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Tatsuhiro Sato Akio Nakashima Lea Guo Fuyuhiko Tamanoi 《The Journal of biological chemistry》2009,284(19):12783-12791
Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway
by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully
reproduced in vitro by using mTORC1 immunoprecipitated by the use of
anti-raptor antibody from mammalian cells starved for nutrients. The low
in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is
dramatically increased by the addition of recombinant Rheb. On the other hand,
the addition of Rheb does not activate mTORC2 immunoprecipitated from
mammalian cells by the use of anti-rictor antibody. The activation of mTORC1
is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42
did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition,
the activation is dependent on the presence of bound GTP. We also find that
the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a
recently proposed mediator of Rheb action, appears not to be involved in the
Rheb-dependent activation of mTORC1 in vitro, because the preparation
of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of
Rheb results in a significant increase of binding of the substrate protein
4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that
competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation
of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated
by Rheb. Rheb does not induce autophosphorylation of mTOR. These results
suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to
regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins
(1). We have shown that Rheb
proteins are conserved and are found from yeast to human
(2). Although yeast and fruit
fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or
simply Rheb) and Rheb2 (RhebL1)
(2). Structurally, these
proteins contain G1-G5 boxes, short stretches of amino acids that define the
function of the Ras superfamily G-proteins including guanine nucleotide
binding (1,
3,
4). Rheb proteins have a
conserved arginine at residue 15 that corresponds to residue 12 of Ras
(1). The effector domain
required for the binding with downstream effectors encompasses the G2 box and
its adjacent sequences (1,
5). Structural analysis by
x-ray crystallography further shows that the effector domain is exposed to
solvent, is located close to the phosphates of GTP especially at residues
35–38, and undergoes conformational change during GTP/GDP exchange
(6). In addition, all Rheb
proteins end with the CAAX (C is cysteine, A is an aliphatic amino
acid, and X is the C-terminal amino acid) motif that signals
farnesylation. In fact, we as well as others have shown that these proteins
are farnesylated
(7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling
pathway that plays central roles in regulating protein synthesis and growth in
response to nutrient, energy, and growth conditions
(10–14).
Rheb is down-regulated by a TSC1·TSC2 complex that acts as a
GTPase-activating protein for Rheb
(15–19).
Recent studies established that the GAP domain of TSC2 defines the functional
domain for the down-regulation of Rheb
(20). Mutations in the
Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms
include the appearance of benign tumors called hamartomas at different parts
of the body as well as neurological symptoms
(21,
22). Overexpression of Rheb
results in constitutive activation of mTOR even in the absence of nutrients
(15,
16). Two mTOR complexes,
mTORC1 and mTORC2, have been identified
(23,
24). Whereas mTORC1 is
involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is
involved in the phosphorylation of Akt in response to insulin. It has been
suggested that Rheb is involved in the activation of mTORC1 but not mTORC2
(25).Although Rheb is clearly involved in the activation of mTOR, the mechanism
of activation has not been established. We as well as others have suggested a
model that involves the interaction of Rheb with the TOR complex
(26–28).
Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was
reported (29). Rheb has been
shown to interact with mTOR
(27,
30), and this may involve
direct interaction of Rheb with the kinase domain of mTOR
(27). However, this Rheb/mTOR
interaction is a weak interaction and is not dependent on the presence of GTP
bound to Rheb (27,
28). Recently, a different
model proposing that FKBP38 (FK506-binding protein
38) mediates the activation of
mTORC1 by Rheb was proposed
(31,
32). In this model, FKBP38
binds mTOR and negatively regulates mTOR activity, and this negative
regulation is blocked by the binding of Rheb to FKBP38. However, recent
reports dispute this idea
(33).To further characterize Rheb activation of mTOR, we have utilized an in
vitro system that reproduces activation of mTORC1 by the addition of
recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved
cells using anti-raptor antibody and have shown that its kinase activity
against 4E-BP1 is dramatically increased by the addition of recombinant Rheb.
Importantly, the activation of mTORC1 is specific to Rheb and is dependent on
the presence of bound GTP as well as an intact effector domain. FKBP38 is not
detected in our preparation and further investigation suggests that FKBP38 is
not an essential component for the activation of mTORC1 by Rheb. Our study
revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1
rather than increasing the kinase activity of mTOR. 相似文献
3.
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
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Tzung-Ju Wu Yi-Hsuan Chiang Yi-Chien Lin Chang-Ru Tsai Tai-Yuan Yu Ming-Ta Sung Yan-Hwa Wu Lee Jing-Jer Lin 《The Journal of biological chemistry》2009,284(19):12801-12808
Ku is a heterodimeric protein involved in nonhomologous end-joining of the
DNA double-stranded break repair pathway. It binds to the double-stranded DNA
ends and then activates a series of repair enzymes that join the broken DNA.
In addition to its function in DNA repair, the yeast Saccharomyces
cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes
that affect telomere function. The yeast telomeres are composed of duplex
C1–3(A/T)G1–3 telomeric DNA repeats plus
single-stranded TG1–3 telomeric DNA tails. Here we show that
Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that
mimics the structure of telomeres. Addition of Cdc13p, a single-stranded
telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of
a ternary complex with Cdc13p binding to the single-stranded tail of the DNA
substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail
inhibits the binding of Yku, the results suggested that loading of Yku and
Cdc13p to telomeres is sequential. Through generating a double-stranded break
near telomeric DNA sequences, we found that Ku protein appears to bind to the
de novo synthesized telomeres earlier than that of Cdc13p in
vivo. Thus, our results indicated that Yku interacts directly with
telomeres and that sequential loading of Yku followed by Cdc13p to telomeres
is required for both proteins to form a ternary complex on telomeres. Our
results also offer a mechanism that the binding of Cdc13p to telomeres might
prevent Yku from initiating DNA double-stranded break repair pathway on
telomeres.DNA damages in the form of double-stranded breaks
(DSBs)4 compromise the
integrity of genomes. Failure in repairing or mis-repairing double-stranded
breaks can lead to chromosome instability and eventually cell death or cancer
(1). Double-stranded breaks are
repaired by two main pathways, the homologous recombination and nonhomologous
DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to
bind to the DNA ends to initiate the repair pathway
(2). Upon binding, Ku then
recruits a series of repair enzymes to join the broken ends
(2). Ku is a heterodimeric
protein composed of 70- and ∼80-kDa subunits. In Saccharomyces
cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical
configuration of the broken ends could be very diverse on DSBs, Ku binds to
double-stranded ends in a sequence- and energy-independent manner. It is
capable of binding to DNA ends with blunt 3′-overhangs or
5′-overhangs as well as double-stranded DNA with nicks, gaps, or
internal loops
(3–7).
However, Ku does not have high affinity to single-stranded DNA. The crystal
structure of human Ku heterodimer indicates that it forms a ring structure
that encircles duplex DNA (7).
This unique structure feature enables Ku to recognize DNA ends and achieves
its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to
be a component of telomeric protein-DNA complex in yeast and mammals
(8–10).
Telomeres are terminal structures of chromosomes composed of short tandem
repeated sequences (11,
12). Mutation of
YKU70 or YKU80 causes defects in telomere structure
(13–15),
telomere silencing
(16–19),
and replication timing of telomeres
(20). The function of yeast Ku
(Yku) on telomeres could mediate through protein-protein interaction with
Sir4p or protein-RNA interaction with Tlc1 RNA
(21,
22). For example, through the
interaction with Sir4p, Yku selectively affects telomeres silencing but not
the silent mating type loci
(17). Yku could also bind to
telomerase Tlc1 RNA for telomere length maintenance
(22). Judged by the DNA
binding activity of Yku, it is reasonable to suggest that it may bind directly
to telomeric DNA. Indeed, it was shown that human Ku is capable of binding
directly to telomeric DNA in vitro
(15). Moreover, because the
deletion of SIR4 in budding yeast
(23) or Taz1 in
fission yeast (24) does not
abolish the association of Ku with chromosomal ends, this suggests that Ku
might bind directly to telomeric DNA in cells. However, because yeast
telomeres have a short 12–14-mer single-stranded tail
(25), it is uncertain whether
Yku could pass the single-stranded region to reach its binding site. The
direct binding of Yku to telomeric DNA has not been experimentally
determined.In contrast to double-stranded breaks, the ends of linear chromosomes are
not recognized by repair enzymes as DNA damage. In S. cerevisiae,
Cdc13p is the single-stranded TG1–3 DNA-binding protein that
enables cells to differentiate whether the ends of a linear DNA are telomeres
or broken ends
(26–29).
Thus, although the mechanism of how cells prevent the activation of DSB repair
pathway in telomere is unclear, it is likely that binding of Cdc13p to
telomeres might inhibit the initiation of DNA damage response by the Ku
protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA
sequences to mimic telomere structure, we showed that Yku binds directly to
this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our
results also showed that Yku loaded to a de novo synthesized telomere
earlier than Cdc13p in vivo. These results support the direct binding
of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block
the activation of DSB repair pathway on telomeres. 相似文献
7.
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Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
10.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
11.
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
12.
13.
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
14.
Akina Saitoh Hye-Won Shin Akane Yamada Satoshi Waguri Kazuhisa Nakayama 《The Journal of biological chemistry》2009,284(20):13948-13957
ArfGAP1 is a prototype of GTPase-activating proteins for ADP-ribosylation
factors (ARFs) and has been proposed to be involved in retrograde transport
from the Golgi apparatus to the endoplasmic reticulum (ER) by regulating the
uncoating of coat protein I (COPI)-coated vesicles. Depletion of ArfGAP1 by
RNA interference, however, causes neither a discernible phenotypic change in
the COPI localization nor a change in the Golgi-to-ER retrograde transport.
Therefore, we also examined ArfGAP2 and ArfGAP3, closely related homologues of
ArfGAP1. Cells in which ArfGAP1, ArfGAP2, and ArfGAP3 are simultaneously
knocked down show an increase in the GTP-bound ARF level. Furthermore, in
these cells proteins resident in or cycling through the cis-Golgi,
including ERGIC-53, β-COP, and GM130, accumulate in the ER-Golgi
intermediate compartment, and Golgi-to-ER retrograde transport is blocked. The
phenotypes observed in the triple ArfGAP knockdown cells are similar to those
seen in β-COP-depleted cells. Both the triple ArfGAP- and
β-COP-depleted cells accumulate characteristic vacuolar structures that
are visible under electron microscope. Furthermore, COPI is concentrated at
rims of the vacuolar structures in the ArfGAP-depleted cells. On the basis of
these observations, we conclude that ArfGAP1, ArfGAP2, and ArfGAP3 have
overlapping roles in regulating COPI function in Golgi-to-ER retrograde
transport.The ADP-ribosylation factors
(ARFs)3 are a family
of small GTPases. Once associated with organellar membranes in their GTP-bound
form, these proteins trigger formation of coated carrier vesicles,
e.g. coat protein I (COPI)-coated vesicles. ARFs cycle between a
GDP-bound inactive state and a GTP-bound active state; in the latter form they
recruit various effectors, including the COPI coat
(1,
2). Exchange of bound GDP for
GTP is catalyzed by guanine-nucleotide exchange factors, which constitute a
large family of proteins that share a Sec7-like catalytic domain
(3,
4). GTP hydrolysis in turn is
stimulated by GTPase-activating proteins (GAPs), which constitute a large
family that share a zinc finger-like catalytic domain
(3,
5).COPI-coated vesicles mediate retrograde transport from the
cis-Golgi or endoplasmic reticulum (ER)-Golgi intermediate
compartment (ERGIC) to the ER and probably intra-Golgi transport as well. In
budding yeasts two ARF-GAPs, Gcs1 and Glo3, have been shown to play
overlapping roles in COPI-mediated transport processes
(6,
7). According to the prevailing
view, ARF-GAPs (in particular, ArfGAP1, which is the founding member of
mammalian ARF-GAPs and the counterpart of yeast Gcs1)
(8) either induce dissociation
of the coat from COPI-coated vesicles or antagonize formation of vesicles (for
review, see Ref. 5). This view
is based on several lines of evidence; first, blocking GTP hydrolysis on ARF1
by adding GTPγS or a GTPase-defective ARF1 mutant inhibits uncoating of
COPI-coated vesicles in a cell-free reconstitution system
(9), indirectly suggesting a
role for ARF-GAP in vesicle uncoating; second, overexpression of the
GTPase-defective ARF1 mutant stabilizes the COPI coat on Golgi membranes
(10); third, overexpression of
ArfGAP1 results in a phenotype similar to that induced by inhibiting
ARF-guanine-nucleotide exchange factors; that is, cytosolic distribution of
the COPI coat and disintegration of the Golgi apparatus
(11); fourth, the addition of
ArfGAP1 to an in vitro system inhibits formation of COPI-coated
vesicles and induces uncoating of pre-existing vesicles
(12); finally,
ArfGAP1-mediated GTP hydrolysis is stimulated by the addition of the COPI coat
in vitro (13,
14).However, additional evidence suggests roles of ArfGAP1 beyond that of a
simple inactivator of ARFs (for review, see Ref.
5); first, GTP hydrolysis on
ARF is required for proper sorting of cargo molecules into COPI-coated
vesicles
(15-17);
second, ArfGAP1 promotes COPI-coated vesicle formation by coupling cargo
sorting to vesicle formation
(18-20);
third, imaging studies have suggested that ArfGAP1 undergoes ARF1-dependent
cycling between the cytosol and Golgi membranes independent of vesicle budding
(21,
22); finally, Antonny and
co-workers (23,
24) have proposed a model in
which ArfGAP1 and Gcs1 sense the curvature of budding vesicles through a motif
outside of their catalytic domain.Despite the critical roles of ArfGAP1 in COPI-coated vesicle formation,
most of the available data regarding their function have been obtained by
in vitro experiments. We, therefore, attempted to determine the
function of ArfGAP1 in the cell by exploiting RNA interference (RNAi).
However, we could not detect any phenotypic change in ArfGAP1 knockdown cells.
Because there are two poorly characterized mammalian ArfGAPs, ArfGAP2 and
ArfGAP3 (25), both of which
are more similar to Glo3 than Gcs1
(26-29),
we then set out to determine the intracellular roles of these ArfGAPs. Here,
we show that ArfGAP1, ArfGAP2, and ArfGAP3 play overlapping roles in
COPI-mediated transport and in maintaining Golgi organization. 相似文献
15.
Obidimma C. Ezezika Noah S. Younger Jia Lu Donald A. Kaiser Zachary A. Corbin Bradley J. Nolen David R. Kovar Thomas D. Pollard 《The Journal of biological chemistry》2009,284(4):2088-2097
Expression of human profilin-I does not complement the
temperature-sensitive cdc3-124 mutation of the single profilin gene
in fission yeast Schizosaccharomyces pombe, resulting in death from
cytokinesis defects. Human profilin-I and S. pombe profilin have
similar affinities for actin monomers, the FH1 domain of fission yeast formin
Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001)
Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not
stimulate actin filament elongation by formin Cdc12p like S. pombe
profilin. Two crystal structures of S. pombe profilin and homology
models of S. pombe profilin bound to actin show how the two profilins
bind to identical surfaces on animal and yeast actins even though 75% of the
residues on the profilin side of the interaction differ in the two profilins.
Overexpression of human profilin-I in fission yeast expressing native profilin
also causes cytokinesis defects incompatible with viability. Human profilin-I
with the R88E mutation has no detectable affinity for actin and does not have
this dominant overexpression phenotype. The Y6D mutation reduces the affinity
of human profilin-I for poly-l-proline by 1000-fold, but
overexpression of Y6D profilin in fission yeast is lethal. The most likely
hypotheses to explain the incompatibility of human profilin-I with Cdc12p are
differences in interactions with the proline-rich sequences in the FH1 domain
of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of
actin monomers ready to elongate actin filament barbed ends
(2), but it also binds to type
II poly-l-proline helices
(3,
4). The actin
(5) and
poly-l-proline
(6–8)
binding sites are on opposite sides of the profilin molecule, so profilin can
link actin to proline-rich targets. Viability of fission yeast depends
independently on profilin binding to both actin and poly-l-proline,
although cells survive >10-fold reductions in affinity for either ligand
(1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p
(9,
10) and profilin
(11) to assemble actin
filaments for the cytokinetic contractile ring. Formins are multidomain
proteins that nucleate and assemble unbranched actin filaments
(12). Formin FH2 domains form
homodimers that can associate processively with the barbed ends of growing
actin filaments (13,
14). FH2 dimers slow the
elongation of barbed ends
(15). Most formin proteins
have an FH1 domain linked to the FH2 domain. Binding profilin-actin to
multiple polyproline sites in an FH1 domain concentrates actin near the barbed
end of an actin filament associated with a formin FH2 homodimer. Actin
transfers very rapidly from the FH1 domains onto the filament end
(16) allowing profilin to
stimulate elongation of the filament
(15,
17).We tested the ability of human (Homo sapiens,
Hs)7 profilin-I to
complement the temperature-sensitive cdc3-124 mutation
(11) in the single fission
yeast profilin gene with the aim of using yeast to characterize human profilin
mutations. The failure of expression of Hs profilin-I to complement the
cdc3-124 mutation prompted us to compare human and fission yeast
profilins more carefully. We report here a surprising incompatibility of Hs
profilin-I with fission yeast formin Cdc12p, a crystal structure of fission
yeast profilin, which allowed a detailed comparison with Hs profilin, and
mutations that revealed how overexpression of Hs profilin-I compromises the
viability of wild-type fission yeast. 相似文献
16.
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
17.
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
18.
19.
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
20.