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1.
Yong Zhang Yong-Gang Wang Qi Zhang Xiu-Jie Liu Xuan Liu Li Jiao Wei Zhu Zhao-Huan Zhang Xiao-Lin Zhao Cheng He 《The Journal of biological chemistry》2009,284(18):12469-12479
TrkA receptor signaling is essential for nerve growth factor (NGF)-induced
survival and differentiation of sensory neurons. To identify possible
effectors or regulators of TrkA signaling, yeast two-hybrid screening was
performed using the intracellular domain of TrkA as bait. We identified
muc18-1-interacting protein 2 (Mint2) as a novel TrkA-binding protein and
found that the phosphotyrosine binding domain of Mint2 interacted with TrkA in
a phosphorylation- and ligand-independent fashion. Coimmunoprecipitation
assays showed that endogenous TrkA interacted with Mint2 in rat tissue
homogenates, and immunohistochemical evidence revealed that Mint2 and TrkA
colocalized in rat dorsal root ganglion neurons. Furthermore, Mint2
overexpression inhibited NGF-induced neurite outgrowth in both PC12 and
cultured dorsal root ganglion neurons, whereas inhibition of Mint2 expression
by RNA interference facilitated NGF-induced neurite outgrowth. Moreover, Mint2
was found to promote the retention of TrkA in the Golgi apparatus and inhibit
its surface sorting. Taken together, our data provide evidence that Mint2 is a
novel TrkA-regulating protein that affects NGF-induced neurite outgrowth,
possibly through a mechanism involving retention of TrkA in the Golgi
apparatus.The neurotrophin family member nerve growth factor
(NGF)3 is
essential for proper development, patterning, and maintenance of nervous
systems (1,
2). NGF has two known
receptors; TrkA, a single-pass transmembrane receptor-tyrosine kinase that
binds selectively to NGF, and p75, a transmembrane glycoprotein that binds all
members of the neurotrophin family
(3,
4). NGF binding activates the
kinase domain of TrkA, leading to autophosphorylation
(5). The resulting
phosphotyrosines become docking sites for adaptor proteins involved in signal
transduction pathways that lead to the activation of Ras, Rac,
phosphatidylinositol 3-kinase, phospholipase Cγ, and other effectors
(2,
6). Many of these
TrkA-interacting adaptor proteins have been identified and include, Grb2, APS,
SH2B, fibroblast growth factor receptor substrate 2 (FRS-2), Shc, and human
tumor imaginal disc 1 (TID1)
(7-10).
The identification of these binding partners has contributed greatly to our
understanding of the mechanisms underlying the functional diversity of
NGF-TrkA signaling.Studies have indicated that the transmission of NGF signaling in neurons
involves retrograde transport of NGF-TrkA complexes from the neurite tip to
the cell body
(11-14).
TrkA associates with components of cytoplasmic dynein, and it is thought that
vesicular trafficking of neurotrophins occurs via direct interaction of Trk
receptors with the dynein motor machinery
(14). Furthermore, the
atypical protein kinase C-interacting protein, p62, associates with TrkA and
plays a novel role in connecting receptor signals with the endosomal signaling
network required for mediating TrkA-induced differentiation
(15). Recently, the
membrane-trafficking protein Pincher has been found to mediate
macroendocytosis underlying retrograde signaling by TrkA
(16). Despite the progress
made to date in understanding Trk complex internalization and trafficking, the
mechanisms remain poorly understood.Mint2 (muc18-1-interacting protein 2) belongs to the Mint protein family,
which consists of three members, Mint1, Mint2, and Mint3. Mint proteins were
first identified as interacting proteins of the synaptic vesicle-docking
protein Munc18-1 (17,
18). Mint1 is also sometimes
referred to as mLIN-10, as it is the mammalian orthologue of the
Caenorhabditis elegans LIN-10
(19). Additionally, Mint1,
Mint2, and Mint3 are also referred to as X11α or X11, X11β or X11L
(X11-like), and X11γ or X11L2 (X11-like 2), respectively
(20). All Mint proteins
contain a conserved central phosphotyrosine binding (PTB) domain and two
contiguous C-terminal PDZ domains (repeated sequences in the brain-specific
protein PSD-95, the Drosophila septate junction protein Discs large,
and the epithelial tight junction protein ZO-1)
(17,
18,
21). Mint1 and Mint2 are
expressed only in neuronal tissue
(17), whereas Mint3 is
ubiquitously expressed (18).
Although the function of Mints proteins is not fully clear, their interactions
with the docking and exocytosis factors Mun18 -1 and CASK, ADP-ribosylation
factor (Arf) GTPases involved in vesicle budding
(22), and other synaptic
adaptor proteins, such as neurabin-II/spinophilin
(23), tamalin
(24), and kalirin-7
(25), all suggest possible
roles for Mints in synaptic vesicle docking and exocytosis. Mint proteins have
also been implicated in the trafficking and/or processing of β-amyloid
precursor protein (β-APP). Through their PTB domains, all three Mints
bind to a motif within the cytoplasmic domain of β-APP
(21,
26-29),
and Mint1 and Mint2 can stabilize β-APP, affect β-APP processing,
and inhibit the production and secretion of Aβ
(28,
30-32).
Although the mechanisms by which Mints inhibit β-APP processing are not
yet well known, Mints and their binding partners have emerged as potential
therapeutic targets for the treatment of Alzheimer disease.To uncover new TrkA-interacting factors and gain insight into the
mechanisms that guide TrkA intracellular trafficking and other aspects of TrkA
signaling, we conducted a yeast two-hybrid screen of a brain cDNA library
using the intracellular domain of TrkA as bait. The screen identified several
candidate TrkA-interacting proteins, one of which was Mint2. Follow-up binding
assays showed that the PTB domain of Mint2 alone was necessary and sufficient
for mediating the interaction with TrkA. Endogenous Mint2 was also
coimmunoprecipitated and colocalized with TrkA in rat DRG tissue.
Overexpression and knockdown studies showed that Mint2 could significantly
inhibit NGF-induced neurite outgrowth in both TrkA-expressing PC12 cells and
DRG neurons. Moreover, Mint2 was found to induce the retention of TrkA in the
Golgi apparatus and inhibit its surface sorting. Our results suggest that
Mint2 is a novel regulator of TrkA receptor signaling. 相似文献
2.
3.
Michael S. Friedman Sivan M. Oyserman Kurt D. Hankenson 《The Journal of biological chemistry》2009,284(21):14117-14125
Wnt11 signals through both canonical (β-catenin) and non-canonical
pathways and is up-regulated during osteoblast differentiation and fracture
healing. In these studies, we evaluated the role of Wnt11 during
osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases
β-catenin accumulation and promotes bone morphogenetic protein
(BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11
dramatically increases expression of the osteoblast-associated genes
Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating
endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also
increases expression of Rspo2 (R-spondin 2), a secreted factor known
to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing
Dmp1, Phex, and Bsp expression and promotes bone
morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates
Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor
(Tcf)/β-catenin signaling with dominant negative Tcf blocks
Wnt11-mediated expression of Dmp1, Phex, and Rspo2
and decreases mineralization. However, dominant negative Tcf fails to block
the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11
signals through β-catenin, activating Rspo2 expression, which is
then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and
maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b
enhances osteoblast differentiation
(1). Canonical Wnt signaling
through β-catenin has also been shown to enhance the chondroinductive and
osteoinductive properties of
BMP22
(2,
3). During BMP2-induced
osteoblast differentiation of mesenchymal stem cell lines, cross-talk between
BMP and Wnt pathways converges through the interaction of Smad4 with
β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and
homeostasis. During limb development, expression of Wnt3a in the apical
ectodermal ridge of limb buds maintains cells in a highly proliferative and
undifferentiated state (4,
5). Disruption of canonical Wnt
signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and
digit-patterning defects (6).
Wnt signaling is also involved in the maintenance of post-natal bone mass.
Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass,
whereas loss of Lrp5 function is associated with decreased bone mass and
osteoporosis pseudoglioma syndrome
(7,
8). Mice with increased Wnt10b
expression have increased trabecular bone, whereas Wnt10b-deficient mice have
reduced trabecular bone (9).
Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased
trabecular bone accrual throughout adulthood
(10).Although canonical Wnt signaling regulates osteoblastogenesis and bone
formation, the profile of endogenous Wnts that play a role in osteoblast
differentiation and maturation is not well described. During development,
Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum
(11). Wnt11 expression is
increased during glucocorticoid-induced osteogenesis
(12), indicating a potential
role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates
both β-catenin-dependent as well as β-catenin-independent signaling
pathways (13). Targeted
disruption of Wnt11 results in late embryonic/early post-natal death because
of cardiac dysfunction (14).
Although these mice have no reported skeletal developmental abnormalities,
early lethality obfuscates a detailed examination of post-natal skeletal
modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of
R-spondin 2 (Rspo2) in the apical ectodermal ridge
(11,
15). R-spondins are a novel
family of proteins that share structural features, including two conserved
cysteinerich furin-like domains and a thrombospondin type I repeat
(16). The four R-spondin
family members can activate canonical Wnt signaling
(15,
17–19).
Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling.
Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting
Dkk-mediated receptor internalization
(20). Rspo1 was also shown to
potentiate Wnt3a-mediated osteoblast differentiation
(21). Rspo2 knock-out
mice, which die at birth, have limb patterning defects associated with altered
β-catenin signaling
(22–24).
However, the role of Rspo2 in osteoblast differentiation and maturation
remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts
activates β-catenin and augments BMP-induced osteoblast maturation and
mineralization. Wnt11 increases the expression of Rspo2.
Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced
osteoblast maturation and mineralization. Although antagonism of
Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2
rescues this block, and knockdown of Rspo2 shows that it is required for
Wnt11-mediated osteoblast maturation and mineralization. These studies
identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and
mineralization. 相似文献
4.
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
5.
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
6.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
7.
8.
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
9.
Christopher P. Gayer Lakshmi S. Chaturvedi Shouye Wang David H. Craig Thomas Flanigan Marc D. Basson 《The Journal of biological chemistry》2009,284(4):2001-2011
The intestinal epithelium is repetitively deformed by shear, peristalsis,
and villous motility. Such repetitive deformation stimulates the proliferation
of intestinal epithelial cells on collagen or laminin substrates via ERK, but
the upstream mediators of this effect are poorly understood. We hypothesized
that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this
mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β
(GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10%
deformation, and pharmacologic blockade of these molecules or reduction by
small interfering RNA (siRNA) prevented the mitogenic effect of strain in
Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required
PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that
AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain
mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera
including the PH domain and hinge region of AKT2 and the catalytic domain and
C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression
of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the
catalytic domain and C-tail of AKT2 did not. These data delineate a role for
PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is
required for both ERK and AKT2 activation, whereas AKT2 is sequentially
required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic
domain and tail region. Manipulating this pathway may prevent mucosal atrophy
and maintain the mucosal barrier in conditions such as ileus, sepsis, and
prolonged fasting when peristalsis and villous motility are decreased and the
mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment.
Numerous different forces deform these cells including shear stress from
endoluminal chyme, bowel peristalsis, and villous motility
(1,
2). During normal bowel
function the mucosa is subjected to injury that must be repaired to maintain
the mucosal barrier (3,
4). Deformation patterns of the
bowel are altered in conditions such as prolonged fasting, post-surgical
ileus, and sepsis states, resulting in profoundly reduced mucosal deformation.
When such states are prolonged, proliferation slows, the mucosa becomes
atrophic, and bacterial translocation may ensue as the mucosal barrier of the
gut breaks down
(5–7).In vitro, repetitive deformation is trophic for intestinal
epithelial cells (8) cultured
on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial
cells (9), non-transformed rat
IEC-6 intestinal epithelial cells
(10), and primary human
intestinal epithelial cells isolated from surgical specimens
(11) proliferate more rapidly
in response to cyclic strain
(12) unless substantial
quantities of fibronectin are added to the media or matrix
(11) to mimic the acute phase
reaction of acute or chronic inflammation and injury. Cyclic strain also
stimulates proliferation in HCT 116 colon cancer cells
(13) and differentiation of
Caco-2 cells cultured on a collagen substrate
(9). This phenomenon has also
been observed in vivo
(14). Thus, repetitive
deformation may help to maintain the normal homeostasis of the gut mucosa
under non-inflammatory conditions. Previous work in our laboratory has
implicated Src, focal adhesion kinase, and the mitogen-activated protein
kinase (MAPK)2
extracellular signal-related kinase (ERK) in the mitogenic effect of strain
(10). Although p38 is also
activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its
activity is not required for the mitogenic effect of strain
(12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to
the MAPK, this is not always the case. Protein kinase C isoenzymes
differentially modulate thrombin effect on MAPK-dependent retinal pigment
epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT
inhibition prevented thrombin-induced ERK activation and RPE proliferation
(15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT
(16), have been implemented in
intestinal epithelial cell proliferation in numerous cell systems not
involving strain
(17–19)
including uncontrolled proliferation in gastrointestinal cancers
(20–22).
Mechanical forces activate this pathway as well. PI3K and AKT are required for
increased extracellular pressure to stimulate colon cancer cell adhesion
(23), although the pathway by
which pressure stimulates colon cancer cells in suspension differs from the
response of adherent intestinal epithelial cells to repetitive deformation
(24), and GSK is not involved
in this effect.3
Repetitive strain also stimulates vascular endothelial cell proliferation via
PI3K and AKT (25,
26), whereas respiratory
strain stimulates angiogenic responses via PI3K
(27). We, therefore,
hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic
effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically
deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm
key results. A frequency of 10 cycles per min was used, which is similar in
order of magnitude to the frequency that the intestinal mucosa might be
deformed by peristalsis or villous motility in vivo
(28,
29). Mechanical forces such as
repetitive deformation are likely cell-type and frequency-specific, as
different cell types respond to different frequencies. Vascular endothelial
cells respond to frequencies of 60–80 cycles/min
(25), whereas intestinal
epithelial cells may actually decrease proliferation in response to
frequencies of 5 cycles/min
(30). We characterized PI3K,
AKT, and GSK phosphorylation with strain, blocked these molecules
pharmacologically or by siRNA, and delineated the specificity of the AKT
effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We
also characterized the interaction of this pathway with the activation of ERK
by strain, which has previously been implicated in the mitogenic response
(12). 相似文献
10.
11.
Zinaida Dubeykovskaya Alexander Dubeykovskiy Joel Solal-Cohen Timothy C. Wang 《The Journal of biological chemistry》2009,284(6):3650-3662
The secreted trefoil factor family 2 (TFF2) protein contributes to the
protection of the gastrointestinal mucosa from injury by strengthening and
stabilizing mucin gels, stimulating epithelial restitution, and restraining
the associated inflammation. Although trefoil factors have been shown to
activate signaling pathways, no cell surface receptor has been directly linked
to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide
to activate signaling via the CXCR4 chemokine receptor in cancer cell lines.
We found that both mouse and human TFF2 proteins (at ∼0.5
μm) activate Ca2+ signaling in lymphoblastic Jurkat
cells that could be abrogated by receptor desensitization (with SDF-1α)
or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4
antibody. TFF2 pretreatment of Jurkat cells decreased Ca2+ rise and
chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric
epithelial cell line AGS became highly responsive to TFF2 treatment upon
expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated
protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1,
respectively, was also dependent on the presence of the CXCR4 receptor.
Finally we demonstrate a distinct proliferative effect of TFF2 protein on an
AGS gastric cancer cell line that expresses CXCR4. Overall these data identify
CXCR4 as a bona fide signaling receptor for TFF2 and suggest a
mechanism through which TFF2 may modulate immune and tumorigenic responses
in vivo.Trefoil factor 2
(TFF2),2 previously
known as spasmolytic polypeptide, is a unique member of the trefoil family
that is expressed primarily in gastric mucous neck cells and is up-regulated
in the setting of chronic inflammation. Experimental induction of ulceration
in the rat stomach leads to rapid up-regulation of TFF2 expression with high
levels observed 30 min after ulceration with persistence for up to 10 days
(1). TFF2 is secreted into the
mucus layer of the gastrointestinal tract of mammals where it stabilizes the
mucin gel layer and stimulates migration of epithelial cells
(2–4),
suggesting an important role in restitution and in maintenance of the
integrity of the gut. Exogenous administration of recombinant TFF2, either
orally or intravenously, provides mucosal protection in several rodent models
of acute gastric or intestinal injury
(5,
6). A TFF2-/-
knock-out mouse model has confirmed the importance of TFF2 in the protection
of gastrointestinal mucosa against chronic injury
(7).It is widely accepted that trefoil factors exert their biological action
through a cell surface receptor. This suggestion comes from studies on binding
of 125I-labeled TFF2 that demonstrated specific binding sites in
the gastric glands, intestine, and colon that could be displaced by
non-radioactive TFF2 (6,
8–10).
Structural studies have revealed potential binding sites for receptors for all
members of the trefoil factor family
(11,
12). In concordance with this
hypothesis, several membrane proteins were found to interact with TFF2. First
it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa
peptide from membrane fractions of rat jejunum and two human adenocarcinoma
cell lines, MCF-7 and Colony-29
(13). Later it was found that
recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein
from the membrane of rat small intestinal cells
(14). However, these 28- and
50-kDa proteins were characterized only by their molecular size without
further identification. Two TFF2-binding proteins that have been characterized
include a 140-kDa protein, the β subunit of the fibronectin receptor, and
a 224-kDa protein called muclin
(15). Another TFF2-binding
protein was isolated by probing two-dimensional blots of mouse stomach with a
murine TFF2 fusion protein, leading to the identification of the gastric
foveolar protein blottin, a murine homolog of the human peptide
TFIZ1(16). Although these
three proteins have now been well characterized, none of them has been shown
to mediate responses to TFF2, and no activated signaling cascades have been
shown.Despite the absence of an identified cell surface receptor for TFF2, there
is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal
transduction pathways (17,
18). TFF3 prevents cell death
via activation of the serine/threonine kinase AKT in colon cancer cell lines
(19). The TFF3 protein also
activates STAT3 signaling in human colorectal cancer cells, thus providing
cells with invasion potential
(20). TFF3 treatment leads to
EGF receptor activation and β-catenin phosphorylation in HT-29 cells
(21) and to transient
phosphorylation of ERK1/2 in oral keratinocytes
(22). With respect to TFF2,
recombinant peptide enhances the migration of human bronchial epithelial cell
line BEAS-2B (4). TFF2 has been
shown to induce phosphorylation of c-Jun NH2-terminal kinase (JNK)
and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is
significantly inhibited by antagonists of ERK kinases and protein kinase C but
not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is
believed that the motogenic effect of trefoil factors and of TFF2 in
particular, could contribute to in vivo restitution of gastric
epithelium by enhancing cell migration.Although previous studies have suggested that TFF2 functions primarily in
cytoprotection, accumulating evidence now suggests that TFF2 may also play a
role in the regulation of host immunity. For example, recombinant TFF2 reduces
inflammation in rat and mouse models of colitis
(23,
24). In addition, TFF2 was
detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow)
(25). Recently we and others
found TFF2 mRNA expression in primary and secondary lymphopoietic organs
(26,
27). These data suggest that
TFF2 may play some function in the immune system. In concordance with these
findings, we detected an exacerbated inflammatory response to acute injury in
TFF2 knock-out animals (27,
28). These observations
prompted us to look at the possible function of TFF2 in immune cells.
Unexpectedly we found that TFF2 modulates Ca2+ and AKT signaling in
lymphoblastic Jurkat cells and that these effects appear to be mediated
through the CXCR4 receptor. 相似文献
12.
Xiaojun Li C. T. Ranjith-Kumar Monica T. Brooks S. Dharmaiah Andrew B. Herr Cheng Kao Pingwei Li 《The Journal of biological chemistry》2009,284(20):13881-13891
The RIG-I-like receptors (RLRs), RIG-I and MDA5, recognize single-stranded
RNA with 5′ triphosphates and double-stranded RNA (dsRNA) to initiate
innate antiviral immune responses. LGP2, a homolog of RIG-I and MDA5 that
lacks signaling capability, regulates the signaling of the RLRs. To establish
the structural basis of dsRNA recognition by the RLRs, we have determined the
2.0-Å resolution crystal structure of human LGP2 C-terminal domain bound
to an 8-bp dsRNA. Two LGP2 C-terminal domain molecules bind to the termini of
dsRNA with minimal contacts between the protein molecules. Gel filtration
chromatography and analytical ultracentrifugation demonstrated that LGP2 binds
blunt-ended dsRNA of different lengths, forming complexes with 2:1
stoichiometry. dsRNA with protruding termini bind LGP2 and RIG-I weakly and do
not stimulate the activation of RIG-I efficiently in cells. Surprisingly,
full-length LGP2 containing mutations that abolish dsRNA binding retained the
ability to inhibit RIG-I signaling.The innate immune response is the first line of defense against invading
pathogens; it is the ubiquitous system of defense against microbial infections
(1). Toll-like receptors
(TLRs)3 and RIG-I
(retinoic acid-inducible gene
1)-like receptors (RLRs) play key roles in innate immune response
toward viral infection
(2-5).
Toll-like receptors TLR3, TLR7, and TLR8 sense viral RNA released in the
endosome following phagocytosis of the pathogens
(6). RIG-I-like receptors RIG-I
and MDA5 detect viral RNA from replicating viruses in infected cells
(3,
7,
8). Stimulation of these
receptors leads to the induction of type I interferons (IFNs) and other
proinflammatory cytokines, conferring antiviral activity to the host cells and
activating the acquired immune responses
(4,
9).RIG-I discriminates between viral and host RNA through specific recognition
of the uncapped 5′-triphosphate of single-stranded RNA (5′ ppp
ssRNA) generated by viral RNA polymerases
(10,
11). In addition, RIG-I also
recognizes double-stranded RNA generated during RNA virus replication
(7,
12). Transfection of cells
with synthetic double-stranded RNA stimulates the activation of RIG-I
(13,
14). Synthetic dsRNA mimics,
such as polyinosinic-polycytidylic acid (poly(I·C)), can activate MDA5
when introduced into the cytoplasm of cells. Digestion of poly(I·C)
with RNase III transforms poly(I·C) from a ligand for MDA5 into a
ligand for RIG-I, suggesting that MDA5 recognizes long dsRNA, whereas RIG-I
recognizes short dsRNA (15).
Studies of RIG-I and MDA5 knock-out mice confirmed the essential roles of
these receptors in antiviral immune responses and demonstrated that they sense
different sets of RNA viruses
(12,
16).RIG-I and MDA5 contain two caspase recruiting domains (CARDs) at their N
termini, a DEX(D/H) box RNA helicase domain, and a C-terminal
regulatory or repressor domain (CTD). The helicase domain and the CTD are
responsible for viral RNA binding, whereas the CARDs are required for
signaling (3,
8). The current model of RIG-I
activation suggests that under resting conditions RIG-I is in a suppressed
conformation, and viral RNA binding triggers a conformation change that leads
to the exposure of the CARDs for the recruitment of the downstream protein
IPS-1 (also known as MAVS, Cardif, or VISA)
(14,
17). Limited proteolysis of
the RIG-I·dsRNA complex showed that RIG-I residues 792-925 of the CTD
are involved in dsRNA and 5′ ppp ssRNA binding
(14). The CTD of RIG-I
overlaps with the C terminus of the previously identified repressor domain
(18). The structures of RIG-I
and LGP2 (laboratory of genetics and
physiology 2) CTD in isolation have been determined by
x-ray crystallography and NMR spectroscopy
(14,
19,
20). A large, positively
charged surface on RIG-I recognizes the 5′ triphosphate group of viral
ssRNA (14,
19). RNA binding studies by
titrating RIG-I CTD with dsRNA and 5′ ppp ssRNA suggested that
overlapping sets of residues on this charged surface are involved in RNA
binding (14). Mutagenesis of
several positively charged residues on this surface either reduces or disrupts
RNA binding by RIG-I, and these mutations also affect the induction of
IFN-β in vivo
(14,
19). However, the exact nature
of how the RLRs recognize viral RNA and how RNA binding activates these
receptors remains to be established.LGP2 is a homolog of RIG-I and MDA5 that lacks the CARDs and thus has no
signaling capability (21,
22). The expression of LGP2 is
inducible by dsRNA or IFN treatment as well as virus infection
(21). Overexpression of LGP2
inhibits Sendai virus and Newcastle disease virus signaling
(21). When coexpressed with
RIG-I, LGP2 can inhibit RIG-I signaling through the interaction of its CTD
with the CARD and the helicase domain of RIG-I
(18). LGP2 could suppress
RIG-I signaling by three possible ways
(23): 1) binding RNA with high
affinity, thereby sequestering RNA ligands from RIG-I; 2) interacting directly
with RIG-I to block the assembly of the signaling complex; and 3) competing
with IKKi (IκB kinase ε) in the NF-κB signaling pathway for a
common binding site on IPS-1. To elucidate the structural basis of dsRNA
recognition by the RLRs, we have crystallized human LGP2 CTD (residues
541-678) bound to an 8-bp double-stranded RNA and determined the structure of
the complex at 2.0 Å resolution. The structure revealed that LGP2 CTD
binds to the termini of dsRNA. Mutagenesis and functional studies showed that
dsRNA binding is likely not required for the inhibition of RIG-I signaling by
LGP2. 相似文献
13.
14.
Ho-Sup Lee Chinten James Lim Wilma Puzon-McLaughlin Sanford J. Shattil Mark H. Ginsberg 《The Journal of biological chemistry》2009,284(8):5119-5127
Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule
(RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to
promote talin-dependent integrin activation. Here, we show that MRL proteins
function as scaffolds that connect the membrane targeting sequences in Ras
GTPases to talin, thereby recruiting talin to the plasma membrane and
activating integrins. The MRL proteins bound directly to talin via short,
N-terminal sequences predicted to form amphipathic helices. RIAM-induced
integrin activation required both its capacity to bind to Rap1 and to talin.
Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the
talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A.
This minimized Rap-RIAM module was sufficient to target talin to the plasma
membrane and to mediate integrin activation, even in the absence of Rap1
activity. We identified a short talin binding sequence in Lamellipodin (Lpd),
another MRL protein; talin binding Lpd sequence joined to a Rap1
membrane-targeting sequence is sufficient to recruit talin and activate
integrins. These data establish the mechanism whereby MRL proteins interact
with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is
central to physiological events such as cell migration, assembly of the
extracellular matrix, the immune response, and hemostasis
(1). Each integrin comprises a
type I transmembrane α and β subunit, each of which has a large
extracellular domain, a single transmembrane domain, and a cytoplasmic domain
(tail). Talin binds to most integrin β cytoplasmic domains and the
binding of talin to the integrin β tail initiates integrin activation
(2–4).
A small, PTB-like domain of talin mediates activation via a two-site
interaction with integrin β tails
(5), and this PTB domain is
functionally masked in the intact talin molecule
(6). A central question in
integrin biology is how the talin-integrin interaction is regulated to control
integrin activation; recent work has implicated Ras GTPases as critical
signaling modules in this process
(7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound
active form and the GDP-bound inactive form. Guanine nucleotide exchange
factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas
GTPase-activating proteins
(GAPs)3 enhance the
hydrolysis of Ras-bound GTP to GDP (for review, see Ref.
8). The Ras subfamily members
Rap1A and Rap1B stimulate integrin activation
(9,
10). For example, expression
of constitutively active Rap1 activates integrin αMβ2 in
macrophage, and inhibition of Rap1 abrogated integrin activation induced by
inflammatory agonists
(11–13).
Murine T-cells expressing constitutively active Rap1 manifest enhanced
integrin dependent cell adhesion
(14). In platelets, Rap1 is
rapidly activated by platelet agonists
(15,
16). A knock-out of Rap1B
(17) or of the Rap1GEF,
RasGRP2 (18), resulted in
impairment of αIIbβ3-dependent platelet aggregation, highlighting
the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1
GTPases play important roles in the activation of several integrins in
multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation
(19–21).
Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a
member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins
(20). RIAM contains Ras
association (RA) and pleckstrin homology (PH) domains and proline-rich
regions, which are defining features of the MRL protein family. In Jurkat
cells, RIAM overexpression induces β1 and β2 integrin-mediated cell
adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion
(20), indicating RIAM is a
downstream regulator of Rap1-dependent signaling. RIAM regulates actin
dynamics as RIAM expression induces cell spreading; conversely, its depletion
reduces cellular F-actin content
(20). Whereas RIAM is greatly
enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in
fibroblasts and other somatic cells
(22).Recently we used forward, reverse, and synthetic genetics to engineer and
order an integrin activation pathway in Chinese hamster ovary cells expressing
a prototype activable integrin, platelet αIIbβ3. We found that Rap1
induced formation of an “integrin activation complex” containing
RIAM and talin (23). Here, we
have established the mechanism whereby Ras GTPases cooperate with MRL family
proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL
proteins function as scaffolds that connect the membrane targeting sequences
in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma
membrane. 相似文献
15.
Matthias Gralle Michelle Gralle Botelho Fred S. Wouters 《The Journal of biological chemistry》2009,284(22):15016-15025
The amyloid precursor protein (APP) is implied both in cell growth and
differentiation and in neurodegenerative processes in Alzheimer disease.
Regulated proteolysis of APP generates biologically active fragments such as
the neuroprotective secreted ectodomain sAPPα and the neurotoxic
β-amyloid peptide. Furthermore, it has been suggested that the intact
transmembrane APP plays a signaling role, which might be important for both
normal synaptic plasticity and neuronal dysfunction in dementia. To understand
APP signaling, we tracked single molecules of APP using quantum dots and
quantitated APP homodimerization using fluorescence lifetime imaging
microscopy for the detection of Förster resonance energy transfer in
living neuroblastoma cells. Using selective labeling with synthetic
fluorophores, we show that the dimerization of APP is considerably higher at
the plasma membrane than in intracellular membranes. Heparan sulfate
significantly contributes to the almost complete dimerization of APP at the
plasma membrane. Importantly, this technique for the first time structurally
defines the initiation of APP signaling by binding of a relevant physiological
extracellular ligand; our results indicate APP as receptor for neuroprotective
sAPPα, as sAPPα binding disrupts APP dimers, and this disruption
of APP dimers by sAPPα is necessary for the protection of neuroblastoma
cells against starvation-induced cell death. Only cells expressing reversibly
dimerized wild-type, but not covalently dimerized mutant APP are protected by
sAPPα. These findings suggest a potentially beneficial effect of
increasing sAPPα production or disrupting APP dimers for neuronal
survival.The amyloid precursor protein
(APP)4 is known both
for its important role in the development and plasticity of the nervous system
(1–6)
and for its involvement in Alzheimer disease (AD)
(7,
8). Despite intensive research
efforts, the initial events that lead to the prevalent sporadic, i.e.
non-familial, forms of AD are still unclear. Furthermore, although a higher
gene dose of APP (9) or the
presence of pathological APP mutations is sufficient to induce familial AD
(for review, see Ref. 10), the
exact pathological mechanism that is triggered by APP is still under
debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are
thought to contribute to synaptic dysfunction and neurotoxicity
(11,
12). On the other hand, the
α-secretase-derived extracellular fragment of APP (sAPPα), which
is present at lower levels in AD patients than in controls
(13), has been shown to be
beneficial for memory function, to possess neuroprotective properties, and to
counteract the effects of Aβ
(14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration
in AD
(19–24);
however, the signal transduction pathway for transmembrane APP remains
unknown, although several potential regulatory proteins, glycosaminoglycans,
and metal ions are known to bind with high affinity to APP and sAPPα
(25,
26). The most common form of
signal transduction for single-pass transmembrane proteins is the
ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the
dimerization of transmembrane APP has been implied several times in the past.
Several studies have investigated the effects of presumed dimer-breaking
perturbations on biological read-outs, such as the production of Aβ
(27,
28), but without directly
measuring the APP aggregation state, or have investigated the aggregation
state of APP subdomains, often reconstituted in cell-free systems
(27–32).
Dimerization interfaces in both the extracellular and the transmembrane domain
have been suggested.In the studies investigating the aggregation state of full-length APP, most
of the employed methods, such as chemical cross-linking and
co-immunoprecipitation, do not lend themselves readily to a rigorous
quantitative analysis of the abundance of potentially instable dimers
(31,
33), whereas in other cases
the use of chimeras may have influenced the dimerization potential or
precluded the search for a natural stimulus
(23,
34). The only previously
reported direct observation of APP dimerization by Förster resonance
energy transfer (FRET) microscopy uses an assay in which the FRET efficiency
varies with the level of overexpression
(35). Therefore, a
concentration-dependent FRET component due to nonspecific stochastic
encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the
selective detection of APP dimers on the surface of live cells, where they
would encounter ligands, they could not differentiate between subpopulations
of APP. This may be one reason why no natural ligand of APP has ever been
shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective
sAPPα
(36–39).
The ligand-dependent dimerization of sAPPα in solution
(40) and its origination from
transmembrane APP suggest that APP might serve as receptor for sAPPα,
but this binding has never been experimentally shown. 相似文献
16.
17.
18.
Zhemin Zhou Yoshiteru Hashimoto Michihiko Kobayashi 《The Journal of biological chemistry》2009,284(22):14930-14938
19.
Yiliang Chen Ting Cai Haojie Wang Zhichuan Li Elizabeth Loreaux Jerry B. Lingrel Zijian Xie 《The Journal of biological chemistry》2009,284(22):14881-14890
Recent studies have ascribed many non-pumping functions to the Na/K-ATPase.
We show here that graded knockdown of cellular Na/K-ATPase α1 subunit
produces a parallel decrease in both caveolin-1 and cholesterol in light
fractions of LLC-PK1 cell lysates. This observation is further substantiated
by imaging analyses, showing redistribution of cholesterol from the plasma
membrane to intracellular compartments in the knockdown cells. Moreover, this
regulation is confirmed in α1+/– mouse liver.
Functionally, the knockdown-induced redistribution appears to affect the
cholesterol sensing in the endoplasmic reticulum, because it activates the
sterol regulatory element-binding protein pathway and increases expression of
hydroxymethylglutaryl-CoA reductase and low density lipoprotein receptor in
the liver. Consistently, we detect a modest increase in hepatic cholesterol as
well as a reduction in the plasma cholesterol. Mechanistically,
α1+/– livers show increases in cellular Src and ERK
activity and redistribution of caveolin-1. Although activation of Src is not
required in Na/K-ATPase-mediated regulation of cholesterol distribution, the
interaction between the Na/K-ATPase and caveolin-1 is important for this
regulation. Taken together, our new findings demonstrate a novel function of
the Na/K-ATPase in control of the plasma membrane cholesterol distribution.
Moreover, the data also suggest that the plasma membrane
Na/K-ATPase-caveolin-1 interaction may represent an important sensing
mechanism by which the cells regulate the sterol regulatory element-binding
protein pathway.The Na/K-ATPase, also called the sodium pump, is an ion transporter that
mediates active transport of Na+ and K+ across the
plasma membrane by hydrolyzing ATP
(1,
2). The functional sodium pump
is mainly composed of α and β subunits. The α subunit is the
catalytic component of the holoenzyme; it contains both the nucleotide and the
cation binding sites (3). So
far, four isoforms of α subunit have been discovered, and each one shows
a distinct tissue distribution pattern
(4,
5). Interestingly, studies
during the past few years have uncovered many non-pumping functions of
Na/K-ATPase
(6–10).
Recently, we have demonstrated that more than half of the Na/K-ATPase may
actually perform cellular functions other than ion pumping at least in LLC-PK1
cells (11). Moreover, the
non-pumping pool of Na/K-ATPase mainly resides in caveolae and interacts with
a variety of proteins such as Src, inositol 1,4,5-trisphosphate receptor, and
caveolin-1
(12–14).
While the interaction between Na/K-ATPase and inositol 1,4,5-trisphosphate
receptor facilitates Ca2+ signaling
(13) the dynamic association
between Na/K-ATPase and Src appears to be an essential step for ouabain to
stimulate cellular kinases
(15). More recently, we report
that the interaction between the Na/K-ATPase and caveolin-1 plays an important
role for the membrane trafficking of caveolin-1. Knockdown of the Na/K-ATPase
leads to altered subcellular distribution of caveolin-1 and increases the
mobility of caveolin-1-containing vesicles
(16).Caveolin is a protein marker for caveolae
(17). Caveolae are
flask-shaped vesicular invaginations of plasma membrane and are enriched in
cholesterol, glycosphingolipids, and sphingomyelin
(18). There are three genes
and six isoforms of caveolin. Caveolin-1 is a 22-kDa protein and is expressed
in many types of cells, including epithelial and endothelial cells. In
addition to their role in biogenesis of caveolae
(19), accumulating evidence
has implicated caveolin proteins in cellular cholesterol homeostasis
(20). For instance, caveolin-1
directly binds to cholesterol in a 1:1 ratio
(21). It was also found to be
an integral member of the intracellular cholesterol trafficking machinery
between internal membranes and plasma membrane
(22,
23). The expression of
caveolin-1 appears to be under control of
SREBPs,2 the master
regulators of intracellular cholesterol level
(24). Furthermore, knockout of
caveolin-1 significantly affected cholesterol metabolism in mouse embryonic
fibroblasts and mouse peritoneal macrophages
(25). Because we found that
the Na/K-ATPase regulates cellular distribution of caveolin-1, we propose that
it may also affect intracellular cholesterol distribution and metabolism. To
test our hypothesis, we have investigated whether sodium pump α1
knockdown affects cholesterol distribution and metabolism both in
vitro and in vivo. Our results indicate that sodium pump
α1 expression level plays a role in the proper distribution of
intracellular cholesterol. Down-regulation of sodium pump α1 not only
redistributes cholesterol between the plasma membrane and cytosolic
compartments, but also alters cholesterol metabolism in mice. 相似文献