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1.
Nanako Masada Antonio Ciruela David A. MacDougall Dermot M. F. Cooper 《The Journal of biological chemistry》2009,284(7):4451-4463
Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified.
Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are
stimulated by Ca2+/calmodulin (CaM), have discrete neuronal
functions. Although the Ca2+ sensitivity of AC1 is higher than that
of AC8, precisely how these two ACs are regulated by Ca2+/CaM
remains elusive, and the basis for their diverse physiological roles is quite
unknown. Distinct localization of the CaM binding domains within the two
enzymes may be essential to differential regulation of the ACs by
Ca2+/CaM. In this study we compare in detail the regulation of AC1
and AC8 by Ca2+/CaM both in vivo and in vitro and
explore the different role of each Ca2+-binding lobe of CaM in
regulating the two enzymes. We also assess the relative dependence of AC1 and
AC8 on capacitative Ca2+ entry. Finally, in real-time fluorescence
resonance energy transfer-based imaging experiments, we examine the effects of
dynamic Ca2+ events on the production of cAMP in cells expressing
AC1 and AC8. Our data demonstrate distinct patterns of regulation and
Ca2+ dependence of AC1 and AC8, which seems to emanate from their
mode of regulation by CaM. Such distinctive properties may contribute
significantly to the divergent physiological roles in which these ACs have
been implicated.Nine membrane-bound mammalian adenylyl cyclases
(ACs),2 AC1–AC9,
have been identified (1). They
possess a common predicted structure
(2)3
and are stimulated by forskolin (FSK; except AC9) and Gsα,
although they are distributed and regulated differently
(1,
3,
4). Four ACs are regulated by
physiological concentrations of Ca2+ and thereby provide a critical
link between the Ca2+- and cAMP-signaling pathways
(3,
5); AC5 and AC6 are directly
inhibited by Ca2+, whereas AC1 and AC8 are stimulated by
Ca2+ in a calmodulin (CaM)-dependent manner
(5). AC3 is also regulated by
CaM in vitro, although this requires supramicromolar concentration of
Ca2+ (6), and in
vivo AC3 is inhibited by Ca2+ via CaM kinase II
(7), unlike AC1 and AC8.AC1 is closely related in sequence to the Ca2+/CaM-stimulable
rutabaga AC from Drosophila, which is important in
Drosophila learning tasks
(8–10).
AC1 and the other Ca2+/CaM-stimulable mammalian AC, AC8, have also
been implicated in learning and memory
(11). As a means of
establishing their proposed roles, single and/or double AC1 and AC8 knockout
mice have been generated. Mouse models have demonstrated that
Ca2+/CaM-stimulable ACs are involved in long-term potentiation and
long-term memory (12).
However, despite the general view that AC1 and AC8 can behave similarly,
discrete physiological actions of each isoform are becoming apparent. Recent
studies by Zhuo''s group demonstrated that AC1 specifically participates in
N-methyl-d-aspartic acid receptor-induced neuronal
excitotoxicity (13) and an
increase in GluR1 synthesis induced by blocking AMPA receptors
(14). Furthermore, Nicol and
colleagues (15,
16) showed a contribution of
AC1, but not AC8, in axon terminal refinement in the retina. On the other
hand, AC8 specifically was seen to be responsible for retrieval from adaptive
presynaptic silencing (17) and
the acquiring of new spatial information
(18). These differences in
physiological roles must reflect not only differences in their distributions
but also presumably in their regulatory properties. Both enzymes are expressed
in brain; AC1 is neuro-specific, whereas the expression of AC8 is more
widespread (1,
12). Within the central
nervous system, AC1 is abundant in the hippocampus, the cerebral cortex, and
the granule cells of the cerebellum, whereas AC8 has a high expression level
in the thalamus and the cerebral cortex
(19). Studies of mouse brain
revealed that AC1 is distributed pre-synaptically and AC8 post-synaptically
(18,
20).Although physiological differences in the roles of these two enzymes are
suggested from the studies outlined above, the regulatory mechanisms that
might underlie these differences are not. AC1 is more sensitive to
Ca2+ than is AC8 in vitro
(21,
22), yet details on how these
two enzymes are regulated by Ca2+/CaM are sparse. In non-excitable
cells, a Ca2+ elevation caused by capacitative Ca2+
entry (CCE), the mode of Ca2+ entry triggered by emptying
Ca2+ from internal stores
(23), preferentially
stimulates AC1 and AC8 (21).
Although stimulation of AC8 by CCE has been shown to be at least partially
dependent on its localization at lipid rafts
(24), whether AC1 is also
targeted to this region of plasma membranes has never been addressed. In
addition, CaM regulation of AC1 and AC8 has not been compared in detail,
although CaM appears to bind to different domains of the two enzymes. AC8
utilizes two CaM binding domains: a classic amphipathic “1-5-8-14”
motif at the N terminus and an IQ-like motif in the C2b domain
(25). A recent study indicates
that CaM pre-associates with the N terminus of AC8, where it becomes fully
saturated upon a Ca2+ rise, and activates the enzyme via a
C-terminally mediated relief of auto-inhibitory mechanisms
(26). By contrast, only
residues 495–522 of the C1b region of AC1 have been shown to bind CaM in
a Ca2+-dependent manner
(27,
28). With the presence of only
one CaM binding domain in AC1, a simpler mechanism of CaM regulation might be
expected.CaM mediates the regulation of numerous Ca2+-dependent processes
in eukaryotic cells (29). The
protein possesses N- and C-terminal lobes, both of which contain two
Ca2+ binding EF hands (EF1 and EF2 in the N lobe, and EF3 and EF4
in the C lobe (30)). Mutations
in the EF hands have been valuable for investigating the interaction of CaM
with its targets. Alanine substitutions in the EF12 pair or EF34 pair have
generated CaM12 and CaM34 to investigate the independent
function of the C and N lobes of CaM, respectively
(31,
32).Against the background of the distinct physiological roles carried out by
AC1 and AC8, we performed a detailed comparison of the two enzymes expressed
in HEK 293 cells. Their sensitivity to Ca2+/CaM was compared both
in vitro and in vivo; the possibility that they might be
expressed in different domains of the plasma membrane was addressed; and
putative lobe-specific CaM regulation was assessed using
Ca2+-binding mutants of CaM. Single cell measurements using a
FRET-based cAMP sensor were performed to compare the kinetic responses of the
enzymes to physiological elevations of [Ca2+]i.
The results demonstrate superficial similarities in the regulation of AC1 and
AC8 but critical disparities in their mechanism of activation by the lobes of
CaM and in the speed and pattern of their responsiveness to
[Ca2+]i. These discrete behaviors provide a
physiological opportunity for different outcomes to elevation of
[Ca2+]i, depending on the AC that is expressed
in particular contexts. 相似文献
2.
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
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.
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
5.
Congmin Li Jenny Chan Franciose Haeseleer Katsuhiko Mikoshiba Krzysztof Palczewski Mitsuhiko Ikura James B. Ames 《The Journal of biological chemistry》2009,284(4):2472-2481
Calcium-binding protein 1 (CaBP1), a neuron-specific member of the
calmodulin (CaM) superfamily, modulates Ca2+-dependent activity of
inositol 1,4,5-trisphosphate receptors (InsP3Rs). Here we present
NMR structures of CaBP1 in both Mg2+-bound and
Ca2+-bound states and their structural interaction with
InsP3Rs. CaBP1 contains four EF-hands in two separate domains. The
N-domain consists of EF1 and EF2 in a closed conformation with Mg2+
bound at EF1. The C-domain binds Ca2+ at EF3 and EF4, and exhibits
a Ca2+-induced closed to open transition like that of CaM. The
Ca2+-bound C-domain contains exposed hydrophobic residues
(Leu132, His134, Ile141, Ile144,
and Val148) that may account for selective binding to
InsP3Rs. Isothermal titration calorimetry analysis reveals a
Ca2+-induced binding of the CaBP1 C-domain to the N-terminal region
of InsP3R (residues 1-587), whereas CaM and the CaBP1 N-domain did
not show appreciable binding. CaBP1 binding to InsP3Rs requires
both the suppressor and ligand-binding core domains, but has no effect on
InsP3 binding to the receptor. We propose that CaBP1 may regulate
Ca2+-dependent activity of InsP3Rs by promoting
structural contacts between the suppressor and core domains.Calcium ion (Ca2+) in the cell functions as an important
messenger that controls neurotransmitter release, gene expression, muscle
contraction, apoptosis, and disease processes
(1). Receptor stimulation in
neurons promotes large increases in intracellular Ca2+ levels
controlled by Ca2+ release from intracellular stores through
InsP3Rs (2). The
neuronal type-1 receptor
(InsP3R1)2
is positively and negatively regulated by cytosolic Ca2+
(3-6),
important for the generation of repetitive Ca2+ transients known as
Ca2+ spikes and waves
(1). Ca2+-dependent
activation of InsP3R1 contributes to the fast rising phase of
Ca2+ signaling known as Ca2+-induced Ca2+
release (7).
Ca2+-induced inhibition of InsP3R1, triggered at higher
cytosolic Ca2+ levels, coordinates the temporal decay of
Ca2+ transients (6).
The mechanism of Ca2+-dependent regulation of InsP3Rs is
complex (8,
9), and involves direct
Ca2+ binding sites
(5,
10) as well as remote sensing
by extrinsic Ca2+-binding proteins such as CaM
(11,
12), CaBP1
(13,
14), CIB1
(15), and NCS-1
(16).Neuronal Ca2+-binding proteins (CaBP1-5
(17)) represent a new
sub-branch of the CaM superfamily
(18) that regulate various
Ca2+ channel targets. Multiple splice variants and isoforms of
CaBPs are localized in different neuronal cell types
(19-21)
and perform specialized roles in signal transduction. CaBP1, also termed
caldendrin (22), has been
shown to modulate the Ca2+-sensitive activity of InsP3Rs
(13,
14). CaBP1 also regulates
P/Q-type voltage-gated Ca2+ channels
(23), L-type channels
(24), and the transient
receptor potential channel, TRPC5
(25). CaBP4 regulates
Ca2+-dependent inhibition of L-type channels in the retina and may
be genetically linked to retinal degeneration
(26). Thus, the CaBP proteins
are receiving increased attention as a family of Ca2+ sensors that
control a variety of Ca2+ channel targets implicated in neuronal
degenerative diseases.CaBP proteins contain four EF-hands, similar in sequence to those found in
CaM and troponin C (18)
(Fig. 1). By analogy to CaM
(27), the four EF-hands are
grouped into two domains connected by a central linker that is four residues
longer in CaBPs than in CaM. In contrast to CaM, the CaBPs contain
non-conserved amino acids within the N-terminal region that may confer target
specificity. Another distinguishing property of CaBPs is that the second
EF-hand lacks critical residues required for high affinity Ca2+
binding (17). CaBP1 binds
Ca2+ only at EF3 and EF4, whereas it binds Mg2+ at EF1
that may serve a functional role
(28). Indeed, changes in
cytosolic Mg2+ levels have been detected in cortical neurons after
treatment with neurotransmitter
(29). Other neuronal
Ca2+-binding proteins such as DREAM
(30), CIB1
(31), and NCS-1
(32) also bind Mg2+
and exhibit Mg2+-induced physiological effects. Mg2+
binding in each of these proteins helps stabilize their Ca2+-free
state to interact with signaling targets.Open in a separate windowFIGURE 1.Amino acid sequence alignment of human CaBP1 with CaM. Secondary
structural elements (α-helices and β-strands) were derived from NMR
analysis. The four EF-hands (EF1, EF2, EF3, and EF4) are highlighted
green, red, cyan, and yellow. Residues in the 12-residue
Ca2+-binding loops are underlined and chelating residues
are highlighted bold. Non-conserved residues in the hydrophobic patch
are colored red.Despite extensive studies on CaBP1, little is known about its structure and
target binding properties, and regulation of InsP3Rs by CaBP1 is
somewhat controversial and not well understood. Here, we present the NMR
solution structures of both Mg2+-bound and Ca2+-bound
conformational states of CaBP1 and their structural interactions with
InsP3R1. These CaBP1 structures reveal important
Ca2+-induced structural changes that control its binding to
InsP3R1. Our target binding analysis demonstrates that the C-domain
of CaBP1 exhibits Ca2+-induced binding to the N-terminal cytosolic
region of InsP3R1. We propose that CaBP1 may regulate
Ca2+-dependent channel activity in InsP3Rs by promoting
a structural interaction between the N-terminal suppressor and ligand-binding
core domains that modulates Ca2+-dependent channel gating
(8,
33,
34). 相似文献
6.
7.
8.
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
9.
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
10.
11.
12.
Yuusuke Maruyama Toshihiko Ogura Kazuhiro Mio Kenta Kato Takeshi Kaneko Shigeki Kiyonaka Yasuo Mori Chikara Sato 《The Journal of biological chemistry》2009,284(20):13676-13685
The Ca2+ release-activated Ca2+ channel is a
principal regulator of intracellular Ca2+ rise, which conducts
various biological functions, including immune responses. This channel,
involved in store-operated Ca2+ influx, is believed to be composed
of at least two major components. Orai1 has a putative channel pore and
locates in the plasma membrane, and STIM1 is a sensor for luminal
Ca2+ store depletion in the endoplasmic reticulum membrane. Here we
have purified the FLAG-fused Orai1 protein, determined its tetrameric
stoichiometry, and reconstructed its three-dimensional structure at 21-Å
resolution from 3681 automatically selected particle images, taken with an
electron microscope. This first structural depiction of a member of the Orai
family shows an elongated teardrop-shape 150Å in height and 95Å in
width. Antibody decoration and volume estimation from the amino acid sequence
indicate that the widest transmembrane domain is located between the round
extracellular domain and the tapered cytoplasmic domain. The cytoplasmic
length of 100Å is sufficient for direct association with STIM1. Orifices
close to the extracellular and intracellular membrane surfaces of Orai1 seem
to connect outside the molecule to large internal cavities.Ca2+ is an intracellular second messenger that plays important
roles in various physiological functions such as immune response, muscle
contraction, neurotransmitter release, and cell proliferation. Intracellular
Ca2+ is mainly stored in the endoplasmic reticulum
(ER).2 This ER system
is distributed through the cytoplasm from around the nucleus to the cell
periphery close to the plasma membrane. In non-excitable cells, the ER
releases Ca2+ through the inositol 1,4,5-trisphosphate
(IP3) receptor channel in response to various signals, and the
Ca2+ store is depleted. Depletion of Ca2+ then induces
Ca2+ influx from outside the cell to help in refilling the
Ca2+ stores and to continue Ca2+ rise for several
minutes in the cytoplasm (1,
2). This Ca2+ influx
was first proposed by Putney
(3) and was named
store-operated Ca2+ influx. In the immune system, store-operated
Ca2+ influx is mainly mediated by the Ca2+
release-activated Ca2+ (CRAC) current, which is a highly
Ca2+-selective inwardly rectified current with low conductance
(4,
5). Pathologically, the loss of
CRAC current in T cells causes severe combined immunodeficiency
(6) where many Ca2+
signal-dependent gene expressions, including cytokines, are interrupted
(7). Therefore, CRAC current is
necessary for T cell functions.Recently, Orai1 (also called CRACM1) and STIM1 have been physiologically
characterized as essential components of the CRAC channel
(8–12).
They are separately located in the plasma membrane and in the ER membrane;
co-expression of these proteins presents heterologous CRAC-like currents in
various types of cells (10,
13–15).
Both of them are shown to be expressed ubiquitously in various tissues
(16–18).
STIM1 senses Ca2+ depletion in the ER through its EF hand motif
(19) and transmits a signal to
Orai1 in the plasma membrane. Although Orai1 is proposed as a regulatory
component for some transient receptor potential canonical channels
(20,
21), it is believed from the
mutation analyses to be the pore-forming subunit of the CRAC channel
(8,
22–24).
In the steady state, both Orai1 and STIM1 molecules are dispersed in each
membrane. When store depletion occurs, STIM1 proteins gather into clusters to
form puncta in the ER membrane near the plasma membrane
(11,
19). These clusters then
trigger the clustering of Orai1 in the plasma membrane sites opposite the
puncta (25,
26), and CRAC channels are
activated (27).Orai1 has two homologous genes, Orai2 and Orai3
(8). They form the Orai family
and have in common the four transmembrane (TM) segments with relatively large
N and C termini. These termini are demonstrated to be in the cytoplasm,
because both N- and C-terminally introduced tags are immunologically detected
only in the membrane-permeabilized cells
(8,
9). The subunit stoichiometry
of Orai1 is as yet controversial: it is believed to be an oligomer, presumably
a dimer or tetramer even in the steady state
(16,
28–30).Despite the accumulation of biochemical and electrophysiological data,
structural information about Orai1 is limited due to difficulties in
purification and crystallization. In this study, we have purified Orai1 in its
tetrameric form and have reconstructed the three-dimensional structure from
negatively stained electron microscopic (EM) images. 相似文献
13.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
14.
15.
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
16.
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
17.
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
18.
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
19.
Il-Ha Lee Craig R. Campbell Sung-Hee Song Margot L. Day Sharad Kumar David I. Cook Anuwat Dinudom 《The Journal of biological chemistry》2009,284(19):12663-12669
It has recently been shown that the epithelial Na+ channel
(ENaC) is compartmentalized in caveolin-rich lipid rafts and that
pharmacological depletion of membrane cholesterol, which disrupts lipid raft
formation, decreases the activity of ENaC. Here we show, for the first time,
that a signature protein of caveolae, caveolin-1 (Cav-1), down-regulates the
activity and membrane surface expression of ENaC. Physical interaction between
ENaC and Cav-1 was also confirmed in a coimmunoprecipitation assay. We found
that the effect of Cav-1 on ENaC requires the activity of Nedd4-2, a ubiquitin
protein ligase of the Nedd4 family, which is known to induce ubiquitination
and internalization of ENaC. The effect of Cav-1 on ENaC requires the
proline-rich motifs at the C termini of the β- and γ-subunits of
ENaC, the binding motifs that mediate interaction with Nedd4-2. Taken
together, our data suggest that Cav-1 inhibits the activity of ENaC by
decreasing expression of ENaC at the cell membrane via a mechanism that
involves the promotion of Nedd4-2-dependent internalization of the
channel.Amiloride-sensitive epithelial Na+ channels
(ENaC)3 are membrane
proteins that are expressed in salt-absorptive epithelia, including the distal
collecting tubules of the kidney, the mucosa of the distal colon, the
respiratory epithelium, and the excretory ducts of sweat and salivary glands
(1–4).
Na+ absorption via ENaC is critical to the normal regulation of
Na+ and fluid homeostasis and is important for maintaining blood
pressure (5) and the volume of
fluid in the respiratory passages
(6). Increased ENaC activity
has been implicated in the salt-sensitive inherited form of hypertension,
Liddle''s syndrome (7), and
dehydration of the surface of the airway epithelium in the pathology
associated with cystic fibrosis lung disease
(8).Expression of ENaC at the cell membrane surface is regulated by the E3
ubiquitin protein ligase, Nedd4-2 (neural precursor cell
expressed developmentally down-regulated
protein 4) (9). Interaction
between the WW domains of Nedd4-2 and the proline-rich PY motifs
(PPPXY) on ENaC is essential for Nedd4-2 to exert a negative effect
on the channel (10,
11). This interaction leads to
ubiquitination-dependent internalization of ENaC
(12,
13). Several regulators of
ENaC exert their effects on the channel by modulating the action of Nedd4-2.
For instance, serum and glucocorticoid-dependent protein kinase
(14), protein kinase B
(15), and G protein-coupled
receptor kinase (16)
up-regulate activity of ENaC by inhibiting Nedd4-2. Although the details of
cellular mechanisms that underlie internalization of ENaC remain to be
elucidated, the physiological significance of Nedd4-dependent internalization
of the channel has been well established. For instance, heritable mutations
that delete the cytosolic termini of the β-or γ-subunit of ENaC,
which contain the proline-rich motifs, are known to cause hyperactivity of
ENaC in the kidney (17) and
increase cell surface expression of the channel
(7,
18).The plasma membranes of most cell types contain lipid raft microdomains
that are enriched with glycosphingolipid and cholesterol
(19), that have distinctive
biophysical properties, and that selectively include or exclude signaling
molecules (20). These
microdomains promote clustering of an array of integral membrane proteins in
the membrane leaflets (21) and
may be important for organizing cascades of signaling molecules
(22,
23). Processes in which raft
microdomains are involved include the intracellular transport of proteins and
lipids to the cell membrane
(24), the endocytotic
retrieval of membrane proteins
(25,
26), and signal transduction
(27,
28). In addition, segregation
of signaling molecules within lipid rafts may facilitate cross-talk between
signal transduction pathways
(29), a phenomenon that may be
important in ensuring rapid and efficient integration of multiple cellular
signaling events (30,
31). Of particular interest is
the subpopulation of lipid rafts enriched with caveolin proteins. Caveolin-1
(Cav-1), a major caveolin isoform expressed in nonmuscle cells, has been
identified as being involved in diverse cellular functions, such as vesicular
transport, cholesterol homeostasis, and signal transduction
(32). Cav-1 also regulates the
activity and membrane expression of ion channels and transporters
(28).In epithelia, the majority of lipid rafts exist at the apical membrane
surface (22). Pools of ENaC
(33–36)
and several proteins that regulate activity of ENaC, such as Nedd4
(37), protein kinase B
(38), protein kinase C
(39), Go
(40), and the G
protein-coupled receptor kinase
(41), have been identified in
detergent-insoluble and cholesterol-rich membrane fractions from a variety of
cell types, consistent with localization of these proteins in lipid rafts.
Furthermore, detergent-free buoyant density separation of lipid rafts has
revealed the presence of Cav-1 with ENaC in the lipid raft-rich membrane
fraction (35). The
physiological role of lipid rafts in the regulation of ENaC has been the
subject of many recent investigations. Most of these studies used a
pharmacological agent, methyl-β-cyclodextrin (MβCD), to promote
redistribution of proteins away from the cholesterol-enriched membrane
domains. The results were, however, inconclusive. In some studies, MβCD
treatment was found to inhibit open probability
(42) or cell surface
expression of ENaC (35),
whereas others found no direct effect of MβCD on the channel
(33,
43).Despite a number of studies into the role of lipid rafts on the regulation
of ENaC, little is known about the physiological relevance of caveolins to the
function of this ion channel. In the present study, we use gene interference
and gene expression techniques to determine the role of Cav-1 in the
regulation of ENaC activity. We provide evidence of the association of Cav-1
with ENaC and evidence that Cav-1 negatively regulates both activity and
abundance of ENaC at the surface of epithelial cells. Importantly, we
demonstrate, for the first time, that the mechanism by which Cav-1 regulates
activity of ENaC involves the E3 ubiquitin protein ligase, Nedd4-2. 相似文献
20.
Benjamin E. L. Lauffer Stanford Chen Cristina Melero Tanja Kortemme Mark von Zastrow Gabriel A. Vargas 《The Journal of biological chemistry》2009,284(4):2448-2458
Many G protein-coupled receptors (GPCRs) recycle after agonist-induced
endocytosis by a sequence-dependent mechanism, which is distinct from default
membrane flow and remains poorly understood. Efficient recycling of the
β2-adrenergic receptor (β2AR) requires a C-terminal PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin
cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth
factor-regulated substrate). The PDZbd is thought to link receptors to actin
through a series of protein interaction modules present in NHERF/EBP50
(Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein
of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not
known, however, if such actin connectivity is sufficient to recapitulate the
natural features of sequence-dependent recycling. We addressed this question
using a receptor fusion approach based on the sufficiency of the PDZbd to
promote recycling when fused to a distinct GPCR, the δ-opioid receptor,
which normally recycles inefficiently in HEK293 cells. Modular domains
mediating actin connectivity promoted receptor recycling with similarly high
efficiency as the PDZbd itself, and recycling promoted by all of the domains
was actin-dependent. Regulation of receptor recycling by Hrs, however, was
conferred only by the PDZbd and not by downstream interaction modules. These
results suggest that actin connectivity is sufficient to mimic the core
recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors
(GPCRs)2 comprise the
largest family of transmembrane signaling receptors expressed in animals and
transduce a wide variety of physiological and pharmacological information.
While these receptors share a common 7-transmembrane-spanning topology,
structural differences between individual GPCR family members confer diverse
functional and regulatory properties
(1-4).
A fundamental mechanism of GPCR regulation involves agonist-induced
endocytosis of receptors via clathrin-coated pits
(4). Regulated endocytosis can
have multiple functional consequences, which are determined in part by the
specificity with which internalized receptors traffic via divergent downstream
membrane pathways
(5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed
by the δ-opioid receptor (δOR), contributes to proteolytic
down-regulation of receptor number and produces a prolonged attenuation of
subsequent cellular responsiveness to agonist
(8,
9). Trafficking of internalized
GPCRs via a rapid recycling pathway, a major route traversed by the
β2-adrenergic receptor (β2AR), restores the complement of functional
receptors present on the cell surface and promotes rapid recovery of cellular
signaling responsiveness (6,
10,
11). When co-expressed in the
same cells, the δOR and β2AR are efficiently sorted between these
divergent downstream membrane pathways, highlighting the occurrence of
specific molecular sorting of GPCRs after endocytosis
(12).Recycling of various integral membrane proteins can occur by default,
essentially by bulk membrane flow in the absence of lysosomal sorting
determinants (13). There is
increasing evidence that various GPCRs, such as the β2AR, require
distinct cytoplasmic determinants to recycle efficiently
(14). In addition to requiring
a cytoplasmic sorting determinant, sequence-dependent recycling of the
β2AR differs from default recycling in its dependence on an intact actin
cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs
(hepatocyte growth factor receptor substrate)
(11,
14). Compared with the present
knowledge regarding protein complexes that mediate sorting of GPCRs to
lysosomes (15,
16), however, relatively
little is known about the biochemical basis of sequence-directed recycling or
its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called
PDZbd), and PDZ-mediated protein association(s) with this sequence appear to
be primarily responsible for its endocytic sorting activity
(17-20).
Fusion of this sequence to the cytoplasmic tail of the δOR effectively
re-routes endocytic trafficking of engineered receptors from lysosomal to
recycling pathways, establishing the sufficiency of the PDZbd to function as a
transplantable sorting determinant
(18). The β2AR-derived
PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ
proteins (21,
22). A well-established
biochemical function of NHERF/EBP50 family proteins is to associate integral
membrane proteins with actin-associated cytoskeletal elements. This is
achieved through a series of protein-interaction modules linking NHERF/EBP50
family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to
actin filaments
(23-26).
Such indirect actin connectivity is known to mediate other effects on plasma
membrane organization and function
(23), however, and NHERF/EBP50
family proteins can bind to additional proteins potentially important for
endocytic trafficking of receptors
(23,
25). Thus it remains unclear
if actin connectivity is itself sufficient to promote sequence-directed
recycling of GPCRs and, if so, if such connectivity recapitulates the normal
cellular regulation of sequence-dependent recycling. In the present study, we
took advantage of the modular nature of protein connectivity proposed to
mediate β2AR recycling
(24,
26), and extended the opioid
receptor fusion strategy used successfully for identifying diverse recycling
sequences in GPCRs
(27-29),
to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can
be effectively bypassed by linking receptors to ERM family proteins in the
absence of the PDZbd itself. Further, we establish that the protein
connectivity network can be further simplified by fusing receptors to an
interaction module that binds directly to actin filaments. We found that
bypassing the PDZ-mediated interaction using either domain is sufficient to
mimic the ability of the PDZbd to promote efficient, actin-dependent recycling
of receptors. Hrs-dependent regulation, however, which is characteristic of
sequence-dependent recycling of wild-type receptors, was recapitulated only by
the fused PDZbd and not by the proposed downstream interaction modules. These
results support a relatively simple architecture of protein connectivity that
is sufficient to mimic the core recycling activity of the β2AR-derived
PDZbd, but not its characteristic cellular regulation. Given that an
increasing number of GPCRs have been shown to bind PDZ proteins that typically
link directly or indirectly to cytoskeletal elements
(17,
27,
30-32),
the present results also suggest that actin connectivity may represent a
common biochemical principle underlying sequence-dependent recycling of
various GPCRs. 相似文献