共查询到20条相似文献,搜索用时 46 毫秒
1.
2.
Jacamo R Sinnett-Smith J Rey O Waldron RT Rozengurt E 《The Journal of biological chemistry》2008,283(19):12877-12887
Protein kinase D (PKD) is a serine/threonine protein kinase rapidly
activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C
(PKC)-dependent pathway. Recently, PKD has been implicated in the regulation
of long term cellular activities, but little is known about the mechanism(s)
of sustained PKD activation. Here, we show that cell treatment with the
preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid
(1–5-min) PKD activation induced by bombesin stimulation, but this
inhibition was greatly diminished at later times of bombesin stimulation
(e.g. 45 min). These results imply that GPCR-induced PKD activation
is mediated by early PKC-dependent and late PKC-independent mechanisms.
Western blot analysis with site-specific antibodies that detect the
phosphorylated state of the activation loop residues Ser744 and
Ser748 revealed striking PKC-independent phosphorylation of
Ser748 as well as Ser744 phosphorylation that remained
predominantly but not completely PKC-dependent at later times of bombesin or
vasopressin stimulation (20–90 min). To determine the mechanisms
involved, we examined activation loop phosphorylation in a set of PKD mutants,
including kinase-deficient, constitutively activated, and PKD forms in which
the activation loop residues were substituted for alanine. Our results show
that PKC-dependent phosphorylation of the activation loop Ser744
and Ser748 is the primary mechanism involved in early phase PKD
activation, whereas PKD autophosphorylation on Ser748 is a major
mechanism contributing to the late phase of PKD activation occurring in cells
stimulated by GPCR agonists. The present studies identify a novel mechanism
induced by GPCR activation that leads to late, PKC-independent PKD
activation.A rapid increase in the synthesis of lipid-derived second messengers with
subsequent activation of protein phosphorylation cascades has emerged as a
fundamental signal transduction mechanism triggered by multiple extracellular
stimuli, including hormones, neurotransmitters, chemokines, and growth factors
(1). Many of these agonists
bind to G protein-coupled receptors
(GPCRs),4 activate
heterotrimeric G proteins and stimulate isoforms of the phospholipase C
family, including β, γ, δ, and ε (reviewed in Refs.
1 and
2). Activated phospholipase Cs
catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce
the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG).
Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular
stores (3,
4) whereas DAG directly
activates the classic (α, β, and γ) and novel (δ,
ε, η, and θ) isoforms of PKC
(5–7).
Although it is increasingly recognized that each PKC isozyme has specific
functions in vivo
(5–8),
the mechanisms by which PKC-mediated signals are propagated to critical
downstream targets remain incompletely defined.PKD, also known initially as PKCμ
(9,
10), and two recently
identified serine protein kinases termed PKD2
(11) and PKCν/PKD3
(12,
13), which are similar in
overall structure and primary amino acid sequence to PKD
(14), constitute a new protein
kinase family within the Ca2+/calmodulin-dependent protein kinase
group (15) and separate from
the previously identified PKCs
(14). Salient features of PKD
structure include an N-terminal regulatory region containing a tandem repeat
of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain)
that confers high affinity binding to phorbol esters and DAG
(9,
16,
17), followed by a pleckstrin
homology (PH) domain that negatively regulates catalytic activity
(18,
19). The C-terminal region of
the PKDs contains its catalytic domain, which is distantly related to
Ca2+-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity
maintained by the N-terminal domain, which represses the catalytic activity of
the enzyme by autoinhibition. Consistent with this model, deletions or single
amino acid substitutions in the PH domain result in constitutive kinase
activity
(18–20).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(21). In response to cellular
stimuli, PKD is converted from a low activity form into a persistently active
form that is retained during isolation from cells, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(21,
22). PKD activation has been
demonstrated in response to engagement of specific GPCRs either by regulatory
peptides
(23–30)
or lysophosphatidic acid (27,
31,
32); signaling through
Gq, G12, Gi, and Rho
(27,
31–34);
activation of receptor tyrosine kinases, such as the platelet-derived growth
factor receptor (23,
35,
36); cross-linking of B-cell
receptor and T-cell receptor in B and T lymphocytes, respectively
(37–40);
and oxidative stress
(41–44).Throughout these studies, multiple lines of evidence indicated that PKC
activity is necessary for rapid PKD activation within intact cells. For
example, rapid PKD activation was selectively and potently blocked by cell
treatment with preferential PKC inhibitors (e.g. GF 109203X or
Gö 6983) that do not directly inhibit PKD catalytic activity
(21,
22), implying that PKD
activation in intact cells is mediated, directly or indirectly, through PKCs.
In line with this conclusion, cotransfection of PKD with active mutant forms
of “novel” PKCs (PKCs δ, ε, η, and θ)
resulted in robust PKD activation in the absence of cell stimulation
(21,
44–46).
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
in response to multiple GPCR agonists in a broad range of cell types,
including normal and cancer cells (reviewed in Ref.
14). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as the activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation (reviewed in Ref.
14). Collectively, these
findings demonstrated the existence of rapidly activated PKC-PKD protein
kinase cascade(s) and raised the possibility that some PKC-dependent
biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular
activities and processes, including signal transduction
(30,
47–49),
chromatin modification (50),
Golgi organization and function
(51,
52), c-Jun function
(47,
53,
54), NFκB-mediated gene
expression (43,
55,
56), and cell survival,
migration, and differentiation and DNA synthesis and proliferation (reviewed
in Ref. 14). Thus, mounting
evidence indicates that PKD has a remarkable diversity of both its signal
generation and distribution and its potential for complex regulatory
interactions with multiple downstream pathways, leading to multiple responses,
including long term cellular events. Despite increasing recognition of its
importance, very little is known about the mechanism(s) of sustained PKD
activation as opposed to the well documented rapid, PKC-dependent PKD
activation.The results presented here demonstrate that prolonged GPCR-induced PKD
activation is mediated by sequential PKC-dependent and PKC-independent phases
of regulation. We report here, for the first time, that PKD
autophosphorylation on Ser748 is a major mechanism contributing to
the late phase of PKD activation occurring in cells stimulated by GPCR
agonists. The present studies expand previous models of PKD regulation by
identifying a novel mechanism induced by GPCR activation that leads to late,
PKC-independent PKD activation. 相似文献
3.
4.
Ivano Bertini Marco Fragai Claudio Luchinat Maxime Melikian Efstratios Mylonas Niko Sarti Dmitri I. Svergun 《The Journal of biological chemistry》2009,284(19):12821-12828
The presence of extensive reciprocal conformational freedom between the
catalytic and the hemopexin-like domains of full-length matrix
metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray
scattering experiments. This finding is discussed in relation to the
essentiality of the hemopexin-like domain for the collagenolytic activity of
MMP-1. The conformational freedom experienced by the present system, having
the shortest linker between the two domains, when compared with similar
findings on MMP-12 and MMP-9 having longer and the longest linker within the
family, respectively, suggests this type of conformational freedom to be a
general property of all MMPs.Matrix metalloproteinases
(MMP)2 are
extracellular hydrolytic enzymes involved in a variety of processes including
connective tissue cleavage and remodeling
(1–3).
All 23 members of the family are able to cleave simple peptides derived from
connective tissue components such as collagen, gelatin, elastin, etc. A subset
of MMPs is able to hydrolyze more resistant polymeric substrates, such as
cross-linked elastin, and partially degraded collagen forms, such as gelatin
and type IV collagens (4).
Intact triple helical type I–III collagen is only attacked by
collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14
(5–12).
Although the detailed mechanism of cleavage of single chain peptides by MMP
has been largely elucidated
(13–19),
little is known about the process of hydrolysis of triple helical collagen. In
fact, triple helical collagen cannot be accommodated in the substrate-binding
groove of the catalytic site of MMPs
(9).All MMPs (but MMP-7) in their active form are constituted by a catalytic
domain (CAT) and a hemopexin-like domain (HPX)
(20–22).
The CAT domain contains two zinc ions and one to three calcium ions. One zinc
ion is at the catalytic site and is responsible for the activity, whereas the
other metal ions have structural roles. The isolated CAT domains retain full
catalytic activity toward simple peptides and single chain polymeric
substrates such as elastin, whereas hydrolysis of triple helical collagen also
requires the presence of the HPX domain
(9,
23–25).
It has been shown that the isolated CAT domain regains a small fraction of the
activity of the full-length (FL) protein when high amounts of either
inactivated full-length proteins or isolated HPX domains are added to the
assay solution (9). Finally, it
has been shown that the presence of the HPX domain alone alters the CD
spectrum of triple helical collagen in a way that suggests its partial
unwinding (26,
27). It is tempting to
speculate that full-length collagenases attack collagen by first locally
unwinding the triple helical structure with the help of the HPX domain and
then cleaving the resulting, exposed, single filaments
(9,
28).Until 2007, three-dimensional structures of full-length MMPs had been
reported only for collagenase MMP-1
(29–31)
and gelatinase MMP-2 (32). The
structures of the two proteins are very similar and show a compact arrangement
of the two domains, which are connected by a short linker (14 and 20 amino
acids, respectively). It is difficult to envisage that rigid and compact
molecules of this type can interact with triple helical collagen in a way that
can lead to first unwinding and then cleavage of individual filaments. It has
been recently suggested that such concerted action could occur much more
easily if the two domains could enjoy at least a partial conformational
independence (9). Slight
differences in the reciprocal orientation of the CAT and HPX domains of MMP-1
in the presence (29) and
absence (30,
31) of the prodomain were
indeed taken as a hint that the two domains could experience relative mobility
(29).Two recent solution studies have shown that conformational independence is
indeed occurring in gelatinase MMP-9
(33) and elastase MMP-12
(34), whereas the x-ray
structure of the latter (34)
is only slightly less compact than those of MMP-1
(29–31)
and MMP-2 (32). Among MMPs,
MMP-9 features an exceptionally long linker (68 amino acid)
(33,
35), which in fact constitutes
a small domain by itself (the O-glycosylated domain)
(33), and therefore, this
inspiring observation can hardly be taken as evidence that conformational
freedom is a general characteristic of the two-domain MMPs. MMP-12 features a
much more normal 16-amino acid linker, thereby making more probable a general
functional role for this conformational freedom
(34). However, both MMP-9 and
MMP-12 retain their full catalytic activity against their substrates even when
deprived of the HPX domain (9).
Therefore, the question remains of whether conformational freedom is also a
required characteristic for those MMPs that are only active as full-length
proteins, i.e. collagenases. Interestingly, the three collagenases
(MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all
MMPs. Demonstrating or negating the presence of conformational freedom in one
of these collagenases would therefore constitute a significant step forward to
formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution
(34) have shown that a
combination of NMR relaxation studies and small angle x-ray scattering (SAXS)
is enough to show the presence and the extent of the relative conformational
freedom of the two domains of MMPs. Here we apply the same strategy to
full-length MMP-1 and show that sizable conformational freedom is indeed
experienced even by this prototypical collagenase, although somewhat less
pronounced than that observed for MMP-12. 相似文献
5.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
6.
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
7.
8.
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
9.
10.
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
11.
12.
13.
Lifu Wang John C. Lawrence Jr. Thomas W. Sturgill Thurl E. Harris 《The Journal of biological chemistry》2009,284(22):14693-14697
mTORC1 contains multiple proteins and plays a central role in cell growth
and metabolism. Raptor (regulatory-associated protein of mammalian target of
rapamycin (mTOR)), a constitutively binding protein of mTORC1, is essential
for mTORC1 activity and critical for the regulation of mTORC1 activity in
response to insulin signaling and nutrient and energy sufficiency. Herein we
demonstrate that mTOR phosphorylates raptor in vitro and in
vivo. The phosphorylated residues were identified by using phosphopeptide
mapping and mutagenesis. The phosphorylation of raptor is stimulated by
insulin and inhibited by rapamycin. Importantly, the site-directed mutation of
raptor at one phosphorylation site, Ser863, reduced mTORC1 activity
both in vitro and in vivo. Moreover, the Ser863
mutant prevented small GTP-binding protein Rheb from enhancing the
phosphorylation of S6 kinase (S6K) in cells. Therefore, our findings indicate
that mTOR-mediated raptor phosphorylation plays an important role on
activation of mTORC1.Mammalian target of rapamycin
(mTOR)2 has been shown
to function as a critical controller in cellular growth, survival, metabolism,
and development (1). mTOR, a
highly conserved Ser-Thr phosphatidylinositol 3-kinase-related protein kinase,
structurally forms two distinct complexes, mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2), each of which catalyzes the phosphorylation of different
substrates (1). The best
characterized substrates for mTORC1 are eIF4E-binding protein (4E-BP, also
known as PHAS) and p70 S6 kinase (S6K)
(1), whereas mTORC2
phosphorylates the hydrophobic and turn motifs of protein kinase B
(Akt/protein kinase B) (2) and
protein kinase C (3,
4). mTORC1 constitutively
consists of mTOR, raptor, and mLst8/GβL
(1), whereas the proline-rich
Akt substrate of 40 kDa (PRAS40) is a regulatory component of mTORC1 that
disassociates after growth factor stimulation
(5,
6). Raptor is essential for
mTORC1 activity by providing a substrate binding function
(7) but also plays a regulatory
role on mTORC1 with stimuli of growth factors and nutrients
(8). In response to insulin,
raptor binding to substrates is elevated through the release of the
competitive inhibitor PRAS40 from mTORC1
(9,
10) because PRAS40 and the
substrates of mTORC1 (4E-BP and S6K) appear to bind raptor through a consensus
sequence, the TOR signaling (TOS) motif
(10–14).
In response to amino acid sufficiency, raptor directly interacts with a
heterodimer of Rag GTPases and promotes mTORC1 localization to the
Rheb-containing vesicular compartment
(15).mTORC1 integrates signaling pathways from growth factors, nutrients,
energy, and stress, all of which generally converge on the tuberous sclerosis
complex (TSC1-TSC2) through the phosphorylation of TSC2
(1). Growth factors inhibit the
GTPase-activating protein activity of TSC2 toward the small GTPase Rheb via
the PI3K/Akt pathway (16,
17), whereas energy depletion
activates TSC2 GTPase-activating protein activity by stimulating AMP-activated
protein kinase (AMPK) (18).
Rheb binds directly to mTOR, albeit with very low affinity
(19), and upon charging with
GTP, Rheb functions as an mTORC1 activator
(6). mTORC1 complexes isolated
from growth factor-stimulated cells show increased kinase activity yet do not
contain detectable levels of associated Rheb. Therefore, how Rheb-GTP binding
to mTOR leads to an increase in mTORC1 activity toward substrates, and what
the role of raptor is in this activation is currently unknown. More recently,
the AMPK and p90 ribosomal S6 kinase (RSK) have been reported to directly
phosphorylate raptor and regulate mTORC1 activity. The phosphorylation of
raptor directly by AMPK reduced mTORC1 activity, suggesting an alternative
regulation mechanism independent of TSC2 in response to energy supply
(20). RSK-mediated raptor
phosphorylation enhances mTORC1 activity and provides a mechanism whereby
stress may activate mTORC1 independent of the PI3K/Akt pathway
(21). Therefore, the
phosphorylation status of raptor can be critical for the regulation of mTORC1
activity.In this study, we investigated phosphorylation sites in raptor catalyzed by
mTOR. Using two-dimensional phosphopeptide mapping, we found that
Ser863 and Ser859 in raptor were phosphorylated by mTOR
both in vivo and in vitro. mTORC1 activity in vitro
and in vivo is associated with the phosphorylation of
Ser863 in raptor. 相似文献
14.
15.
16.
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
17.
Rebecca M. Dixon Jack R. Mellor Jonathan G. Hanley 《The Journal of biological chemistry》2009,284(21):14230-14235
Oxygen and glucose deprivation (OGD) induces delayed cell death in
hippocampal CA1 neurons via Ca2+/Zn2+-permeable,
GluR2-lacking AMPA receptors (AMPARs). Following OGD, synaptic AMPAR currents
in hippocampal neurons show marked inward rectification and increased
sensitivity to channel blockers selective for GluR2-lacking AMPARs. This
occurs via two mechanisms: a delayed down-regulation of GluR2 mRNA expression
and a rapid internalization of GluR2-containing AMPARs during the OGD insult,
which are replaced by GluR2-lacking receptors. The mechanisms that underlie
this rapid change in subunit composition are unknown. Here, we demonstrate
that this trafficking event shares features in common with events that mediate
long term depression and long term potentiation and is initiated by the
activation of N-methyl-d-aspartic acid receptors. Using
biochemical and electrophysiological approaches, we show that peptides that
interfere with PICK1 PDZ domain interactions block the OGD-induced switch in
subunit composition, implicating PICK1 in restricting GluR2 from synapses
during OGD. Furthermore, we show that GluR2-lacking AMPARs that arise at
synapses during OGD as a result of PICK1 PDZ interactions are involved in
OGD-induced delayed cell death. This work demonstrates that PICK1 plays a
crucial role in the response to OGD that results in altered synaptic
transmission and neuronal death and has implications for our understanding of
the molecular mechanisms that underlie cell death during stroke.Oxygen and glucose deprivation
(OGD)3 associated with
transient global ischemia induces delayed cell death, particularly in
hippocampal CA1 pyramidal cells
(1–3),
a phenomenon that involves Ca2+/Zn2+-permeable,
GluR2-lacking AMPARs (4).
AMPARs are heteromeric complexes of subunits GluR1–4
(5), and most AMPARs in the
hippocampus contain GluR2, which renders them calcium-impermeable and results
in a marked inward rectification in their current-voltage relationship
(6–8).
Ischemia induces a delayed down-regulation of GluR2 mRNA and protein
expression (4,
9–11),
resulting in enhanced AMPAR-mediated Ca2+ and Zn2+
influx into CA1 neurons (10,
12). In these neurons,
AMPAR-mediated postsynaptic currents (EPSCs) show marked inward rectification
1–2 days following ischemia and increased sensitivity to 1-naphthyl
acetyl spermine (NASPM), a channel blocker selective for GluR2-lacking AMPARs
(13–16).
Blockade of these channels at 9–40 h following ischemia is
neuroprotective, indicating a crucial role for Ca2+-permeable
AMPARs in ischemic cell death
(16).In addition to delayed changes in AMPAR subunit composition as a result of
altered mRNA expression, it was recently reported that
Ca2+-permable, GluR2-lacking AMPARs are targeted to synaptic sites
via membrane trafficking at much earlier times during OGD
(17). This subunit
rearrangement involves endocytosis of AMPARs containing GluR2 complexed with
GluR1/3, followed by exocytosis of GluR2-lacking receptors containing GluR1/3
(17). However, the molecular
mechanisms behind this trafficking event are unknown, and furthermore, it is
not known whether these trafficking-mediated changes in AMPAR subunit
composition contribute to delayed cell death.AMPAR trafficking is a well studied phenomenon because of its crucial
involvement in long term depression (LTD) and long term potentiation (LTP),
activity-dependent forms of synaptic plasticity thought to underlie learning
and memory. AMPAR endocytosis, exocytosis, and more recently subunit-switching
events (brought about by trafficking that involves endo/exocytosis) are
central to the necessary changes in synaptic receptor complement
(7,
18–20).
It is possible that similar mechanisms regulate AMPAR trafficking during
OGD.PICK1 is a PDZ and BAR (Bin-amphiphysin-Rus) domain-containing protein that
binds, via the PDZ domain, to a number of membrane proteins including AMPAR
subunits GluR2/3. This interaction is required for AMPAR internalization from
the synaptic plasma membrane in response to Ca2+ influx via NMDAR
activation in hippocampal neurons
(21–23).
This process is the major mechanism that underlies the reduction in synaptic
strength in LTD. Furthermore, PICK1-mediated trafficking has recently emerged
as a mechanism that regulates the GluR2 content of synaptic receptors, which
in turn determines their Ca2+ permeability
(7,
20). This is likely to be of
profound importance in both plasticity and pathological mechanisms.
Importantly, PICK1 overexpression has been shown to induce a shift in synaptic
AMPAR subunit composition in hippocampal CA1 neurons, resulting in inwardly
rectifying AMPAR EPSCs via reduced surface GluR2 and no change in GluR1
(24). This suggests that PICK1
may mediate the rapid switch in subunit composition occurring during OGD
(17). Here, we demonstrate
that the OGD-induced switch in AMPAR subunit composition is dependent on PICK1
PDZ interactions, and importantly, that this early trafficking event that
occurs during OGD contributes to the signaling that results in delayed
neuronal death. 相似文献
18.
A Role for the Proton-coupled Folate Transporter (PCFT-SLC46A1) in Folate
Receptor-mediated
Endocytosis 总被引:1,自引:0,他引:1
Rongbao Zhao Sang Hee Min Yanhua Wang Estela Campanella Philip S. Low I. David Goldman 《The Journal of biological chemistry》2009,284(7):4267-4274
Recently, this laboratory identified a proton-coupled folate transporter
(PCFT), with optimal activity at low pH. PCFT is critical to intestinal folate
absorption and transport into the central nervous system because there are
loss-of-function mutations in this gene in the autosomal recessive disorder,
hereditary folate malabsorption. The current study addresses the role PCFT
might play in another transport pathway, folate receptor (FR)-mediated
endocytosis. FRα cDNA was transfected into novel PCFT+ and
PCFT– HeLa sublines. FRα was shown to bind and trap
folates in vesicles but with minimal export into the cytosol in
PCFT– cells. Cotransfection of FRα and PCFT resulted in
enhanced folate transport into cytosol as compared with transfection of
FRα alone. Probenecid did not inhibit folate binding to FR, but
inhibited PCFT-mediated transport at endosomal pH, and blocked
FRα-mediated transport into the cytosol. FRα and PCFT co-localized
to the endosomal compartment. These observations (i) indicate that PCFT plays
a role in FRα-mediated endocytosis by serving as a route of export of
folates from acidified endosomes and (ii) provide a functional role for PCFT
in tissues in which it is expressed, such as the choroid plexus, where the
extracellular milieu is at neutral pH.Loss of function mutations of the proton-coupled folate transporter
(PCFT),2 which
functions optimally at low pH, are the molecular basis for the autosomal
recessive disorder, hereditary folate malabsorption (HFM)
(1–4).
Infants present with this disorder several months after birth with marked
folate deficiency anemia, hypogammaglobulinemia with immune deficiency and
infections, neurological deficits, and often seizures
(5). PCFT is highly expressed
at the apical brush-border membrane of the duodenum and proximal jejunum
(6–9)
where the pH at the microclimate of the surface of this epithelium is low (pH
5.8–6.0), and folates are absorbed
(1,
7,
10,
11). Hence, the failure to
absorb folates in the absence of this transporter in HFM is expected. However,
PCFT expression, and its associated folate transport activity at low pH, is
observed in many tissues where the transport interface is presumed to be at pH
7.4 (12). Of particular
interest is the mechanism by which PCFT mediates transport of folates into the
central nervous system (CNS) where this transporter is expressed in brain and
choroid plexus (1,
7,
13). Transport into the CNS is
impaired in patients with HFM who have very low cerebrospinal fluid (CSF)
folate levels and marked reversal of the blood:CSF folate gradient which is
normally 2–3:1 (5).Folates are also transported into cells by a receptor-mediated process.
Folate receptor-α (FRα) is anchored to cell membranes via a
glycosylphosphatidylinositol domain. Uptake begins with folate binding to
receptor at the cell surface followed by invagination of the membrane and the
formation of endosomes that traffic along microtubules to a perinuclear
compartment before returning to the plasma membrane
(14–16).
During transit in the cytoplasm, endosomes acidify to a pH of
∼6.0–6.5 (17),
folate is released from the receptor and exported from the intact endosome
into the cytoplasm. This putative exporter was shown to require a
trans-endosomal pH gradient
(18–20).The current report addresses the hypothesis that PCFT is an endosomal
folate exporter and thereby plays a role in FRα-mediated endocytosis
(1,
2,
21,
22), that the ubiquitous
expression of PCFT in mammalian tissues may be related to this function, and
that loss of this function may be a basis for the low CSF folate levels in
HFM. The experimental approach utilized a series of HeLa sublines, developed
in this laboratory, in which constitutive expression of FRα is
negligible. HeLa R5 cells lack reduced folate carrier (RFC) function due to a
genomic deletion of this gene
(23). A derivative of R5
cells, HeLa R1-11 cells lack, in addition, PCFT expression, while an R1-11
revertant re-expresses PCFT
(24). The impact of PCFT on
FRα-mediated endocytosis, achieved by transfection of the receptor into
these cell lines, was assessed under conditions in which there was negligible
PCFT-mediated transport directly across the plasma membrane into cells. 相似文献
19.
Greg Brown Alexander Singer Vladimir V. Lunin Michael Proudfoot Tatiana Skarina Robert Flick Samvel Kochinyan Ruslan Sanishvili Andrzej Joachimiak Aled M. Edwards Alexei Savchenko Alexander F. Yakunin 《The Journal of biological chemistry》2009,284(6):3784-3792
Gluconeogenesis is an important metabolic pathway, which produces glucose
from noncarbohydrate precursors such as organic acids, fatty acids, amino
acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of
gluconeogenesis, is found in all organisms, and five different classes of
these enzymes have been identified. Here we demonstrate that Escherichia
coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which
show different catalytic properties. We present the first crystal structure of
a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and
in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor
(phosphate). The crystal structure of the ligand-free GlpX revealed a compact,
globular shape with two α/β-sandwich domains. The core fold of GlpX
is structurally similar to that of Li+-sensitive phosphatases
implying that they have a common evolutionary origin and catalytic mechanism.
The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that
the active site is located between two domains and accommodates several
conserved residues coordinating two metal ions and the substrate. The third
metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate
strongly inhibited activity of both GlpX and YggF, and the crystal structure
of the GlpX complex with phosphate demonstrated that the inhibitor molecule
binds to the active site. Alanine replacement mutagenesis of GlpX identified
12 conserved residues important for activity and suggested that
Thr90 is the primary catalytic residue. Our data provide insight
into the molecular mechanisms of the substrate specificity and catalysis of
GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase
(FBPase,2 EC
3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of
fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It
is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis,
and the product, fructose 6-phosphate, is an important precursor in various
biosynthetic pathways (1). In
all organisms, gluconeogenesis is an important metabolic pathway that allows
the cells to synthesize glucose from noncarbohydrate precursors, such as
organic acids, amino acids, and glycerol. FBPases are members of the large
superfamily of lithium-sensitive phosphatases, which includes three families
of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167
sequences, Pfam data base). These enzymes show metal-dependent and
lithium-sensitive phosphomonoesterase activity and include inositol
polyphosphate 1-phosphatases, inositol monophosphatases (IMPases),
3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting
on both inositol 1,4-bisphosphate and PAP (PIPases)
(2). They possess a common
structural core with the active site lying between α+β and
α/β domains (3).
Li+-sensitive phosphatases are putative targets for lithium therapy
in the treatment of manic depressive patients
(4), whereas FBPases are
targets for the development of drugs for the treatment of noninsulin-dependent
diabetes (5,
6). In addition, FBPase is
required for virulence in Mycobacterium tuberculosis and
Leishmania major and plays an important role in the production of
lysine and glutamate by Corynebacterium glutamicum
(7,
8).Presently, five different classes of FBPases have been proposed based on
their amino acid sequences (FBPases I to V)
(9–11).
Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in
various prokaryotes. Types I, II, and III are primarily in bacteria, type IV
in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in
thermophilic prokaryotes from both domains
(11). Many organisms have more
than one FBPase, mostly the combination of types I + II or II + III, but no
bacterial genome has a combination of types I and III FBPases
(9). The type I FBPase is the
most widely distributed among living organisms and is the primary FBPase in
Escherichia coli, most bacteria, a few archaea, and all
eukaryotes (9,
11–15).
The type II FBPases are represented by the E. coli GlpX and FBPase
F-I from Synechocystis PCC6803
(9,
16); type III is represented
by the Bacillus subtilis FBPase
(17); type IV is represented
by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus
furiosus (18), MJ0109
from Methanococcus jannaschii
(19), and AF2372 from
Archaeoglobus fulgidus
(20); and type V is
represented by the FBPases TK2164 from Pyrococcus
(Thermococcus) kodakaraensis and ST0318 from Sulfolobus
tokodai (10,
21).Three-dimensional structures of the type I (from pig kidney, spinach
chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V
(ST0318) FBPases have been solved
(10,
11,
19,
20,
22,
23). FBPases I and IV and
inositol monophosphatases share a common sugar phosphatase fold organized in
five layered interleaved α-helices and β-sheets
(α-β-α-β-α)
(2,
19,
24). ST0318 (an FBPase V
enzyme) is composed of one domain with a completely different four-layer
α-β-β-α fold
(10). The FBPases from these
three classes (I, IV, and V) require divalent cations for activity
(Mg2+, Mn2+, or Zn2+), and their structures
have revealed the presence of three or four metal ions in the active site.E. coli has five Li+-sensitive phosphatases as follows:
CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase
II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a
3′-phosphoadenosine 5′-phosphatase involved in the cysteine
biosynthesis pathway (25,
26), whereas SuhB is an
inositol monophosphatase (IMPase) that is also known as a suppressor of
temperature-sensitive growth phenotypes in E. coli
(27,
28). Fbp is required for
growth on gluconeogenic substrates and probably represents the main
gluconeogenic FBPase (12).
This enzyme has been characterized both biochemically and structurally and
shown to be inhibited by low concentrations of AMP (IC50 15
μm) (11,
29,
30). The E. coli
GlpX, a class II enzyme FBPase, has been shown to possess a
Mn2+-dependent FBPase activity
(9). The increased expression
of glpX from a multicopy plasmid complemented the Fbp-
phenotype; however, the glpX knock-out strain grew normally on
gluconeogenic substrates (succinate or glycerol)
(9).In this study, we present the first structure of a class II FBPase, the
E. coli GlpX, in a free state and in the complex with FBP + metals or
phosphate. We have demonstrated that the fold of GlpX is similar to that of
the lithium-sensitive phosphatases. We have identified the GlpX residues
important for activity and proposed a catalytic mechanism. We have also showed
that YggF is a third FBPase in E. coli, which has distinct catalytic
properties and is more sensitive than GlpX to the inhibition by lithium or
phosphate. 相似文献
20.
Sharon Barone Stacey L. Fussell Anurag Kumar Singh Fred Lucas Jie Xu Charles Kim Xudong Wu Yiling Yu Hassane Amlal Ursula Seidler Jian Zuo Manoocher Soleimani 《The Journal of biological chemistry》2009,284(8):5056-5066
The identity of the transporter responsible for fructose absorption in the
intestine in vivo and its potential role in fructose-induced
hypertension remain speculative. Here we demonstrate that Glut5 (Slc2a5)
deletion reduced fructose absorption by ∼75% in the jejunum and decreased
the concentration of serum fructose by ∼90% relative to wild-type mice on
increased dietary fructose. When fed a control (60% starch) diet,
Glut5-/- mice had normal blood pressure and displayed normal weight
gain. However, whereas Glut5+/+ mice showed enhanced salt
absorption in their jejuna in response to luminal fructose and developed
systemic hypertension when fed a high fructose (60% fructose) diet for 14
weeks, Glut5-/- mice did not display fructose-stimulated salt
absorption in their jejuna, and they experienced a significant impairment of
nutrient absorption in their intestine with accompanying hypotension as early
as 3–5 days after the start of a high fructose diet. Examination of the
intestinal tract of Glut5-/- mice fed a high fructose diet revealed
massive dilatation of the caecum and colon, consistent with severe
malabsorption, along with a unique adaptive up-regulation of ion transporters.
In contrast to the malabsorption of fructose, Glut5-/- mice did not
exhibit an absorption defect when fed a high glucose (60% glucose) diet. We
conclude that Glut5 is essential for the absorption of fructose in the
intestine and plays a fundamental role in the generation of fructose-induced
hypertension. Deletion of Glut5 results in a serious nutrient-absorptive
defect and volume depletion only when the animals are fed a high fructose diet
and is associated with compensatory adaptive up-regulation of ion-absorbing
transporters in the colon.Fructose is a monosaccharide and is one of the three most important blood
sugars along with glucose and galactose
(1–3).
It plays an essential role in vital metabolic functions in the body, including
glycolysis and gluconeogenesis
(4–6).
Fructose is predominantly metabolized in the liver. A high flux of fructose to
the liver perturbs glucose metabolism and leads to a significantly enhanced
rate of triglyceride synthesis. In addition, fructose can be metabolized in
the liver to uric acid, a potent antioxidant
(7,
8).The classic model of sugar absorption indicates that sodium glucose
cotransporter 1
(Sglt1)3 and Glut5
absorb glucose and fructose, respectively, from intestinal lumen to cytosol,
and Glut2 transports both glucose and fructose from the cytosol to the blood
(9–19).
Glut2 has high affinity for glucose and a moderate affinity for fructose,
whereas Glut5 predominantly transports fructose with very low affinity for
glucose
(9–19;
reviews in Refs. 14,
17–19).
The expression of Glut5 or Glut2 in the small intestine increases in rats or
mice fed a diet high in fructose or perfused with increased fructose
concentration
(11–14,
18,
19).Glut2 is predominantly found on the basolateral membrane and in the
cytoplasm of enterocytes at basal state but is thought to be recruited to the
apical membrane in the presence of increased glucose or fructose in the
intestinal lumen (11,
19). Given the fact that both
Glut5 and Glut2 can transport fructose in vitro and given the ability
of Glut2 to traffic to the apical membrane, the contribution of Glut5 to the
absorption of fructose in vivo and systemic fructose homeostasis
remains speculative.The marked increase in dietary fructose consumption in the form of high
fructose corn syrup, a common sweetener used in the food industry, table
sugar, and fruits correlates with the increased incidence of metabolic
syndrome, which is reaching an epidemic proportion in developed countries and
is a major contributor to premature morbidity and mortality in our society
(20–22).
Increased dietary fructose intake recapitulates many aspects of metabolic
syndrome, including dyslipidemia, insulin resistance, and hypertension in rat
and mouse
(23–26).
Recent studies demonstrate that fructose-induced hypertension is initiated by
increased absorption of salt and fructose in the intestine
(27); however, the one or more
molecules (Glut2, Glut5, Glut7, or Sglt1) that are responsible for the
absorption of fructose in the intestine remain speculative. Further, although
Glut7, Glut5, and Glut2 can transport fructose in vitro, the role of
Glut5 in in vivo fructose absorption remains unknown. To ascertain
the role of Glut5 in fructose absorption in the intestine in vivo and
fructose-induced hypertension, mice lacking the Glut5 gene
(Glut5-/-) were placed on either high fructose or normal diet and
compared with their wild-type littermates (Glut5+/+). 相似文献