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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.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
4.
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
5.
6.
7.
8.
As obligate intracellular parasites, viruses exploit diverse cellular
signaling machineries, including the mitogen-activated protein-kinase pathway,
during their infections. We have demonstrated previously that the open reading
frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90
ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities
(Kuang, E., Tang, Q., Maul, G. G., and Zhu, F.
(2008) J. Virol. 82
,1838
-1850). Here, we define the
mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45
to RSK increases the association of extracellular signal-regulated kinase
(ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass
protein complexes. We further demonstrated that the complexes shielded active
pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK
and ERK were activated and sustained at high levels. Finally, we provide
evidence that this mechanism contributes to the sustained activation of ERK
and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase
(ERK)2
mitogen-activated protein kinase (MAPK) signaling pathway has been implicated
in diverse cellular physiological processes including proliferation, survival,
growth, differentiation, and motility
(1-4)
and is also exploited by a variety of viruses such as Kaposi
sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human
immunodeficiency virus, respiratory syncytial virus, hepatitis B virus,
coxsackie, vaccinia, coronavirus, and influenza virus
(5-17).
The MAPK kinases relay the extracellular signaling through sequential
phosphorylation to an array of cytoplasmic and nuclear substrates to elicit
specific responses (1,
2,
18). Phosphorylation of MAPK
is reversible. The kinetics of deactivation or duration of signaling dictates
diverse biological outcomes
(19,
20). For example, sustained
but not transient activation of ERK signaling induces the differentiation of
PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells
(20-22).
During viral infection, a unique biphasic ERK activation has been observed for
some viruses (an early transient activation triggered by viral binding or
entry and a late sustained activation correlated with viral gene expression),
but the responsible viral factors and underlying mechanism for the sustained
ERK activation remain largely unknown
(5,
8,
13,
23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine
kinases that lie at the terminus of the ERK pathway
(1,
24-26).
In mammals, four isoforms are known, RSK1 to RSK4. Each one has two
catalytically functional kinase domains, the N-terminal kinase domain (NTKD)
and C-terminal kinase domain (CTKD) as well as a linker region between the
two. The NTKD is responsible for phosphorylation of exogenous substrates, and
the CTKD and linker region regulate RSK activation
(1,
24,
25). In quiescent cells ERK
binds to the docking site in the C terminus of RSK
(27-29).
Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase
(MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker
region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD
activation loop. The activated CTKD then phosphorylates Ser-380 in the linker
region, creating a docking site for 3-phosphoinositide-dependent protein
kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates
Ser-221 of RSK in the activation loop and activates the NTKD. The activated
NTKD autophosphorylates the serine residue near the ERK docking site, causing
a transient dissociation of active ERK from RSK
(25,
26,
28). The stimulation of
quiescent cells by a mitogen such as epidermal growth factor or a phorbol
ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually
results in a transient RSK activation that lasts less than 30 min. RSKs have
been implicated in regulating cell survival, growth, and proliferation.
Mutation or aberrant expression of RSK has been implicated in several human
diseases including Coffin-Lowry syndrome and prostate and breast cancers
(1,
24,
25,
30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma,
primary effusion lymphoma, and a subset of multicentric Castleman disease
(33,
34). Infection and
reactivation of KSHV activate multiple MAPK pathways
(6,
12,
35). Noticeably, the ERK/RSK
activation is sustained late during KSHV primary infection and reactivation
from latency (5,
6,
12,
23), but the mechanism of the
sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45,
an immediate early and also virion tegument protein of KSHV, interacts with
RSK1 and RSK2 and strongly stimulates their kinase activities
(23). We also demonstrated
that the activation of RSK plays an essential role in KSHV lytic replication
(23). In the present study we
determined the mechanism of ORF45-induced sustained ERK/RSK activation. We
found that ORF45 increases the association of RSK with ERK and protects them
from dephosphorylation, causing sustained activation of both ERK and RSK. 相似文献
9.
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
10.
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
11.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
12.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
13.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
14.
Jenny Erales Sabrina Lignon Brigitte Gontero 《The Journal of biological chemistry》2009,284(19):12735-12744
A new role is reported for CP12, a highly unfolded and flexible protein,
mainly known for its redox function with A4
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both reduced and oxidized
CP12 can prevent the in vitro thermal inactivation and aggregation of
GAPDH from Chlamydomonas reinhardtii. This mechanism is thus not
redox-dependent. The protection is specific to CP12, because other proteins,
such as bovine serum albumin, thioredoxin, and a general chaperone, Hsp33, do
not fully prevent denaturation of GAPDH. Furthermore, CP12 acts as a specific
chaperone, since it does not protect other proteins, such as catalase, alcohol
dehydrogenase, or lysozyme. The interaction between CP12 and GAPDH is
necessary to prevent the aggregation and inactivation, since the mutant C66S
that does not form any complex with GAPDH cannot accomplish this protection.
Unlike the C66S mutant, the C23S mutant that lacks the N-terminal bridge is
partially able to protect and to slow down the inactivation and aggregation.
Tryptic digestion coupled to mass spectrometry confirmed that the S-loop of
GAPDH is the interaction site with CP12. Thus, CP12 not only has a redox
function but also behaves as a specific “chaperone-like protein”
for GAPDH, although a stable and not transitory interaction is observed. This
new function of CP12 may explain why it is also present in complexes involving
A2B2 GAPDHs that possess a regulatory C-terminal
extension (GapB subunit) and therefore do not require CP12 to be
redox-regulated.CP12 is a small 8.2-kDa protein present in the chloroplasts of most
photosynthetic organisms, including cyanobacteria
(1,
2), higher plants
(3), the diatom
Asterionella formosa
(4,
5), and green
(1) and red algae
(6). It allows the formation of
a supramolecular complex between phosphoribulokinase (EC 2.7.1.19) and
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH),3 two key
enzymes of the Calvin cycle pathway, and was recently shown to interact with
fructose bisphosphate aldolase, another enzyme of the Calvin cycle pathway
(7). The
phosphoribulokinase·GAPDH·CP12 complex has been extensively
studied in Chlamydomonas reinhardtii
(8,
9) and in Arabidopsis
thaliana (10,
11). In the green alga C.
reinhardtii, the interaction between CP12 and GAPDH is strong
(8). GAPDH may exist as a
homotetramer composed of four GapA subunits (A4) in higher plants,
cyanobacteria, and green and red algae
(6,
12), but in higher plants, it
can also exist as a heterotetramer (A2B2), composed of
two subunits, GapA and GapB
(13,
14). GapB, up to now, has
exclusively been found in Streptophyta, but recently two
prasinophycean green algae, Ostreococcus tauri and Ostreococcus
lucimarinus, were also shown to possess a GapB gene, whereas
CP12 is missing (15).
The GapB subunit is similar to the GapA subunit but has a C-terminal extension
containing two redox-regulated cysteine residues
(16). Thus, although the
A4 GAPDHs lack these regulatory cysteine residues
(13,
14,
17–20),
they are also redox-regulated through its interaction with CP12, since the C
terminus of this small protein resembles the C-terminal extension of the GapB
subunit. The regulatory cysteine residues for GapA are thus supplied by CP12,
as is well documented in the literature
(1,
8,
11,
16).CP12 belongs to the family of intrinsically unstructured proteins (IUPs)
(21–26).
The amino acid composition of these proteins causes them to have no or few
secondary structures. Their total or partial lack of structure and their high
flexibility allow them to be molecular adaptors
(27,
28). They are often able to
bind to several partners and are involved in most cellular functions
(29,
30). Recently, some IUPs have
been described in photosynthetic organisms
(31,
32).There are many functional categories of IUPs
(22,
33). They can be, for
instance, involved in permanent binding and have (i) a scavenger role,
neutralizing or storing small ligands; (ii) an assembler role by forming
complexes; and (iii) an effector role by modulating the activity of a partner
molecule (33). These functions
are not exclusive; thus, CP12 can form a stable complex with GAPDH, regulating
its redox properties (8,
34,
35), and can also bind a metal
ion (36,
37). IUPs can also bind
transiently to partners, and some of them have been found to possess a
chaperone activity (31,
38). This chaperone function
was first shown for α-synuclein
(39) and for α-casein
(40), which are fully
disordered. The amino acid composition of IUPs is less hydrophobic than those
of soluble proteins; hence, they lack hydrophobic cores and do not become
insoluble when heated. Since CP12 belongs to this family, we tested if it was
resistant to heat treatment and finally, since it is tightly bound to GAPDH,
if it could prevent aggregation of its partner, GAPDH, an enzyme well known
for its tendency to aggregate
(41–44)
and consequently a substrate commonly used in chaperone studies
(45,
46).Unlike chaperones, which form transient, dynamic complexes with their
protein substrates through hydrophobic interactions
(47,
48), CP12 forms a stable
complex with GAPDH. The interaction involves the C-terminal part of the
protein and the presence of negatively charged residues on CP12
(35). However, only a
site-directed mutagenesis has been performed to characterize the interaction
site on GAPDH. Although the mutation could have an indirect effect, the
residue Arg-197 was shown to be a good candidate for the interaction site
(49).In this report, we accordingly used proteolysis experiments coupled with
mass spectrometry to detect which regions of GAPDH are protected by its
association with CP12. To conclude, the aim of this report was to characterize
a chaperone function of CP12 that had never been described before and to map
the interaction site on GAPDH using an approach that does not involve
site-directed mutagenesis. 相似文献
15.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
16.
Hongjie Yuan Katie M. Vance Candice E. Junge Matthew T. Geballe James P. Snyder John R. Hepler Manuel Yepes Chian-Ming Low Stephen F. Traynelis 《The Journal of biological chemistry》2009,284(19):12862-12873
Zinc is hypothesized to be co-released with glutamate at synapses of the
central nervous system. Zinc binds to NR1/NR2A
N-methyl-d-aspartate (NMDA) receptors with high affinity
and inhibits NMDAR function in a voltage-independent manner. The serine
protease plasmin can cleave a number of substrates, including
protease-activated receptors, and may play an important role in several
disorders of the central nervous system, including ischemia and spinal cord
injury. Here, we demonstrate that plasmin can cleave the native NR2A
amino-terminal domain (NR2AATD), removing the functional high
affinity Zn2+ binding site. Plasmin also cleaves recombinant
NR2AATD at lysine 317 (Lys317), thereby producing a
∼40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments
observed in rat brain membrane preparations. A homology model of the
NR2AATD predicts that Lys317 is near the surface of the
protein and is accessible to plasmin. Recombinant expression of NR2A with an
amino-terminal deletion at Lys317 is functional and Zn2+
insensitive. Whole cell voltage-clamp recordings show that Zn2+
inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected
HEK 293 cells and cultured cortical neurons is significantly reduced by
plasmin treatment. Mutating the plasmin cleavage site Lys317 on
NR2A to alanine blocks the effect of plasmin on Zn2+ inhibition.
The relief of Zn2+ inhibition by plasmin occurs in
PAR1-/- cortical neurons and thus is independent of interaction
with protease-activated receptors. These results suggest that plasmin can
directly interact with NMDA receptors, and plasmin may increase NMDA receptor
responses through disruption or removal of the amino-terminal domain and
relief of Zn2+ inhibition.N-Methyl-d-aspartate
(NMDA)2 receptors are
one of three types of ionotropic glutamate receptors that play critical roles
in excitatory neurotransmission, synaptic plasticity, and neuronal death
(1–3).
NMDA receptors are comprised of glycine-binding NR1 subunits in combination
with at least one type of glutamate-binding NR2 subunit
(1,
4). Each subunit contains three
transmembrane domains, one cytoplasmic re-entrant membrane loop, one bi-lobed
domain that forms the ligand binding site, and one bi-lobed amino-terminal
domain (ATD), thought to share structural homology to periplasmic amino
acid-binding proteins
(4–6).
Activation of NMDA receptors requires combined stimulation by glutamate and
the co-agonist glycine in addition to membrane depolarization to overcome
voltage-dependent Mg2+ block of the ion channel
(7). The activity of NMDA
receptors is negatively modulated by a variety of extracellular ions,
including Mg2+, polyamines, protons, and Zn2+ ions,
which can exert tonic inhibition under physiological conditions
(1,
4). Several extracellular
modulators such as Zn2+ and ifenprodil are thought to act at the
ATD of the NMDA receptor
(8–14).Zinc is a transition metal that plays key roles in both catalytic and
structural capacities in all mammalian cells
(15). Zinc is required for
normal growth and survival of cells. In addition, neuronal death in
hypoxia-ischemia and epilepsy has been associated with Zn2+
(16–18).
Abnormal metabolism of zinc may contribute to induction of cytotoxicity in
neurodegenerative diseases, such as Alzheimer''s disease, Parkinson''s disease,
and amyotrophic lateral sclerosis
(19). Zinc is co-released with
glutamate at excitatory presynaptic terminals and inhibits native NMDA
receptor activation (20,
21). Zn2+ inhibits
NMDA receptor function through a dual mechanism, which includes
voltage-dependent block and voltage-independent inhibition
(22–24).
Voltage-independent Zn2+ inhibition at low nanomolar concentrations
(IC50, 20 nm) is observed for NR2A-containing NMDA
receptors
(25–28).
Evidence has accumulated that the amino-terminal domain of the NR2A subunit
controls high-affinity Zn2+ inhibition of NMDA receptors, and
several histidine residues in this region may constitute part of an
NR2A-specific Zn2+ binding site
(8,
9,
11,
12). For the NR2A subunit,
several lines of evidence suggest that Zn2+ acts by enhancing
proton inhibition (8,
11,
29,
30).Serine proteases present in the circulation, mast cells, and elsewhere
signal directly to cells by cleaving protease-activated receptors (PARs),
members of a subfamily of G-protein-coupled receptors. Cleavage exposes a
tethered ligand domain that binds to and activates the cleaved receptors
(31,
32). Protease receptor
activation has been studied extensively in relation to coagulation and
thrombolysis (33). In addition
to their circulation in the bloodstream, some serine proteases and PARs are
expressed in the central nervous system, and have been suggested to play roles
in physiological conditions (e.g. long-term potentiation or memory)
and pathophysiological states such as glial scarring, edema, seizure, and
neuronal death (31,
34–36).Functional interactions between proteases and NMDA receptors have
previously been suggested. Earlier studies reported that the blood-derived
serine protease thrombin potentiates NMDA receptor response more than 2-fold
through activation of PAR1
(37). Plasmin, another serine
protease, similarly potentiates NMDA receptor response
(38). Tissue-plasminogen
activator (tPA), which catalyzes the conversion of the zymogen precursor
plasminogen to plasmin and results in PAR1 activation, also interacts with and
cleaves the ATD of the NR1 subunit of the NMDA receptor
(39,
40). This raises the
possibility that plasmin may also interact directly with the NMDA receptor
subunits to modulate receptor response. We therefore investigated the ability
of plasmin to cleave the NR2A NMDA receptor subunit. We found that nanomolar
concentrations of plasmin can cleave within the ATD, a region that mediates
tonic voltage-independent Zn2+ inhibition of NR2A-containing NMDA
receptors. We hypothesized that plasmin cleavage reduces the
Zn2+-mediated inhibition of NMDA receptors by removing the
Zn2+ binding domain. In the present study, we have demonstrated
that Zn2+ inhibition of agonist-evoked NMDA currents is decreased
significantly by plasmin treatment in recombinant NR1/NR2A-transfected HEK 293
cells and cultured cortical neurons. These concentrations of plasmin may be
pathophysiologically relevant in situations in which the blood-brain barrier
is compromised, which could allow blood-derived plasmin to enter brain
parenchyma at concentrations in excess of these that can cleave NR2A. Thus,
ability of plasmin to potentiate NMDA function through the relief of the
Zn2+ inhibition could exacerbate the harmful actions of NMDA
receptor overactivation in pathological situations. In addition, if newly
cleaved NR2AATD enters the bloodstream during ischemic injury, it
could serve as a biomarker of central nervous system injury. 相似文献
17.
Sophie Pattingre Chantal Bauvy St��phane Carpentier Thierry Levade Beth Levine Patrice Codogno 《The Journal of biological chemistry》2009,284(5):2719-2728
Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated
during periods of nutrient starvation to preserve cell integrity. Ceramide is
a bioactive sphingolipid associated with a large range of cell processes. Here
we show that short-chain ceramides (C2-ceramide and
C6-ceramide) and stimulation of the de novo ceramide
synthesis by tamoxifen induce the dissociation of the complex formed between
the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This
dissociation is required for macroautophagy to be induced either in response
to ceramide or to starvation. Three potential phosphorylation sites,
Thr69, Ser70, and Ser87, located in the
non-structural N-terminal loop of Bcl-2, play major roles in the dissociation
of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal
protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to
stimulate macroautophagy. These findings reveal a new aspect of sphingolipid
signaling in up-regulating a major cell process involved in cell adaptation to
stress.Macroautophagy (referred to below as “autophagy”) is a
vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is
conserved in eukaryotic cells
(1–3).
Autophagy is initiated by the formation of a multimembrane-bound autophagosome
that engulfs cytoplasmic proteins and organelles. The last stage in the
process results in fusion with the lysosomal compartments, where the
autophagic cargo undergoes degradation. Basal autophagy is important in
controlling the quality of the cytoplasm by removing damaged organelles and
protein aggregates. Inhibition of basal autophagy in the brain is deleterious,
and leads to neurodegeneration in mouse models
(4,
5). Stimulation of autophagy
during periods of nutrient starvation is a physiological response present at
birth and has been shown to provide energy in various tissues of newborn pups
(6). In cultured cells,
starvation-induced autophagy is an autonomous cell survival mechanism, which
provides nutrients to maintain a metabolic rate and level of ATP compatible
with cell survival (7). In
addition, starvation-induced autophagy blocks the induction of apoptosis
(8). In other contexts, such as
drug treatment and a hypoxic environment, autophagy has also been shown to be
cytoprotective in cancer cells
(9,
10). However, autophagy is
also part of cell death pathways in certain situations
(11). Autophagy can be a
player in apoptosis-independent type-2 cell death (type-1 cell death is
apoptosis), also known as autophagic cell death. This situation has been shown
to occur when the apoptotic machinery is crippled in mammalian cells
(12,
13). Autophagy can also be
part of the apoptotic program, for instance in tumor necrosis
factor-α-induced cell death when NF-κB is inhibited
(14), or in human
immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T
cells (15). Moreover autophagy
has recently been shown to be required for the externalization of
phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of
apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the
intertwined regulation of these processes
(17,
18). Many signaling pathways
involved in the regulation of autophagy also regulate apoptosis. This
intertwining has recently been shown to occur at the level of the molecular
machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been
shown to inhibit starvation-induced autophagy by interacting with the
autophagy protein Beclin 1
(19). Beclin 1 is one of the
Atg proteins conserved from yeast to humans (it is the mammalian orthologue of
yeast Atg6) and is involved in autophagosome formation
(20). Beclin 1 is a platform
protein that interacts with several different partners, including hVps34
(class III phosphatidylinositol 3-kinase), which is responsible for the
synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is
important for events associated with the nucleation of the isolation membrane
before it elongates and closes to form autophagosomes in response to other Atg
proteins, including the Atg12 and
LC32
(microtubule-associated protein light chain 3 is the mammalian orthologue of
the yeast Atg8) ubiquitin-like conjugation systems
(3,
21). Various partners
associated with the Beclin 1 complex modulate the activity of hVps34. For
instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1,
and Bif-1 all up-regulate it
(22,
23).In view of the intertwining between autophagy and apoptosis, it is
noteworthy that Beclin 1 belongs to the BH3-only family of proteins
(24–26).
However, and unlike most of the proteins in this family, Beclin 1 is not able
to trigger apoptosis when its expression is forced in cells
(27). A BH3-mimetic drug,
ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger
autophagy by mirroring the effect of starvation
(25).The sphingolipids constitute a family of bioactive lipids
(28–32)
of which several members, such as ceramide and sphingosine 1-phosphate, are
signaling molecules. These molecules constitute a “sphingolipid
rheostat” that determines the fate of the cell, because in many settings
ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic
effect (31,
32). However, ceramide is also
engaged in a wide variety of other cell processes, such as the formation of
exosomes (33),
differentiation, cell proliferation, and senescence
(34). Recently we showed that
both ceramide and sphingosine 1-phosphate are able to stimulate autophagy
(35,
36). It has also been shown
that ceramide triggers autophagy in a large panel of mammalian cells
(37–39).
However, elucidation of the mechanism by which ceramide stimulates autophagy
is still in its infancy. We have previously demonstrated that ceramide induces
autophagy in breast and colon cancer cells by inhibiting the Class I
phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central
role in inhibiting autophagy
(36). Inhibition of mTOR is
another hallmark of starvation-induced autophagy
(17). This finding led us to
investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results
presented here show that ceramide is more potent than starvation in
dissociating the Beclin 1-Bcl-2 complex (see Ref.
40). This dissociation is
dependent on three phosphorylation sites (Thr69, Ser70,
and Ser87) located in a non-structural loop of Bcl-2. Ceramide
induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2.
Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation,
and thus the induction of autophagy by ceramide. These findings help to
explain how autophagy is regulated by a major lipid second messenger. 相似文献
18.
19.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
20.