described a
molecular and functional interaction between NS5B, the viral RNA-dependent RNA
polymerase (RdRp), and cyclophilin B (CypB). CypB may be a key regulator in
HCV replication by modulating the affinity of NS5B for RNA. This regulation is
abolished in the presence of cyclosporin A (CsA), an inhibitor of cyclophilins
(6). These results provided for
the first time a molecular mechanism for the early-on observed anti-HCV
activity of CsA
(18–20).
Although this initial report suggests that only CypB would be involved in the
HCV replication process (17),
a growing number of studies have recently pointed out a role for other
cyclophilins
(21–25).In vitro selection of CsA-resistant HCV mutants indicated the
importance of two HCV nonstructural proteins, NS5B and NS5A
(26), with a preponderant
effect for mutations in the C-terminal half of NS5A. NS5A is a large
phosphoprotein (49 kDa), indispensable for HCV replication and particle
assembly
(27–29),
but for which the exact function(s) in the HCV replication cycle remain to be
elucidated. This nonstructural protein is anchored to the cytoplasmic leaflet
of the endoplasmic reticulum membrane via an N-terminal amphipathic
α-helix (residues 1–27)
(30,
31). Its cytoplasmic sequence
can be divided into three domains: D1 (residues 27–213), D2 (residues
250–342), and D3 (residues 356–447), all connected by low
complexity sequences (32). D1,
a zinc-binding domain, adopts a dimeric claw-shaped structure, which is
proposed to interact with RNA
(33,
34). NS5A-D2 is essential for
HCV replication, whereas NS5A-D3 is a key determinant for virus infectious
particle assembly (27,
35). NS5A-D2 and -D3, for
which sequence conservation among HCV genotypes is significantly lower than
for D1, have been proposed to be natively unfolded domains
(28,
32). Molecular and structural
characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered
nature of this domain (36,
37).As it is still not clear which cyclophilins are cofactors for HCV
replication, and as mutations in HCV NS5A protein have been associated with
CsA resistance, we decided to examine the interaction between both CypA and
CypB and domain 2 of the HCV NS5A protein. We first characterized, at the
molecular level, NS5A-D2 from the HCV JFH1 infectious strain (genotype 2a) and
showed by NMR spectroscopy that this natively unfolded domain indeed interacts
with both cyclophilin A and cyclophilin B. Our NMR chemical shift mapping
experiments indicated that the interaction occurs at the level of the
cyclophilin active site, whereas it lacks a precise localization on NS5A-D2. A
peptide derived from the only well conserved amino acid motif in NS5A-D2 did
interact with cyclophilin A but only with a 10-fold lower affinity than the
full domain. We concluded from this that the many proline residues form
multiple anchoring points, especially when they adopt the cis
conformation. NMR exchange spectroscopy further demonstrated that NS5A-D2 is a
substrate for the PPIase activities of both CypA and CypB. Both the
NS5A/cyclophilin interaction and the PPIase activity of the cyclophilins on
NS5A-D2 were abolished by CsA, underscoring the specificity of the
interaction. 相似文献