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1.
2.
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
3.
4.
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
5.
6.
Eun-Yeong Bergsdorf Anselm A. Zdebik Thomas J. Jentsch 《The Journal of biological chemistry》2009,284(17):11184-11193
Members of the CLC gene family either function as chloride channels or as
anion/proton exchangers. The plant AtClC-a uses the pH gradient across the
vacuolar membrane to accumulate the nutrient
in this organelle. When AtClC-a was
expressed in Xenopus oocytes, it mediated
exchange
and less efficiently mediated Cl–/H+ exchange.
Mutating the “gating glutamate” Glu-203 to alanine resulted in an
uncoupled anion conductance that was larger for Cl– than
. Replacing the “proton
glutamate” Glu-270 by alanine abolished currents. These could be
restored by the uncoupling E203A mutation. Whereas mammalian endosomal ClC-4
and ClC-5 mediate stoichiometrically coupled
2Cl–/H+ exchange, their
transport is largely uncoupled from
protons. By contrast, the AtClC-a-mediated
accumulation in plant vacuoles
requires tight
coupling. Comparison of AtClC-a and ClC-5 sequences identified a proline in
AtClC-a that is replaced by serine in all mammalian CLC isoforms. When this
proline was mutated to serine (P160S), Cl–/H+
exchange of AtClC-a proceeded as efficiently as
exchange, suggesting a role of this residue in
exchange. Indeed, when the corresponding serine of ClC-5 was replaced by
proline, this Cl–/H+ exchanger gained efficient
coupling. When inserted into the model Torpedo chloride channel
ClC-0, the equivalent mutation increased nitrate relative to chloride
conductance. Hence, proline in the CLC pore signature sequence is important
for
exchange and conductance both in
plants and mammals. Gating and proton glutamates play similar roles in
bacterial, plant, and mammalian CLC anion/proton exchangers.CLC proteins are found in all phyla from bacteria to humans and either
mediate electrogenic anion/proton exchange or function as chloride channels
(1). In mammals, the roles of
plasma membrane CLC Cl– channels include transepithelial
transport
(2–5)
and control of muscle excitability
(6), whereas vesicular CLC
exchangers may facilitate endocytosis
(7) and lysosomal function
(8–10)
by electrically shunting vesicular proton pump currents
(11). In the plant
Arabidopsis thaliana, there are seven CLC isoforms
(AtClC-a–AtClC-g)2
(12–15),
which may mostly reside in intracellular membranes. AtClC-a uses the pH
gradient across the vacuolar membrane to transport the nutrient nitrate into
that organelle (16). This
secondary active transport requires a tightly coupled
exchange. Astonishingly, however, mammalian ClC-4 and -5 and bacterial EcClC-1
(one of the two CLC isoforms in Escherichia coli) display tightly
coupled Cl–/H+ exchange, but anion flux is largely
uncoupled from H+ when
is transported
(17–21).
The lack of appropriate expression systems for plant CLC transporters
(12) has so far impeded
structure-function analysis that may shed light on the ability of AtClC-a to
perform efficient
exchange. This dearth of data contrasts with the extensive mutagenesis work
performed with CLC proteins from animals and bacteria.The crystal structure of bacterial CLC homologues
(22,
23) and the investigation of
mutants (17,
19–21,
24–29)
have yielded important insights into their structure and function. CLC
proteins form dimers with two largely independent permeation pathways
(22,
25,
30,
31). Each of the monomers
displays two anion binding sites
(22). A third binding site is
observed when a certain key glutamate residue, which is located halfway in the
permeation pathway of almost all CLC proteins, is mutated to alanine
(23). Mutating this gating
glutamate in CLC Cl– channels strongly affects or even
completely suppresses single pore gating
(23), whereas CLC exchangers
are transformed by such mutations into pure anion conductances that are not
coupled to proton transport
(17,
19,
20). Another key glutamate,
located at the cytoplasmic surface of the CLC monomer, seems to be a hallmark
of CLC anion/proton exchangers. Mutating this proton glutamate to
nontitratable amino acids uncouples anion transport from protons in the
bacterial EcClC-1 protein (27)
but seems to abolish transport altogether in mammalian ClC-4 and -5
(21). In those latter
proteins, anion transport could be restored by additionally introducing an
uncoupling mutation at the gating glutamate
(21).The functional complementation by AtClC-c and -d
(12,
32) of growth phenotypes of a
yeast strain deleted for the single yeast CLC Gef1
(33) suggested that these
plant CLC proteins function in anion transport but could not reveal details of
their biophysical properties. We report here the first functional expression
of a plant CLC in animal cells. Expression of wild-type (WT) and mutant
AtClC-a in Xenopus oocytes indicate a general role of gating and
proton glutamate residues in anion/proton coupling across different isoforms
and species. We identified a proline in the CLC signature sequence of AtClC-a
that plays a crucial role in
exchange. Mutating it to serine, the residue present in mammalian CLC proteins
at this position, rendered AtClC-a Cl–/H+ exchange
as efficient as
exchange. Conversely, changing the corresponding serine of ClC-5 to proline
converted it into an efficient
exchanger. When proline replaced the critical serine in Torpedo
ClC-0, the relative conductance of
this model Cl– channel was drastically increased, and
“fast” protopore gating was slowed. 相似文献
7.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
8.
9.
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
10.
Jiang Tian Xin Li Man Liang Lijun Liu Joe X. Xie Qiqi Ye Peter Kometiani Manoranjani Tillekeratne Runming Jin Zijian Xie 《The Journal of biological chemistry》2009,284(22):14921-14929
Here we show that ouabain-induced cell growth regulation is intrinsically
coupled to changes in the cellular amount of Na/K-ATPase via the
phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR)
pathway. Ouabain increases the endocytosis and degradation of Na/K-ATPase in
LLC-PK1, human breast (BT20), and prostate (DU145) cancer cells. However,
ouabain stimulates the PI3K/Akt/mTOR pathway and consequently up-regulates the
expression of Na/K-ATPase in LLC-PK1 but not BT20 and DU145 cells. This
up-regulation is sufficient to replete the plasma membrane pool of Na/K-ATPase
and to stimulate cell proliferation in LLC-PK1 cells. On the other hand,
ouabain causes a gradual depletion of Na/K-ATPase and an increased expression
of cell cycle inhibitor p21cip, which consequently
inhibits cell proliferation in BT20 and DU145 cells. Consistently, we observe
that small interfering RNA-mediated knockdown of Na/K-ATPase is sufficient to
induce the expression of p21cip and slow the proliferation
of LLC-PK1 cells. Moreover, this knockdown converts the growth stimulatory
effect of ouabain to growth inhibition in LLC-PK1 cells. Mechanistically, both
Src and caveolin-1 are required for ouabain-induced activation of Akt and
up-regulation of Na/K-ATPase. Furthermore, inhibition of the PI3K/Akt/mTOR
pathway by rapamycin completely blocks ouabain-induced expression of
Na/K-ATPase and converts ouabain-induced growth stimulation to growth
inhibition in LLC-PK1 cells. Taken together, we conclude that changes in the
expression of Na/K-ATPase dictate the growth regulatory effects of ouabain on
cells.The Na/K-ATPase, a member of P-type ATPase family, was discovered as an
energy transducing ion pump. It transports Na+ and K+
across the cell membrane and maintains ion homeostasis in animal cells
(1,
2). Recent studies indicate
that the Na/K-ATPase is also an important receptor that can transduce ligand
binding into the activation of protein kinase cascades
(3). Specifically, the
Na/K-ATPase interacts with Src, which provides at least two important cellular
regulations (4,
5). First, association with
Na/K-ATPase keeps Src in an inactive state. Thus, the Na/K-ATPase serves as a
native negative Src regulator
(4). Second, this interaction
forms a functional receptor complex for cardiotonic steroids
(CTS)3
(3), a group of well
characterized ligands of the Na/K-ATPase. Cardiotonic steroids include
cardenolides (e.g. ouabain) and bufadienolides (e.g.
marinobufagenin) (6). Although
CTS are known cardiac drugs, some of them have now been identified as
endogenous steroid hormones
(6–8).
Binding of CTS to the receptor complex activates the Na/K-ATPase-associated
Src. Subsequently, the activated Src transactivates other tyrosine kinases,
and together they recruit and further phosphorylate multiple membrane and
soluble proteins, which results in the activation of protein kinase cascades
and the generation of second messengers
(3,
4,
6). Ultimately, this chain of
signaling events would alter cellular functions and cell growth in a
cell-specific manner (5,
9–12).
For instance, we and others have demonstrated that ouabain-induced activation
of ERK and PI3K/Akt/mTOR pathways are responsible for cell growth stimulation
in transformed cell lines, in primary cultures, as well as in vivo
(13–18).It has also been recognized for a long time that CTS inhibit cell growth in
many cancer cells
(19–24).
Of particular significance are studies that indicate the beneficial effects of
CTS therapy in women with breast cancer
(25–29).
Consistently, recent in vitro and in vivo studies have
identified several new CTS compounds that exhibit anti-cancer activities
(30–32).
Oleandrin, for example, is in clinical trials in the United States as an
anti-cancer remedy for human cancers
(31,
33). Although ouabain inhibits
the pumping function of the Na/K-ATPase, it is important to note that the
growth inhibitory effect of ouabain can occur at doses that neither cause
significant changes in intracellular Na+ and K+ nor
affect cell viability. Rather, much like its effect on cell growth
stimulation, ouabain induces cell growth inhibition through the activation of
protein kinases and the generation of second messengers
(19–23,
34). For example, a recent
report showed that these nontoxic concentrations of ouabain stimulated Src,
resulting in activation of the epidermal growth factor receptor/ERK pathway
and induction of the expression of cell cycle inhibitor
p21cip and cell growth arrest
(34). Thus, it becomes
important to understand the molecular mechanisms that govern different fates
of cells in response to CTS stimulation.Prior studies have demonstrated that CTS induce endocytosis of the
Na/K-ATPase and regulate its cellular expression via receptor-mediated signal
transduction (35,
36). Because the Na/K-ATPase
has both pumping and signaling functions, it is conceivable that changes in
the amount of cellular Na/K-ATPase could have significant consequences on cell
growth. Therefore, we have conducted the following experiments to reveal the
role of cellular Na/K-ATPase in ouabain-induced cell growth regulation. 相似文献
11.
John Brognard Matthew Niederst Gloria Reyes Noel Warfel Alexandra C. Newton 《The Journal of biological chemistry》2009,284(22):15215-15223
PHLPP2 (PH domain leucine-rich repeat protein phosphatase 2) terminates Akt
and protein kinase C (PKC) activity by specifically dephosphorylating these
kinases at a key regulatory site, the hydrophobic motif (Ser-473 in Akt1).
Here we identify a polymorphism that results in an amino acid change from a
Leu to Ser at codon 1016 in the phosphatase domain of PHLPP2, which reduces
phosphatase activity toward Akt both in vitro and in cells, in turn
resulting in reduced apoptosis. Depletion of endogenous PHLPP2 variants in
breast cancer cells revealed the Ser-1016 variant is less functional toward
both Akt and PKC. In pair-matched high grade breast cancer samples we observed
retention of only the Ser allele from heterozygous patients (identical results
were observed in a pair-matched normal and tumor cell line). Thus, we have
identified a functional polymorphism that impairs the activity of PHLPP2 and
correlates with elevated Akt phosphorylation and increased PKC levels.Breast cancer is diagnosed in ∼180,000 women and is the cause of 40,000
deaths each year in the
U.S.2 A prevalent
underlying mechanism driving tumorigenesis is aberrant signal transduction
pathways that result in constitutive activation of cell growth, proliferation,
and survival pathways (2). A
well characterized signal transduction pathway in breast cancer that promotes
cellular survival, growth, and proliferation is the phosphatidylinositol
3-kinase/Akt pathway (3). This
pathway is activated by a number of mechanisms, including gene amplification
or gain of function mutations in upstream receptor protein-tyrosine kinases
(4,
5), constitutive activation of
hormone receptors (6),
activating mutations in phosphatidylinositol 3-kinase and Akt
(7,
8), and loss of function
mutations in the regulatory phosphatase
PTEN3 (phosphatase and
tensin homolog on chromosome ten)
(9). Thus, Akt is a major
regulator of breast tumorigenesis.There are three isoforms of Akt present in humans. All three isoforms
contain activating phosphorylation sites in the activation loop (Thr-308 in
Akt1) and in the C-terminal hydrophobic motif (Ser-473 in Akt1)
(10). Upon growth factor
receptor stimulation, phosphatidylinositol 3-kinase becomes activated and
phosphorylates the D3 position of, typically, phosphatidylinositol
(4,
5) bisphosphate to generate
phosphatidylinositol (3,4,5)-trisphosphate
(11). This
3′-phosphorylated lipid recruits Akt to the plasma membrane by binding
to its PH domain, resulting in conformational changes that allow access to the
activation loop phosphorylation site
(11). Constitutively bound
phosphatidylinositol-dependent kinase-1 then phosphorylates Akt at Thr-308,
accompanied by phosphorylation at Ser-473 resulting in a catalytically active
kinase (12). Phosphorylation
of Ser-473 depends on the mTORC2 complex
(13-16).
Signaling through this pathway is terminated by removal of the lipid second
messenger phosphatidylinositol (3,4,5)-trisphosphate catalyzed by the
phosphatase PTEN and by direct dephosphorylation of Akt by the
recently-identified PHLPP family of phosphatases and protein phosphatase
2A-type phosphatases
(17-20).The PHLPP family of phosphatases comprise three variants, the alternatively
spliced PHLPP1α and PHLPP1β, and PHLPP2
(21). PHLPP1 and PHLPP2
specifically dephosphorylate the hydrophobic motif of specific Akt isozymes,
thus decreasing Akt activity and promoting apoptosis
(18,
19). PHLPP2 binds and
dephosphorylates Akt1 and Akt3, whereas PHLPP1 binds and dephosphorylates Akt2
and Akt3 (18,
22). Their role in
inactivating Akt suggests that both PHLPP1 and PHLPP2 could be potential tumor
suppressors. Consistent with such a role, these phosphatases also
dephosphorylate the hydrophobic motif of PKC, resulting in degradation of PKC.
For this kinase, phosphorylation stabilizes the enzyme, so that the effect of
depletion of the PHLPP phosphatases is to increase PKC protein levels
(23). PKC is a well
characterized oncogene, and loss of function of the PHLPP phosphatases could
increase PKC protein levels and promote tumorigenesis
(24). Providing further
rationale that PHLPP2 could be a potential tumor suppressor, the phosphatase
is located on chromosome 16q22.3, a region that encounters frequent loss of
heterozygosity (LOH) in many primary and malignant breast tumors
(25).Here we identify a non-synonymous polymorphism that results in an amino
acid change from a Leu to a Ser at codon 1016 in the PP2C phosphatase domain
of PHLPP2. Overexpression studies reveal the Ser-1016 variant has impaired
phosphatase activity and is less effective at inducing apoptosis than the
Leu-1016 variant. When comparing a pair-matched normal and breast cancer cell
line or pair-matched normal and high grade tumor patient samples that are
heterozygous, we observe preferential loss of the Leu allele in the tumor
tissue or breast cancer cell line. This observation provides evidence that
PHLPP2 could be one of the elusive tumor suppressor genes on chromosome 16q,
and for heterozygous patients, loss of the more catalytically active Leu-1016
may promote breast tumorigenesis. 相似文献
12.
Maria T. Salgado Enika Nagababu Joseph M. Rifkind 《The Journal of biological chemistry》2009,284(19):12710-12718
Nitric oxide (NO) plays a crucial role in human physiology by regulating
vascular tone and blood flow. The short life-span of NO in blood requires a
mechanism to retain NO bioactivity in the circulation. Recent studies have
suggested a mechanism involving the reduction of nitrite back to NO by
deoxyhemoglobin in RBCs. A role for RBCs in transporting NO must, however,
bypass the scavenging of NO in RBCs by hemoglobin. To understand how the
nitrite reaction can deliver bioactive NO to the vasculature, we have studied
the intermediates formed during the reaction. A reliable measure of the total
concentration of heme-associated nitrite/NO intermediates formed was provided
by combining filtration to measure free nitrite by chemiluminescence and
electron paramagnetic resonance to measure the final product Hb(II)NO. By
modifying the chemiluminescence method used to detect NO, we have been able to
identify two intermediates: 1) a heme-associated nitrite complex that is
released as NO in acid solution in the presence of ascorbate and 2) an
intermediate that releases NO at neutral pH in the presence of ferricyanide
when reacted with an Fe(III) ligand like azide. This species designated as
“Hb(II)NO+ ⇆ Hb(III)NO” has properties of both
isomeric forms resulting in a slower NO dissociation rate and much higher
stability than Hb(III)NO, but provides a potential source for bioactive NO,
which can be released from the RBC. This detailed analysis of the nitrite
reaction with deoxyHb provides important insights into the mechanism for
nitrite induced vasodilation by RBCs.Nitric oxide (NO), also known as the endothelium-derived relaxing factor,
is an important messenger molecule involved in the regulation of vascular tone
and blood flow (1). The primary
source for the synthesis of NO in the circulatory system involves endothelial
nitric-oxide synthase (2). This
enzyme requires oxygen for the synthesis of NO and is, therefore, less
effective in the microcirculation where hypoxic vasodilation regulates the
delivery of oxygen. Because nitric oxide has a life-time in blood of <2 ms
(3), a mechanism is required to
allow for more distal and sustained effects of NO at the reduced oxygen
pressures found in the microcirculation. Recent studies have suggested that
the bioactivity of NO can be conserved in the blood by the uptake of NO and/or
nitrite by red blood cells
(RBCs)2 and its
interaction with hemoglobin
(4–7).
However, any role for the red cell in transporting nitric oxide must be able
to avoid the very efficient scavenging of nitric oxide by both oxyhemoglobin
(oxyHb) and deoxyhemoglobin (deoxyHb) that destroy and trap NO, respectively,
preventing a physiological role for RBC NO.In a series of studies, Stamler and co-workers
(7–10)
have hypothesized that NO can bypass this difficulty by being transferred to
the β-93 thiol group of hemoglobin (Hb) forming S-nitrosylated
hemoglobin (SNO-Hb) when partially heme nitrosylated hemoglobin (Hb(II)NO) is
oxygenated. The allosteric quaternary conformational change of hemoglobin at
low oxygen pressure destabilizes the β-93 nitrosylated thiol and results
in the transfer of NO to membrane thiol groups facilitating the release of the
NO to the plasma and the vasculature. However, the extremely low levels of
SNO-Hb (11) found in human
blood and its instability (12)
as a result of intracellular reducing conditions within the RBCs do not
support the SNO-Hb hypothesis as the major mechanism for NO transport
(11–13).The 2003 studies by Rifkind and Gladwin and their collaborators
(4,
5,
14,
15) proposed an alternative
mechanism that involved the reduction of nitrite, formed by the oxidation of
NO, back to NO by a reaction with deoxyHb. Nitrite is present in the blood at
fairly high levels (0.1–0.5 μmol/liter)
(4,
16–18),
and it is much more stable than NO or S-nitrosothiols
(6), making nitrite an ideal
storage pool that can be converted to NO. However, the mechanism by which the
NO produced in the red cell by nitrite reduction is exported without being
trapped or destroyed is still unclear. Recent studies by Rifkind and
co-workers (5,
13,
19) have suggested that the
trapping of NO by deoxyHb and/or oxyHb can be bypassed by the formation of a
metastable intermediate(s) that retains the NO in a state that is not quenched
by reacting with oxyHb or deoxyHb.In this report, we quantitate the two intermediate species that are formed
during the reduction of nitrite by deoxyHb when an excess of hemoglobin is
present. We also demonstrate that one of the intermediate species designated
as “Hb(II)NO+ ⇆ Hb(III)NO” has properties of
Hb(II)NO+ and Hb(III)NO, respectively. This species has a slower NO
dissociation rate and a much higher stability than Hb(III)NO. This
intermediate is a potential source for bioactive NO that can be released from
RBCs. 相似文献
13.
14.
Toru Sugiyama Bruce D. Levy Thomas Michel 《The Journal of biological chemistry》2009,284(19):12691-12700
Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial
isoform of NO synthase (eNOS) catalysis and is an important determinant of
NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells
in the setting of the oxidative stress associated with diabetes. However, the
relative roles of de novo BH4 synthesis and BH4 redox recycling in
the regulation of eNOS bioactivity remain incompletely defined. We used small
interference RNA (siRNA)-mediated “knockdown” GTP cyclohydrolase-1
(GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate
reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin
(BH2)), and studied the effects on eNOS regulation and biopterin metabolism in
cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1
attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and
NO production; these effects were recovered by supplementation with BH4. In
contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR
but not GTPCH1 knockdown increased reactive oxygen species (ROS) production.
The increase in ROS production seen with siRNA-mediated DHFR knockdown was
abolished either by simultaneous siRNA-mediated knockdown of eNOS or by
supplementing with BH4. In contrast, addition of BH2 increased ROS production;
this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1
knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory
site serine 116; these effects were recovered by supplementation with BH4.
These studies demonstrate a striking contrast in the pattern of eNOS
regulation seen by the selective modulation of BH4 salvage/reduction
versus de novo BH4 synthetic pathways. Our findings suggest that the
depletion of BH4 is not sufficient to perturb NO signaling, but rather that
concentration of intracellular BH2, as well as the relative concentrations of
BH4 and BH2, together play a determining role in the redox regulation of
eNOS-modulated endothelial responses.Regulation of endothelial nitric oxide
(NO)2 production
represents a critical mechanism for the modulation of vascular homeostasis. NO
is released by endothelial cells in response to diverse humoral, neural, and
mechanical stimuli
(1–4).
Endothelial cell-derived NO activates guanylate cyclase in vascular smooth
muscle cells, leading to increased levels of cGMP and to smooth muscle
relaxation. Blood platelets represent another key target for the actions of
endothelium-derived NO (5):
platelet aggregation is inhibited by NO-induced guanylate cyclase activation.
Many other effects of NO have been identified in cultured vascular cells and
in vascular tissues, including the regulation of apoptosis, cell adhesion,
angiogenesis, thrombosis, vascular smooth muscle proliferation, and
atherogenesis, among other cellular responses and (patho)physiological
processes.The endothelial isoform of NO synthase (eNOS) is a membrane-associated
homodimeric 135-kDa protein that is robustly expressed in endothelial cells
(2,
4,
6,
7). Similar to all the
mammalian NOS isoforms, eNOS functions as an obligate homodimer that includes
a cysteine-complex Zn2+ (zinc-tetrathiolate) at the dimer interface
(8–10).
eNOS is a Ca2+/calmodulin-dependent enzyme that is activated in
response to the stimulation of a variety of Ca2+-mobilizing cell
surface receptors in vascular endothelium and in cardiac myocytes. The
activity of eNOS is also regulated by phosphorylation at multiple sites
(11) that are differentially
modulated following the activation of cell surface receptors by agonists such
as insulin and vascular endothelial growth factor (VEGF)
(12). The phosphorylation of
eNOS at Ser-1179 activates eNOS, but phosphorylation at Thr-497 or Ser-116 is
associated with inhibition of eNOS activity
(13–17).
eNOS is reversibly targeted to plasmalemmal caveolae as a consequence of the
protein''s N-myristoylation and thiopalmitoylation. The generation of
NO by eNOS requires several redox-active cofactors, including nicotinamide
adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD),
flavin mononucleotide (FMN), calmodulin, and tetrahydrobiopterin (BH4), which
have key roles in the electron flow required for eNOS catalysis. If the flow
of electrons within eNOS is disrupted, the enzyme is uncoupled from NO
production and other redox-active products are generated, including hydrogen
peroxide and superoxide anion radical
(18,
19).In vascular disease states such as diabetes, endothelial dysfunction is
characterized by a decrease in NO bioactivity and by a concomitant increase in
superoxide formation, while eNOS mRNA and protein levels are maintained or
even increased. “Uncoupled” eNOS generates reactive oxygen species
(ROS), shifting the nitroso-redox balance and having adverse consequences in
the vascular wall (20).
Several enzymes expressed in vascular tissues contribute to the production and
efficient degradation of ROS, and an enhanced activity of oxidant enzymes
and/or reduced activity of antioxidant enzymes may cause oxidative stress.
Various agonists, pathological conditions, and therapeutic interventions lead
to modulated expression and function of oxidant and antioxidant enzymes.
However, the intimate relationship between intracellular redox state, eNOS
regulation, and NO bioavailability remains incompletely characterized.BH4 is a key redox-active cofactor for activity of all NOS enzymes
(21). The exact role of BH4 in
NOS catalysis is not yet completely defined, but this cofactor appears to
facilitate electron transfer from the eNOS reductase domain and maintains the
heme prosthetic group of the enzyme in its redox-active form
(18,
22,
23). Moreover, BH4 promotes
formation of active NOS homodimers
(24) and inhibits the
formation of hydrogen peroxide or superoxide by uncoupled eNOS
(18,
19). It has been reported that
the endothelial dysfunction associated with diabetes is accompanied a decrease
in the abundance of bioactive BH4. Supplementation with BH4 has been shown to
improve endothelial function in the models of diabetes and hypertension
(25,
26,
27). Moreover, BH4 oxidation
is seen in vascular cells in the setting of oxidative stress associated with
diabetes (28) and hypertension
(29).BH4 can be formed either by a de novo biosynthetic pathway or by a
salvage pathway. Guanosine triphosphate cyclohydrolase-1 (GTPCH1) catalyzes
the conversion of GTP to dihydroneopterin triphosphate. BH4 is generated by
further steps catalyzed by 6-pyruvoyltetrahydropterin synthase and sepiapterin
reductase (30). GTPCH1 appears
to be the rate-limiting enzyme in BH4 biosynthesis; overexpression of GTPCH1
is sufficient to augment BH4 levels in cultured endothelial cells
(31). On the other hand,
dihydrofolate reductase (DHFR) catalyzes the regeneration of BH4 from its
oxidized form, 7,8-dihydrobiopterin (BH2), in several cell types
(30,
32). DHFR is mainly involved
in folate metabolism and converts inactive BH2 back to BH4 and plays an
important role in the metabolism of exogenously administered BH4. However, the
relative contributions of endothelial GTPCH1 and DHFR to the modulation of
eNOS-dependent pathways are incompletely understood.In these studies, we have used siRNA-mediated “knockdown” of
GTPCH1 and DHFR to explore the relative roles of BH4 synthesis and recycling
in the modulation of eNOS bioactivity, as well as in the regulation of
NO-dependent signaling pathways in endothelial cells. 相似文献
15.
16.
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
17.
18.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
19.
Michele L. Forster James J. Mahn Billy Tsai 《The Journal of biological chemistry》2009,284(19):13045-13056
Protein-disulfide isomerase (PDI), an endoplasmic reticulum (ER)-resident
protein, is primarily known as a catalyst of oxidative protein folding but
also has a protein unfolding activity. We showed previously that PDI unfolds
the cholera toxin A1 (CTA1) polypeptide to facilitate the ER-to-cytosol
retrotranslocation of the toxin during intoxication. We now provide insight
into the mechanism of this unfoldase activity. PDI includes two redox-active
(a and a′) and two redox-inactive (b and
b′) thioredoxin-like domains, a linker (x), and a
C-terminal domain (c) arranged as
abb′xa′c. Using recombinant PDI
fragments, we show that binding of CTA1 by the continuous
PDIbb′xa′ fragment is necessary and sufficient
to trigger unfolding. The specific linear arrangement of
bb′xa′ and the type a domain
(a′ versus a) C-terminal to
bb′x are additional determinants of activity. These
data suggest a general mechanism for the unfoldase activity of PDI: the
concurrent and specific binding of bb′xa′ to
particular regions along the CTA1 molecule triggers its unfolding.
Furthermore, we show the bb′ domains of PDI are indispensable
to the unfolding reaction, whereas the function of its a′
domain can be substituted partially by the a′ domain from ERp57
(abb′xa′c) or ERp72
(ca°abb′xa′), PDI-like proteins
that do not unfold CTA1 normally. However, the bb′ domains of
PDI were insufficient to convert full-length ERp57 into an unfoldase because
the a domain of ERp57 inhibited toxin binding. Thus, we propose that
generating an unfoldase from thioredoxin-like domains requires the
bb′(x) domains of PDI followed by an a′
domain but not preceded by an inhibitory a domain.Protein-disulfide isomerase
(PDI)2 is a
multifunctional protein that resides in the endoplasmic reticulum (ER) lumen
of all eukaryotic cells (reviewed in Ref.
1). Mammalian PDI was first
identified as a catalyst of oxidative protein folding
(2), but it is now also known
to mediate viral infection (3,
4), antigen processing
(5), collagen assembly
(6), and ER-associated
degradation
(7–9).
To participate in this variety of cellular processes, PDI performs multiple
activities. For example, during oxidative protein folding, PDI catalyzes the
oxidation and isomerization of disulfide bonds and induces conformational
changes in non-native polypeptides
(10). Independently of redox
chemistry, PDI is a molecular chaperone, binding polypeptides to prevent their
aggregation
(11–13).
PDI also acts as a structural subunit of the prolyl 4-hydroxylase (P4H) and
microsomal triglyceride transfer protein complexes; however, this function is
similar to its chaperone activity
(14–19).
In contrast to its protein folding activities, PDI unfolds the catalytic A1
polypeptide of cholera toxin (CTA1) in preparation for the retrotranslocation
of the toxin from the ER lumen into the cytosol
(8,
20).Cholera toxin (CT) is a pathogenic factor that causes secretory diarrhea in
animals (reviewed in Ref. 21).
The holotoxin includes a single catalytic A subunit (CTA) and a homopentameric
B subunit (CTB) joined noncovalently
(22). Upon secretion from the
bacterium Vibrio cholerae, CTA is cleaved into the A1 and A2
polypeptides, which are joined by a disulfide bond and noncovalent
interactions (22,
23). To intoxicate a cell, CTB
binds the ganglioside GM1 on the surface of the cell, and the holotoxin is
transported in a retrograde manner to the ER lumen
(24). In the ER, CTA is
reduced to generate CTA1, and PDI unfolds and dissociates CTA1 from the
holotoxin (20). The unfolded
toxin is subsequently transported across the ER membrane
(25,
26). Upon reaching the
cytosol, CTA1 refolds and induces toxicity
(27,
28).We showed previously that PDI acts as a redox-dependent chaperone to unfold
CTA1 (20). In the reduced
state of PDI, it binds and unfolds the toxin. Subsequent oxidation of PDI by
ER oxidase 1 causes PDI to release unfolded CTA1
(25). Aside from this
information, nothing is known about the mechanism of the unfolding activity of
PDI.PDI is a modular protein comprising two a-type thioredoxin-like
domains (a and a′), two b-type
thioredoxin-like domains (b and b′), a flexible linker
(x), and an extended C-terminal domain (c) arranged as
abb′xa′c
(29–31).
The a-type domains are characterized by the presence of the catalytic
sequence CXXC and are therefore redox-active, whereas the
b-type domains lack this sequence and are redox-inactive
(32). The thioredoxin-like
domains of PDI differ from each other in primary structure despite having a
common fold. The crystal structure of yeast PDI shows the bb′
domains form a rigid base from which the a-type domains extend like
flexible arms (33,
34). This base is thought to
be the core of a substrate-binding groove formed by all four thioredoxin-like
domains (30,
33,
35).To understand the mechanism of the unfoldase activity of PDI, we analyzed
the contribution of each domain to the ability of PDI to bind and unfold CTA1
using recombinant PDI fragments. Unfolded CTA1 was detected by an established
in vitro trypsin sensitivity assay that relies on tryptic cleavage
sites hidden in the folded toxin to be exposed in the unfolded toxin
(20). Because CTA1 likely
mimics a misfolded host cell protein for its recognition and unfolding by PDI
(22,
36,
37), this study has
implications for how PDI unfolds endogenous misfolded proteins in preparation
for their retrotranslocation and subsequent ER-associated degradation.There are nearly 20 mammalian PDI-like proteins, characterized by the
presence of one or more thioredoxin-like domains and ER localization (reviewed
in Refs. 38,
39). We previously
demonstrated that two PDI-like proteins, ERp72 and ERp57, do not facilitate
CTA1 retrotranslocation (8). In
contrast to PDI, ERp72 retains CTA1 in the ER and either stabilizes its native
conformation or renders it more compact
(8). To understand how these
structurally homologous proteins are functionally unique, we tested whether
the various thioredoxin-like domains of ERp57 and ERp72 could functionally
replace the corresponding PDI domains to unfold CTA1. Thus, in addition to
suggesting a general mechanism for the unfoldase activity of PDI, our data
indicate functional similarities and differences among thioredoxin-like
domains of PDI family proteins. 相似文献
20.
Zhemin Zhou Yoshiteru Hashimoto Michihiko Kobayashi 《The Journal of biological chemistry》2009,284(22):14930-14938