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1.
Gaetan Pascreau Frank Eckerdt Andrea L. Lewellyn Claude Prigent James L. Maller 《The Journal of biological chemistry》2009,284(9):5497-5505
p53 is an important tumor suppressor regulating the cell cycle at multiple
stages in higher vertebrates. The p53 gene is frequently deleted or mutated in
human cancers, resulting in loss of p53 activity. This leads to centrosome
amplification, aneuploidy, and tumorigenesis, three phenotypes also observed
after overexpression of the oncogenic kinase Aurora A. Accordingly, recent
studies have focused on the relationship between these two proteins. p53 and
Aurora A have been reported to interact in mammalian cells, but the function
of this interaction remains unclear. We recently reported that
Xenopus p53 can inhibit Aurora A activity in vitro but only
in the absence of TPX2. Here we investigate the interplay between
Xenopus Aurora A, TPX2, and p53 and show that newly synthesized TPX2
is required for nearly all Aurora A activation and for full p53 synthesis and
phosphorylation in vivo during oocyte maturation. In vitro,
phosphorylation mediated by Aurora A targets serines 129 and 190 within the
DNA binding domain of p53. Glutathione S-transferase pull-down
studies indicate that the interaction occurs via the p53 transactivation
domain and the Aurora A catalytic domain around the T-loop. Our studies
suggest that targeting of TPX2 might be an effective strategy for specifically
inhibiting the phosphorylation of Aurora A substrates, including p53.Aurora A is an oncogenic protein kinase that is active in mitosis and plays
important roles in spindle assembly and centrosome function
(1). Overexpression of either
human or Xenopus Aurora A transforms mammalian cells, but only when
the p53 pathway is altered
(2–4).
Aurora A is localized on centrosomes during mitosis, and overexpression of the
protein leads to centrosome amplification and aneuploidy
(2,
3,
5,
6), two likely contributors to
genomic instability (7,
8). Because of its oncogenic
potential and amplification in human tumors, considerable attention has been
focused on the mechanism of Aurora A activation in mitosis. Evidence from
several laboratories indicates that activation occurs as a result of
phosphorylation of a threonine residue in the T-loop of the kinase
(4,
9,
10). Purification of Aurora
A-activating activity from M phase Xenopus egg extracts led to an
apparent activation mechanism in which autophosphorylation at the T-loop is
stimulated by binding of the targeting protein for Xklp2 (TPX2)
(11–14).
On the other hand, it has been shown that Aurora A activity can be inhibited
by interaction with several proteins, including PP1 (protein phosphatase 1),
AIP (Aurora A kinase-interacting protein), and, more recently, p53
(9,
15–17).p53 is a well known tumor suppressor able to drive cell cycle arrest,
apoptosis, or senescence when DNA is damaged or cell integrity is threatened
(18,
19). In human cancers, the p53
gene is frequently deleted or mutated, leading to inactivation of p53
functions (20). p53 protein is
almost undetectable in “normal cells,” mainly due to its
instability. Indeed, during a normal cell cycle, p53 associates with Mdm2 in
the nucleus and thereafter undergoes nuclear exclusion, allowing its
ubiquitination and subsequent degradation
(21). In cells under stress,
p53 is stabilized through the disruption of its interaction with Mdm2
(21), leading to p53
accumulation in the nucleus and triggering different responses, as described
above.Although p53 has mostly been characterized as a nuclear protein, it has
also been shown to localize on centrosomes
(22–24)
and regulate centrosome duplication
(23,
24). Centrosomes are believed
to act as scaffolds that concentrate many regulatory molecules involved in
signal transduction, including multiple protein kinases
(25). Thus, centrosomal
localization of p53 might be important for its own regulation by
phosphorylation/dephosphorylation, and one of its regulators could be the
mitotic kinase Aurora A. Indeed, phenotypes associated with the misexpression
of these two proteins are very similar. For example, overexpression of Aurora
A kinase leads to centrosome amplification, aneuploidy, and tumorigenesis, and
the same effects are often observed after down-regulation of p53
transactivation activity or deletion/mutation of its gene
(26,
27).Several recent studies performed in mammalian models show interplay between
p53 and Aurora A, with each protein having the ability to inhibit the other,
depending on the stage of the cell cycle and the stress level of the cell
(17,
28,
29). These studies reported
that p53 is a substrate of Aurora A, and serines 215 and 315 were demonstrated
to be the two major Aurora A phosphorylation sites in human p53 in
vitro and in vivo. Phosphorylation of Ser-215 within the DNA
binding domain of human p53 inhibited both p53 DNA binding and transactivation
activities (29). Recently, our
group showed that Xenopus p53 is able to inhibit Aurora A kinase
activity in vitro, but this inhibitory effect can be suppressed by
prior binding of Aurora A to TPX2
(9). Contrary to somatic cells,
where p53 is nuclear, unstable, and expressed at a very low level, p53 is
highly expressed in the cytoplasm of Xenopus oocytes and stable until
later stages of development
(30,
31). The high concentration of
both p53 and Aurora A in the oocyte provided a suitable basis for
investigating p53-Aurora A interaction and also evaluating Xenopus
p53 as a substrate of Aurora A. 相似文献
2.
3.
4.
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
5.
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
6.
Dong Han Hamid Y. Qureshi Yifan Lu Hemant K. Paudel 《The Journal of biological chemistry》2009,284(20):13422-13433
In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked
to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms
paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is
phosphorylated at a number of sites, migrates as ∼60-, 64-, and 68-kDa
bands on SDS-gel, and does not promote microtubule assembly. Upon
dephosphorylation, the PHF-tau migrates as ∼50–60-kDa bands on
SDS-gels in a manner similar to tau that is isolated from normal brain and
promotes microtubule assembly. The site(s) that inhibits microtubule
assembly-promoting activity when phosphorylated in the diseased brain is not
known. In this study, when tau was phosphorylated by Cdk5 in vitro,
its mobility shifted from ∼60-kDa bands to ∼64- and 68-kDa bands in a
time-dependent manner. This mobility shift correlated with phosphorylation at
Ser202, and Ser202 phosphorylation inhibited tau
microtubule-assembly promoting activity. When several tau point mutants were
analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17,
but not nonspecific mutations S214A and S262A, promoted Ser202
phosphorylation and mobility shift to a ∼68-kDa band. Furthermore,
Ser202 phosphorylation inhibited the microtubule assembly-promoting
activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17
missense mutations, by promoting phosphorylation at Ser202, inhibit
the microtubule assembly-promoting activity of tau in vitro,
suggesting that Ser202 phosphorylation plays a major role in the
development of NFT pathology in AD and related tauopathies.Neurofibrillary tangles
(NFTs)4 and senile
plaques are the two characteristic neuropathological lesions found in the
brains of patients suffering from Alzheimer disease (AD). The major fibrous
component of NFTs are paired helical filaments (PHFs) (for reviews see Refs.
1–3).
Initially, PHFs were found to be composed of a protein component referred to
as “A68” (4).
Biochemical analysis reveled that A68 is identical to the
microtubule-associated protein, tau
(4,
5). Some characteristic
features of tau isolated from PHFs (PHF-tau) are that it is abnormally
hyperphosphorylated (phosphorylated on more sites than the normal brain tau),
does not bind to microtubules, and does not promote microtubule assembly
in vitro. Upon dephosphorylation, PHF-tau regains its ability to bind
to and promote microtubule assembly
(6,
7). Tau hyperphosphorylation is
suggested to cause microtubule instability and PHF formation, leading to NFT
pathology in the brain
(1–3).PHF-tau is phosphorylated on at least 21 proline-directed and
non-proline-directed sites (8,
9). The individual contribution
of these sites in converting tau to PHFs is not entirely clear. However, some
sites are only partially phosphorylated in PHFs
(8), whereas phosphorylation on
specific sites inhibits the microtubule assembly-promoting activity of tau
(6,
10). These observations
suggest that phosphorylation on a few sites may be responsible and sufficient
for causing tau dysfunction in AD.Tau purified from the human brain migrates as ∼50–60-kDa bands on
SDS-gel due to the presence of six isoforms that are phosphorylated to
different extents (2). PHF-tau
isolated from AD brain, on the other hand, displays ∼60-, 64-, and 68
kDa-bands on an SDS-gel (4,
5,
11). Studies have shown that
∼64- and 68-kDa tau bands (the authors have described the ∼68-kDa tau
band as an ∼69-kDa band in these studies) are present only in brain areas
affected by NFT degeneration
(12,
13). Their amount is
correlated with the NFT densities at the affected brain regions. Moreover, the
increase in the amount of ∼64- and 68-kDa band tau in the brain correlated
with a decline in the intellectual status of the patient. The ∼64- and
68-kDa tau bands were suggested to be the pathological marker of AD
(12,
13). Biochemical analyses
determined that ∼64- and 68-kDa bands are hyperphosphorylated tau, which
upon dephosphorylation, migrated as normal tau on SDS-gel
(4,
5,
11). Tau sites involved in the
tau mobility shift to ∼64- and 68-kDa bands were suggested to have a role
in AD pathology (12,
13). It is not known whether
phosphorylation at all 21 PHF-sites is required for the tau mobility shift in
AD. However, in vitro the tau mobility shift on SDS-gel is sensitive
to phosphorylation only on some sites
(6,
14). It is therefore possible
that in the AD brain, phosphorylation on some sites also causes a tau mobility
shift. Identification of such sites will significantly enhance our knowledge
of how NFT pathology develops in the brain.PHFs are also the major component of NFTs found in the brains of patients
suffering from a group of neurodegenerative disorders collectively called
tauopathies (2,
11). These disorders include
frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17),
corticobasal degeneration, progressive supranuclear palsy, and Pick disease.
Each PHF-tau isolated from autopsied brains of patients suffering from various
tauopathies is hyperphosphorylated, displays ∼60-, 64-, and 68-kDa bands
on SDS-gel, and is incapable of binding to microtubules. Upon
dephosphorylation, the above referenced PHF-tau migrates as a normal tau on
SDS-gel, binds to microtubules, and promotes microtubule assembly
(2,
11). These observations
suggest that the mechanisms of NFT pathology in various tauopathies may be
similar and the phosphorylation-dependent mobility shift of tau on SDS-gel may
be an indicator of the disease. The tau gene is mutated in familial FTDP-17,
and these mutations accelerate NFT pathology in the brain
(15–18).
Understanding how FTDP-17 mutations promote tau phosphorylation can provide a
better understanding of how NFT pathology develops in AD and various
tauopathies. However, when expressed in CHO cells, G272V, R406W, V337M, and
P301L tau mutations reduce tau phosphorylation
(19,
20). In COS cells, although
G272V, P301L, and V337M mutations do not show any significant affect, the
R406W mutation caused a reduction in tau phosphorylation
(21,
22). When expressed in SH-SY5Y
cells subsequently differentiated into neurons, the R406W, P301L, and V337M
mutations reduce tau phosphorylation
(23). In contrast, in
hippocampal neurons, R406W increases tau phosphorylation
(24). When phosphorylated by
recombinant GSK3β in vitro, the P301L and V337M mutations do not
have any effect, and the R406W mutation inhibits phosphorylation
(25). However, when incubated
with rat brain extract, all of the G272V, P301L, V337M, and R406W mutations
stimulate tau phosphorylation
(26). The mechanism by which
FTDP-17 mutations promote tau phosphorylation leading to development of NFT
pathology has remained unclear.Cyclin-dependent protein kinase 5 (Cdk5) is one of the major kinases that
phosphorylates tau in the brain
(27,
28). In this study, to
determine how FTDP-17 missense mutations affect tau phosphorylation, we
phosphorylated four FTDP-17 tau mutants (G272V, P301L, V337M, and R406W) by
Cdk5. We have found that phosphorylation of tau by Cdk5 causes a tau mobility
shift to ∼64- and 68 kDa-bands. Although the mobility shift to a
∼64-kDa band is achieved by phosphorylation at Ser396/404 or
Ser202, the mobility shift to a 68-kDa band occurs only in response
to phosphorylation at Ser202. We show that in
vitro, FTDP-17 missense mutations, by promoting phosphorylation at
Ser202, enhance the mobility shift to ∼64- and 68-kDa bands and
inhibit the microtubule assembly-promoting activity of tau. Our data suggest
that Ser202 phosphorylation is the major event leading to NFT
pathology in AD and related tauopathies. 相似文献
7.
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
8.
James Sinnett-Smith Rodrigo Jacamo Robert Kui YunZu M. Wang Steven H. Young Osvaldo Rey Richard T. Waldron Enrique Rozengurt 《The Journal of biological chemistry》2009,284(20):13434-13445
Rapid protein kinase D (PKD) activation and phosphorylation via protein
kinase C (PKC) have been extensively documented in many cell types cells
stimulated by multiple stimuli. In contrast, little is known about the role
and mechanism(s) of a recently identified sustained phase of PKD activation in
response to G protein-coupled receptor agonists. To elucidate the role of
biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in
these cells potently enhanced duration of ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. Cell treatment with the
preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD
activation induced by bombesin stimulation for <15 min but did not prevent
PKD catalytic activation induced by bombesin stimulation for longer times
(>60 min). The existence of sequential PKC-dependent and PKC-independent
PKD activation was demonstrated in 3T3 cells stimulated with various
concentrations of bombesin (0.3–10 nm) or with vasopressin, a
different Gq-coupled receptor agonist. To gain insight into the
mechanisms involved, we determined the phosphorylation state of the activation
loop residues Ser744 and Ser748. Transphosphorylation
targeted Ser744, whereas autophosphorylation was the predominant
mechanism for Ser748 in cells stimulated with Gq-coupled
receptor agonists. We next determined which phase of PKD activation is
responsible for promoting enhanced ERK activation and DNA synthesis in
response to Gq-coupled receptor agonists. We show, for the first
time, that the PKC-independent phase of PKD activation mediates prolonged ERK
signaling and progression to DNA synthesis in response to bombesin or
vasopressin through a pathway that requires epidermal growth factor
receptor-tyrosine kinase activity. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation
requires the identification of the molecular pathways that govern the
transition of quiescent cells into the S phase of the cell cycle. In this
context the activation and phosphorylation of protein kinase D
(PKD),4 the founding
member of a new protein kinase family within the
Ca2+/calmodulin-dependent protein kinase (CAMK) group and separate
from the previously identified PKCs (for review, see Ref.
1), are attracting intense
attention. In unstimulated cells, PKD is in a state of low catalytic (kinase)
activity maintained by autoinhibition mediated by the N-terminal domain, a
region containing a repeat of cysteinerich zinc finger-like motifs and a
pleckstrin homology (PH) domain
(1–4).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(5–7).
In response to cellular stimuli
(1), including phorbol esters,
growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR)
agonists (6,
8–16)
that signal through Gq, G12, Gi, and Rho
(11,
15–19),
PKD is converted into a form with high catalytic activity, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(5,
20).During these studies multiple lines of evidence indicated that PKC activity
is necessary for rapid PKD activation within intact cells. For example, rapid
PKD activation was selectively and potently blocked by cell treatment with
preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do
not directly inhibit PKD catalytic activity
(5,
20), implying that PKD
activation in intact cells is mediated directly or indirectly through PKCs.
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
induced by multiple GPCR agonists and other receptor ligands in a range of
cell types (for review, see Ref.
1). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation
(1,
4,
7,
17,
21). Collectively, these
findings demonstrated the existence of a rapidly activated PKC-PKD protein
kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD
activation was followed by a late, PKC-independent phase of catalytic
activation and phosphorylation induced by stimulation of the bombesin
Gq-coupled receptor ectopically expressed in COS-7 cells
(22). This study raised the
possibility that PKD mediates rapid biological responses downstream of PKCs,
whereas, in striking contrast, PKD could mediate long term responses through
PKC-independent pathways. Despite its potential importance for defining the
role of PKC and PKD in signal transduction, this hypothesis has not been
tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in
several cellular processes and activities, including signal transduction
(14,
23–25),
chromatin organization (26),
Golgi function (27,
28), gene expression
(29–31),
immune regulation (26), and
cell survival, adhesion, motility, differentiation, DNA synthesis, and
proliferation (for review, see Ref.
1). In Swiss 3T3 fibroblasts, a
cell line used extensively as a model system to elucidate mechanisms of
mitogenic signaling
(32–34),
PKD expression potently enhances ERK activation, DNA synthesis, and cell
proliferation induced by Gq-coupled receptor agonists
(8,
14). Here, we used this model
system to elucidate the role and mechanism(s) of biphasic PKD activation.
First, we show that the Gq-coupled receptor agonists bombesin and
vasopressin, in contrast to phorbol esters, specifically induce PKD activation
through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3
cells. Subsequently, we demonstrate for the first time that the
PKC-independent phase of PKD activation is responsible for promoting ERK
signaling and progression to DNA synthesis through an epidermal growth factor
receptor (EGFR)-dependent pathway. Thus, our results identify a novel
mechanism of Gq-coupled receptor-induced mitogenesis mediated by
sustained PKD activation through a PKC-independent pathway. 相似文献
9.
10.
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
11.
L. Andy Chen Jing Li Scott R. Silva Lindsey N. Jackson Yuning Zhou Hiroaki Watanabe Kirk L. Ives Mark R. Hellmich B. Mark Evers 《The Journal of biological chemistry》2009,284(4):2459-2471
The protein kinase D (PKD) family of serine/threonine kinases, which can be
activated by gastrointestinal hormones, consists of three distinct isoforms
that modulate a variety of cellular processes including intracellular protein
transport as well as constitutive and regulated secretion. Although
isoform-specific functions have been identified in a variety of cell lines,
the expression and function of PKD isoforms in normal, differentiated
secretory tissues is unknown. Here, we demonstrate that PKD isoforms are
differentially expressed in the exocrine and endocrine cells of the pancreas.
Specifically, PKD3 is the predominant isoform expressed in exocrine cells of
the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly
expressed in the pancreatic islets. Within isolated mouse pancreatic acinar
cells, PKD3 undergoes rapid membrane translocation, trans-activating
phosphorylation, and kinase activation after gastrointestinal hormone or
cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs
viaaCa2+-independent, diacylglycerol- and protein kinase
C-dependent mechanism. PKD phosphorylation can also be induced by physiologic
concentrations of secretagogues and by in vivo stimulation of the
pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK,
ribosomal S6 kinase) signaling and significantly enhances
cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a
novel distinction between the exocrine and endocrine cells of the pancreas and
further identify PKD3 as a signaling molecule that promotes hormone-stimulated
amylase secretion.Protein kinase D
(PKD),2 a
serine/threonine kinase family with a catalytic domain homologous to the
Ca2+/calmodulin-dependent kinase domain and two cysteine-rich
phorbol ester binding domains similar to those of protein kinase C (PKC), is a
physiologically important downstream mediator of diacylglycerol (DAG) signal
transduction (1,
2). The mammalian PKDs include
three members, PKD1, PKD2, and PKD3, which demonstrate different expression
patterns and functions depending on the cell type and external signal stimuli.
PKDs are ubiquitously expressed, but levels of individual isoforms vary with
developmental stage and cell type
(3). PKD proteins are reported
to localize in the cytosol, Golgi, nucleus, and vesicle structures
(4-9).
Activation of PKDs results in a dynamic translocation among subcellular
compartments (10,
11). Expression of multiple
isoforms in different cell types and in different subcellular localizations
suggests that individual PKD isoforms may serve specific functions. The
majority of findings demonstrating the diverse expression patterns and
functions of PKD have been described using established cell lines
(4-9,
12). However, little is known
about PKD isoform expression and function in normal differentiated cells and
tissues.Recent functional studies have shown that PKD isoforms differentially
regulate exocytic protein trafficking and cargo specificity
(9,
12-14).
Furthermore, PKD isoforms are differentially activated by oxidative stress
signaling via PKCδ-mediated tyrosine phosphorylation
(15). In each of these
studies, PKD3 was found to have a regulatory mechanism or cellular function
distinct from that of PKD1 and PKD2. Unlike the other two isoforms, PKD3 lacks
the N terminus hydrophobic domain or the C terminus PDZ binding motif and
contains divergent PH (pleckstrin homology) and C1 domains, which are
important for regulating its catalytic activity
(12,
16,
17). Current knowledge of the
physiologic function of PKD3 is limited. It has been demonstrated using
kinase-inactive mutants that PKD3 activity is required for basolateral
exocytosis in Madin-Darby canine kidney cells
(13). PKD3 has also been
implicated in the epigenetic control of chromatin by regulating class II
histone deacetylases in B lymphocytes
(18). Furthermore, PKD3 was
found to be a specific regulator of glucose transport in skeletal muscle cells
(19).The exocrine pancreas is highly specialized for the synthesis, storage, and
exocrine secretion of digestive enzymes and bicarbonate-rich fluid
(20). More than 90% of the
newly synthesized proteins in the pancreas is targeted to the secretory
pathway (21). In addition, the
pancreas contains a variety of endocrine cells localized to the islets which
secrete peptide hormones. Numerous steps in the secretory pathway are
modulated by DAG signaling, which promotes secretion by maintaining Golgi
function and/or activating DAG receptor kinases such as PKCs, which are
regulators of exocytic proteins
(1,
22-25).
PKD is also critical for DAG-mediated secretion, as it is recruited by DAG to
the trans-Golgi network, where it phosphorylates the lipid kinase
phosphatidylinositol 4-kinase to initiate the process of vesicle fission
(9,
26). Gastrointestinal (GI)
hormones such as cholecystokinin (CCK), gastrin, neurotensin (NT), and
bombesin (BBS)/gastrin-releasing peptide are potent regulatory peptides that
modulate pancreatic function
(27,
28). They are known to
activate PKDs to promote cell proliferation and survival in gut epithelial
cells
(29-32);
however, the role of PKDs in modulating the secretory actions of GI hormones
is unknown.Although the PKD isoforms have been reported to be expressed in secretory
tissues such as salivary glands, adrenal glands, intestinal mucosa, and the
pituitary (3,
5,
33), the role of PKD in the
process of regulated secretion remains poorly understood. Previously, we
demonstrated that PKD1 mediates NT peptide secretion from a pancreas-derived
neuroendocrine cell line, BON, and that PKD1 activation is regulated by PKC
and Rho/Rho kinase pathways
(4); PKD1 and PKD2 isoforms are
highly expressed in this endocrine cell line with little to no PKD3
expression, thus suggesting that PKD1/2 may be the predominant isoforms for
endocrine secretion. The distribution and role of PKD isoforms in the
pancreas, an organ with both exocrine and endocrine functions, is not known.
Interestingly, we demonstrate that in both human and mouse pancreas, PKD3 is
the predominant PKD isoform expressed in the exocrine acini, whereas PKD1 and
PKD2 are more highly expressed in endocrine islets. PKD3 is catalytically
activated by GI hormone stimulation of the pancreas, and its activation is
dependent on CCK1/2 receptor binding and on DAG/PKC activity. PKD3
overexpression in mouse pancreatic acinar cells significantly increased
CCK-mediated pancreatic amylase secretion, suggesting that PKD3, in concert
with other signaling molecules, contributes to stimulated amylase secretion.
Our findings reveal a distinct expression pattern in the exocrine and
endocrine cells of the mouse and human pancreas and identify PKD3 as a novel
DAG-activated mediator of the exocrine secretory process in response to GI
hormone signaling. 相似文献
12.
13.
Sophie Pattingre Chantal Bauvy St��phane Carpentier Thierry Levade Beth Levine Patrice Codogno 《The Journal of biological chemistry》2009,284(5):2719-2728
Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated
during periods of nutrient starvation to preserve cell integrity. Ceramide is
a bioactive sphingolipid associated with a large range of cell processes. Here
we show that short-chain ceramides (C2-ceramide and
C6-ceramide) and stimulation of the de novo ceramide
synthesis by tamoxifen induce the dissociation of the complex formed between
the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This
dissociation is required for macroautophagy to be induced either in response
to ceramide or to starvation. Three potential phosphorylation sites,
Thr69, Ser70, and Ser87, located in the
non-structural N-terminal loop of Bcl-2, play major roles in the dissociation
of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal
protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to
stimulate macroautophagy. These findings reveal a new aspect of sphingolipid
signaling in up-regulating a major cell process involved in cell adaptation to
stress.Macroautophagy (referred to below as “autophagy”) is a
vacuolar, lysosomal degradation pathway for cytoplasmic constituents that is
conserved in eukaryotic cells
(1–3).
Autophagy is initiated by the formation of a multimembrane-bound autophagosome
that engulfs cytoplasmic proteins and organelles. The last stage in the
process results in fusion with the lysosomal compartments, where the
autophagic cargo undergoes degradation. Basal autophagy is important in
controlling the quality of the cytoplasm by removing damaged organelles and
protein aggregates. Inhibition of basal autophagy in the brain is deleterious,
and leads to neurodegeneration in mouse models
(4,
5). Stimulation of autophagy
during periods of nutrient starvation is a physiological response present at
birth and has been shown to provide energy in various tissues of newborn pups
(6). In cultured cells,
starvation-induced autophagy is an autonomous cell survival mechanism, which
provides nutrients to maintain a metabolic rate and level of ATP compatible
with cell survival (7). In
addition, starvation-induced autophagy blocks the induction of apoptosis
(8). In other contexts, such as
drug treatment and a hypoxic environment, autophagy has also been shown to be
cytoprotective in cancer cells
(9,
10). However, autophagy is
also part of cell death pathways in certain situations
(11). Autophagy can be a
player in apoptosis-independent type-2 cell death (type-1 cell death is
apoptosis), also known as autophagic cell death. This situation has been shown
to occur when the apoptotic machinery is crippled in mammalian cells
(12,
13). Autophagy can also be
part of the apoptotic program, for instance in tumor necrosis
factor-α-induced cell death when NF-κB is inhibited
(14), or in human
immunodeficiency virus envelope-mediated cell death in bystander naive CD4 T
cells (15). Moreover autophagy
has recently been shown to be required for the externalization of
phosphatidylserine, the eat-me signal for phagocytic cells, at the surface of
apoptotic cells (16).The complex relationship between autophagy and apoptosis reflects the
intertwined regulation of these processes
(17,
18). Many signaling pathways
involved in the regulation of autophagy also regulate apoptosis. This
intertwining has recently been shown to occur at the level of the molecular
machinery of autophagy. In fact the anti-apoptotic protein Bcl-2 has been
shown to inhibit starvation-induced autophagy by interacting with the
autophagy protein Beclin 1
(19). Beclin 1 is one of the
Atg proteins conserved from yeast to humans (it is the mammalian orthologue of
yeast Atg6) and is involved in autophagosome formation
(20). Beclin 1 is a platform
protein that interacts with several different partners, including hVps34
(class III phosphatidylinositol 3-kinase), which is responsible for the
synthesis of phosphatidylinositol 3-phosphate. The production of this lipid is
important for events associated with the nucleation of the isolation membrane
before it elongates and closes to form autophagosomes in response to other Atg
proteins, including the Atg12 and
LC32
(microtubule-associated protein light chain 3 is the mammalian orthologue of
the yeast Atg8) ubiquitin-like conjugation systems
(3,
21). Various partners
associated with the Beclin 1 complex modulate the activity of hVps34. For
instance, Bcl-2 inhibits the activity of this enzyme, whereas UVRAG, Ambra-1,
and Bif-1 all up-regulate it
(22,
23).In view of the intertwining between autophagy and apoptosis, it is
noteworthy that Beclin 1 belongs to the BH3-only family of proteins
(24–26).
However, and unlike most of the proteins in this family, Beclin 1 is not able
to trigger apoptosis when its expression is forced in cells
(27). A BH3-mimetic drug,
ABT-737, is able to dissociate the Beclin 1-Bcl-2 complex, and to trigger
autophagy by mirroring the effect of starvation
(25).The sphingolipids constitute a family of bioactive lipids
(28–32)
of which several members, such as ceramide and sphingosine 1-phosphate, are
signaling molecules. These molecules constitute a “sphingolipid
rheostat” that determines the fate of the cell, because in many settings
ceramide is pro-apoptotic and sphingosine 1-phosphate mitigates this apoptotic
effect (31,
32). However, ceramide is also
engaged in a wide variety of other cell processes, such as the formation of
exosomes (33),
differentiation, cell proliferation, and senescence
(34). Recently we showed that
both ceramide and sphingosine 1-phosphate are able to stimulate autophagy
(35,
36). It has also been shown
that ceramide triggers autophagy in a large panel of mammalian cells
(37–39).
However, elucidation of the mechanism by which ceramide stimulates autophagy
is still in its infancy. We have previously demonstrated that ceramide induces
autophagy in breast and colon cancer cells by inhibiting the Class I
phosphatidylinositol 3-phosphate/mTOR signaling pathway, which plays a central
role in inhibiting autophagy
(36). Inhibition of mTOR is
another hallmark of starvation-induced autophagy
(17). This finding led us to
investigate the effect of ceramide on the Beclin 1-Bcl-2 complex. The results
presented here show that ceramide is more potent than starvation in
dissociating the Beclin 1-Bcl-2 complex (see Ref.
40). This dissociation is
dependent on three phosphorylation sites (Thr69, Ser70,
and Ser87) located in a non-structural loop of Bcl-2. Ceramide
induces the c-Jun N-terminal kinase 1-dependent phosphorylation of Bcl-2.
Expression of a dominant negative form of JNK1 blocks Bcl-2 phosphorylation,
and thus the induction of autophagy by ceramide. These findings help to
explain how autophagy is regulated by a major lipid second messenger. 相似文献
14.
15.
Christopher P. Gayer Lakshmi S. Chaturvedi Shouye Wang David H. Craig Thomas Flanigan Marc D. Basson 《The Journal of biological chemistry》2009,284(4):2001-2011
The intestinal epithelium is repetitively deformed by shear, peristalsis,
and villous motility. Such repetitive deformation stimulates the proliferation
of intestinal epithelial cells on collagen or laminin substrates via ERK, but
the upstream mediators of this effect are poorly understood. We hypothesized
that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this
mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β
(GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10%
deformation, and pharmacologic blockade of these molecules or reduction by
small interfering RNA (siRNA) prevented the mitogenic effect of strain in
Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required
PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that
AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain
mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera
including the PH domain and hinge region of AKT2 and the catalytic domain and
C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression
of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the
catalytic domain and C-tail of AKT2 did not. These data delineate a role for
PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is
required for both ERK and AKT2 activation, whereas AKT2 is sequentially
required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic
domain and tail region. Manipulating this pathway may prevent mucosal atrophy
and maintain the mucosal barrier in conditions such as ileus, sepsis, and
prolonged fasting when peristalsis and villous motility are decreased and the
mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment.
Numerous different forces deform these cells including shear stress from
endoluminal chyme, bowel peristalsis, and villous motility
(1,
2). During normal bowel
function the mucosa is subjected to injury that must be repaired to maintain
the mucosal barrier (3,
4). Deformation patterns of the
bowel are altered in conditions such as prolonged fasting, post-surgical
ileus, and sepsis states, resulting in profoundly reduced mucosal deformation.
When such states are prolonged, proliferation slows, the mucosa becomes
atrophic, and bacterial translocation may ensue as the mucosal barrier of the
gut breaks down
(5–7).In vitro, repetitive deformation is trophic for intestinal
epithelial cells (8) cultured
on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial
cells (9), non-transformed rat
IEC-6 intestinal epithelial cells
(10), and primary human
intestinal epithelial cells isolated from surgical specimens
(11) proliferate more rapidly
in response to cyclic strain
(12) unless substantial
quantities of fibronectin are added to the media or matrix
(11) to mimic the acute phase
reaction of acute or chronic inflammation and injury. Cyclic strain also
stimulates proliferation in HCT 116 colon cancer cells
(13) and differentiation of
Caco-2 cells cultured on a collagen substrate
(9). This phenomenon has also
been observed in vivo
(14). Thus, repetitive
deformation may help to maintain the normal homeostasis of the gut mucosa
under non-inflammatory conditions. Previous work in our laboratory has
implicated Src, focal adhesion kinase, and the mitogen-activated protein
kinase (MAPK)2
extracellular signal-related kinase (ERK) in the mitogenic effect of strain
(10). Although p38 is also
activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its
activity is not required for the mitogenic effect of strain
(12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to
the MAPK, this is not always the case. Protein kinase C isoenzymes
differentially modulate thrombin effect on MAPK-dependent retinal pigment
epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT
inhibition prevented thrombin-induced ERK activation and RPE proliferation
(15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT
(16), have been implemented in
intestinal epithelial cell proliferation in numerous cell systems not
involving strain
(17–19)
including uncontrolled proliferation in gastrointestinal cancers
(20–22).
Mechanical forces activate this pathway as well. PI3K and AKT are required for
increased extracellular pressure to stimulate colon cancer cell adhesion
(23), although the pathway by
which pressure stimulates colon cancer cells in suspension differs from the
response of adherent intestinal epithelial cells to repetitive deformation
(24), and GSK is not involved
in this effect.3
Repetitive strain also stimulates vascular endothelial cell proliferation via
PI3K and AKT (25,
26), whereas respiratory
strain stimulates angiogenic responses via PI3K
(27). We, therefore,
hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic
effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically
deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm
key results. A frequency of 10 cycles per min was used, which is similar in
order of magnitude to the frequency that the intestinal mucosa might be
deformed by peristalsis or villous motility in vivo
(28,
29). Mechanical forces such as
repetitive deformation are likely cell-type and frequency-specific, as
different cell types respond to different frequencies. Vascular endothelial
cells respond to frequencies of 60–80 cycles/min
(25), whereas intestinal
epithelial cells may actually decrease proliferation in response to
frequencies of 5 cycles/min
(30). We characterized PI3K,
AKT, and GSK phosphorylation with strain, blocked these molecules
pharmacologically or by siRNA, and delineated the specificity of the AKT
effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We
also characterized the interaction of this pathway with the activation of ERK
by strain, which has previously been implicated in the mitogenic response
(12). 相似文献
16.
17.
18.
19.
Bertha C. Elias Takuya Suzuki Ankur Seth Francesco Giorgianni Gautam Kale Le Shen Jerrold R. Turner Anjaparavanda Naren Dominic M. Desiderio Radhakrishna Rao 《The Journal of biological chemistry》2009,284(3):1559-1569
Occludin is phosphorylated on tyrosine residues during the oxidative
stress-induced disruption of tight junction, and in vitro
phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the
present study mass spectrometric analyses of C-terminal domain of occludin
identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation
sites, which are located in a highly conserved sequence of occludin, YETDYTT;
Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion
of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and
the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin
also abolished the c-Src-mediated phosphorylation and regulation of ZO-1
binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding
even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A
mutant was localized at the plasma membrane and cell-cell contact sites in
Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at
the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant
occludin in Madin-Darby canine kidney cells was significantly delayed compared
with that of wild type occludin or its T398A/T402A mutant. Furthermore,
expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for
hydrogen peroxide-induced barrier disruption. This study reveals a unique
motif in the occludin sequence that is involved in the regulation of ZO-1
binding by reversible phosphorylation of specific Tyr residues.Epithelial tight junctions
(TJs)2 form a
selective barrier to the diffusion of toxins, allergens, and pathogens from
the external environment into the tissues in the gastrointestinal tract, lung,
liver, and kidney (1).
Disruption of TJs is associated with the gastrointestinal diseases such as
inflammatory bowel disease, celiac disease, infectious enterocolitis, and
colon cancer
(2–4)
as well as in diseases of lung and kidney
(5,
6). Numerous inflammatory
mediators such as tumor necrosis factor α, interferon γ, and
oxidative stress
(7–12)
are known to disrupt the epithelial TJs and the barrier function. Several
studies have indicated that hydrogen peroxide disrupts the TJs in intestinal
epithelium by a tyrosine kinase-dependent mechanism
(11,
12).Four types of integral proteins, occludin, claudins, junctional adhesion
molecules, and tricellulin are associated with TJs. Occludin, claudins, and
tricellulin are tetraspan proteins, and their extracellular domains interact
with homotypic domains of the adjacent cells
(1,
2,
13). The intracellular domains
of these proteins interact with a variety of soluble proteins such as ZO-1,
ZO-2, ZO-3, 7H6, cingulin, and symplekin
(14–23);
this protein complex interacts with the perijunctional actomyosin ring. The
interactions among TJ proteins are essential for the assembly and the
maintenance of TJs. Therefore, regulation of the interactions among TJ
proteins may regulate the TJ integrity. A significant body of evidence
indicates that numerous signaling molecules are associated with the TJs.
Protein kinases and protein phosphatases such as protein kinase Cζ
(PKCζ), PKCι/λ
(24), c-Src
(25), c-Yes
(26,
27), mitogen-activated protein
kinase (28), PP2A, and PP1
(29) interact with TJs,
indicating that TJs are dynamically regulated by intracellular signal
transduction involving protein phosphorylation. Additionally, other signaling
molecules such as calcium
(30), phosphatidylinositol
3-kinase (31), Rho
(32), and Rac
(33) are involved in the
regulation of TJs.Occludin, a ∼65-kDa protein, has been well characterized to be
assembled into the TJs. Although occludin knock-out mice showed the formation
of intact TJs in different epithelia
(34), numerous studies have
emphasized that it plays an important role in the regulation of TJ integrity.
Occludin spans the membrane four times to form two extracellular loops and one
intracellular loop, and the N-terminal and C-terminal domains hang into the
intracellular compartment
(35–37).
In epithelium with intact TJs, occludin is highly phosphorylated on Ser and
Thr residues (38), whereas Tyr
phosphorylation is undetectable. However, the disruption of TJs in Caco-2 cell
monolayers by oxidative stress and acetaldehyde leads to Tyr phosphorylation
of occludin; the tyrosine kinase inhibitors attenuate the disruption of TJs
(39,
40). Furthermore, a previous
in vitro study demonstrated that Tyr phosphorylation of the
C-terminal domain of occludin leads to the loss of its interaction with ZO-1
and ZO-3 (25).In the present study we identified the Tyr residues in occludin that are
phosphorylated by c-Src and determined their role in regulated interaction
between occludin and ZO-1 and its assembly into the TJs. Results show that 1)
Tyr-379 and Tyr-383 in chicken occludin and Tyr-398 and Tyr-402 in human
occludin are the exclusive sites of phosphorylation by c-Src, and these Tyr
residues are located in a highly conserved sequence of occludin, YET-DYTT, 2)
deletion of YEDTYTT or point mutation of Tyr-398 and Tyr-402 in human occludin
attenuates the phosphorylation-dependent regulation of ZO-1 binding, 3)
Y398D/Y402D mutation of human occludin leads to loss of ZO-1 binding and
prevents its translocation to the plasma membrane and cell-cell contact sites
in Rat-1 cells, 4) Y398D/Y402D mutation of occludin delays its assembly into
the intercellular junctions during the calcium-induced assembly of TJs, and 5)
expression of Y398D/Y402D mutant occludin sensitizes cell monolayers for
hydrogen peroxide-induced disruption of barrier function. 相似文献
20.
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