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1.
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
2.
3.
Jaemin Lee Xiaofan Wang Bruno Di Jeso Peter Arvan 《The Journal of biological chemistry》2009,284(19):12752-12761
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin
(Tg) has been identified as critically important in Tg export from the
endoplasmic reticulum. In a number of human kindreds suffering from congenital
hypothyroidism, and in the cog congenital goiter mouse and
rdw rat dwarf models, thyroid hormone synthesis is inhibited because
of mutations in the ChEL domain that block protein export from the endoplasmic
reticulum. We hypothesize that Tg forms homodimers through noncovalent
interactions involving two predicted α-helices in each ChEL domain that
are homologous to the dimerization helices of acetylcholinesterase. This has
been explored through selective epitope tagging of dimerization partners and
by inserting an extra, unpaired Cys residue to create an opportunity for
intermolecular disulfide pairing. We show that the ChEL domain is necessary
and sufficient for Tg dimerization; specifically, the isolated ChEL domain can
dimerize with full-length Tg or with itself. Insertion of an N-linked
glycan into the putative upstream dimerization helix inhibits homodimerization
of the isolated ChEL domain. However, interestingly, co-expression of upstream
Tg domains, either in cis or in trans, overrides the
dimerization defect of such a mutant. Thus, although the ChEL domain provides
a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL
domain actively stabilizes the Tg dimer complex for intracellular
transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of
thyroglobulin (Tg)2 to
the apical luminal cavity of thyroid follicles
(1). Once secreted, Tg is
iodinated via the activity of thyroid peroxidase
(2). A coupling reaction
involving a quinol-ether linkage especially engages di-iodinated tyrosyl
residues 5 and 130 to form thyroxine within the amino-terminal portion of the
Tg polypeptide (3,
4). Preferential iodination of
Tg hormonogenic sites is dependent not on the specificity of the peroxidase
(5) but upon the native
structure of Tg (6,
7). To date, no other thyroidal
proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746
amino acids) involves three regions called I-II-III comprised of
disulfide-rich repeat domains held together by intradomain disulfide bonds
(8,
9). The final 581 amino acids
of Tg are strongly homologous to acetylcholinesterase
(10–12).
Rate-limiting steps in the overall process of Tg secretion involve its
structural maturation within the endoplasmic reticulum (ER)
(13). Interactions between
regions I-II-III and the cholinesterase-like (ChEL) domain have recently been
suggested to be important in this process, with ChEL functioning as an
intramolecular chaperone and escort for I-II-III
(14). In addition, Tg
conformational maturation culminates in Tg homodimerization
(15,
16) with progression to a
cylindrical, and ultimately, a compact ovoid structure
(17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a
commonly affected site of mutation, including the recently described A2215D
(20,
21), R2223H
(22), G2300D, R2317Q
(23), G2355V, G2356R, and the
skipping of exon 45 (which normally encodes 36 amino acids), as well as the
Q2638stop mutant (24) (in
addition to polymorphisms including P2213L, W2482R, and R2511Q that may be
associated with thyroid overgrowth
(25)). As best as is currently
known, all of the congenital hypothyroidism-inducing Tg mutants are defective
for intracellular transport
(26). A homozygous G2300R
mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is
responsible for congenital hypothyroidism in rdw rats
(27,
28), whereas we identified the
Tg-L2263P point mutation as the cause of hypothyroidism in the cog
mouse (29). Such mutations
perturb intradomain structure
(30), and interestingly, block
homodimerization (31).
Acquisition of quaternary structure has long been thought to be required for
efficient export from the ER
(32) as exemplified by
authentic acetylcholinesterase
(33,
34) in which dimerization
enhances protein stability and export
(35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain)
is blocked within the ER (30),
whereas a secretory version of the isolated ChEL domain of Tg devoid of
I-II-III undergoes rapid and efficient intracellular transport and secretion
(14). A striking homology
positions two predicted α-helices of the ChEL domain to the identical
relative positions of the dimerization helices in acetylcholinesterase. This
raises the possibility that ChEL may serve as a homodimerization domain for
Tg, providing a critical function in maturation for Tg transport to the site
of thyroid hormone synthesis
(1).In this study, we provide unequivocal evidence for homodimerization of the
ChEL domain and “hetero”-dimerization of that domain with
full-length Tg, and we provide significant evidence that the predicted ChEL
dimerization helices provide a nidus for Tg assembly. On the other hand, our
data also suggest that upstream Tg regions known to interact with ChEL
(14) actively stabilize the Tg
dimer complex. Together, I-II-III and ChEL provide unique contributions to the
process of intracellular transport of Tg through the secretory pathway. 相似文献
4.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
5.
S��bastien Thomas Brigitte Ritter David Verbich Claire Sanson Lyne Bourbonni��re R. Anne McKinney Peter S. McPherson 《The Journal of biological chemistry》2009,284(18):12410-12419
Intersectin-short (intersectin-s) is a multimodule scaffolding protein
functioning in constitutive and regulated forms of endocytosis in non-neuronal
cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of
Drosophila and Caenorhabditis elegans. In vertebrates,
alternative splicing generates a second isoform, intersectin-long
(intersectin-l), that contains additional modular domains providing a guanine
nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is
expressed in multiple tissues and cells, including glia, but excluded from
neurons, whereas intersectin-l is a neuron-specific isoform. Thus,
intersectin-I may regulate multiple forms of endocytosis in mammalian neurons,
including SV endocytosis. We now report, however, that intersectin-l is
localized to somatodendritic regions of cultured hippocampal neurons, with
some juxtanuclear accumulation, but is excluded from synaptophysin-labeled
axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV
recycling. Instead intersectin-l co-localizes with clathrin heavy chain and
adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces
the rate of transferrin endocytosis. The protein also co-localizes with
F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation
during development. Our data indicate that intersectin-l is indeed an
important regulator of constitutive endocytosis and neuronal development but
that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis
(CME)4 is a
major mechanism by which cells take up nutrients, control the surface levels
of multiple proteins, including ion channels and transporters, and regulate
the coupling of signaling receptors to downstream signaling cascades
(1-5).
In neurons, CME takes on additional specialized roles; it is an important
process regulating synaptic vesicle (SV) availability through endocytosis and
recycling of SV membranes (6,
7), it shapes synaptic
plasticity
(8-10),
and it is crucial in maintaining synaptic membranes and membrane structure
(11).Numerous endocytic accessory proteins participate in CME, interacting with
each other and with core components of the endocytic machinery such as
clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific
modules and peptide motifs
(12). One such module is the
Eps15 homology domain that binds to proteins bearing NPF motifs
(13,
14). Another is the Src
homology 3 (SH3) domain, which binds to proline-rich domains in protein
partners (15). Intersectin is
a multimodule scaffolding protein that interacts with a wide range of
proteins, including several involved in CME
(16). Intersectin has two
N-terminal Eps15 homology domains that are responsible for binding to epsin,
SCAMP1, and numb
(17-19),
a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25
(17,
20,
21), and five SH3 domains in
its C-terminal region that interact with multiple proline-rich domain
proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS
(16,
22-25).
The rich binding capability of intersectin has linked it to various functions
from CME (17,
26,
27) and signaling
(22,
28,
29) to mitogenesis
(30,
31) and regulation of the
actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of
Drosophila and C. elegans where it acts as a scaffold,
regulating the synaptic levels of endocytic accessory proteins
(21,
32-34).
In vertebrates, the intersectin gene is subject to alternative splicing, and a
longer isoform (intersectin-l) is generated that is expressed exclusively in
neurons (26,
28,
35,
36). This isoform has all the
binding modules of its short (intersectin-s) counterpart but also has
additional domains: a DH and a PH domain that provide guanine nucleotide
exchange factor (GEF) activity specific for Cdc42
(23,
37) and a C2 domain at the C
terminus. Through its GEF activity and binding to actin regulatory proteins,
including N-WASP, intersectin-l has been implicated in actin regulation and
the development of dendritic spines
(19,
23,
24). In addition, because the
rest of the binding modules are shared between intersectin-s and -l, it is
generally thought that the two intersectin isoforms have the same endocytic
functions. In particular, given the well defined role for the invertebrate
orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l
performs this role in mammalian neurons, which lack intersectin-s. Defining
the complement of intersectin functional activities in mammalian neurons is
particularly relevant given that the protein is involved in the
pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is
localized on chromosome 21q22.2 and is overexpressed in DS brains
(38). Interestingly,
alterations in endosomal pathways are a hallmark of DS neurons and neurons
from the partial trisomy 16 mouse, Ts65Dn, a model for DS
(39,
40). Thus, an endocytic
trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured
hippocampal neurons. We find that intersectin-l is localized to the
somatodendritic regions of neurons, where it co-localizes with CHC and AP-2
and regulates the uptake of transferrin. Intersectin-l also co-localizes with
actin at dendritic spines and disrupting intersectin-l function alters
dendritic spine development. In contrast, intersectin-l is absent from
presynaptic terminals and has little or no role in SV recycling. 相似文献
6.
Yong Ren Houbo Jiang Fang Yang Kazuhiro Nakaso Jian Feng 《The Journal of biological chemistry》2009,284(6):4009-4017
Mitogen-activated protein kinases, originally known as
microtubule-associated protein (MAP) kinases, are activated in response to a
variety of stimuli. Here we report that microtubule-depolymerizing agents such
as colchicine or nocodazole induced strong activation of MAP kinases including
JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose
mutations are linked to Parkinson disease (PD). Our previous study has shown
that parkin stabilizes microtubules through strong interactions mediated by
three independent domains. We found that each of the three microtubule-binding
domains of parkin was sufficient to reduce MAP kinase activation induced by
microtubule depolymerization. The ability to attenuate microtubule
depolymerization and the ensuing MAP kinase activation was abrogated in
B-lymphocytes and fibroblasts derived from PD patients with parkin mutations
such as exon 4 deletion. Such mutations produced truncated parkin proteins
lacking any microtubule binding domain and prevented parkin from protecting
midbrain dopaminergic neurons against microtubule-depolymerizing toxins such
as rotenone or colchicine. Consistent with these, blocking MAP kinase
activation in midbrain dopaminergic neurons by knocking down MAP kinase
kinases (MKK) significantly reduced the selective toxicity of rotenone or
colchicine. Conversely, overexpression of MAP kinases caused marked toxicities
that were significantly attenuated by parkin. Thus, the results suggest that
parkin protects midbrain dopaminergic neurons against
microtubule-depolymerizing PD toxins such as rotenone by stabilizing
microtubules to attenuate MAP kinase activation.Mitogen-activated protein kinases are a superfamily of kinases that include
the extracellular signal-related kinases
(ERK1/2),2 Jun
N-terminal kinases (JNK1/2/3), and p38 proteins
(p38α/β/γ/δ) in mammalian species
(1). Initially, MAP kinase
stood for microtubule-associated protein kinase because microtubule-associated
proteins such as MAP2 are excellent substrates of MAP kinases
(2,
3). Previous studies have shown
that a significant portion of ERK is associated with microtubules
(4). It has also been shown
that JNK1 is required for the maintenance of microtubule integrity in neurons
through controlling the phosphorylation states of MAP2 and MAP1B
(5). Phosphorylation of tau, an
axon-enriched MAP, by p38δ promotes microtubule assembly
(6). All MAP kinases are
proline-directed kinases, preferring serine or threonine residues followed by
proline. The abundance of such sites on many microtubule-associated proteins
suggests that MAP kinases play critical roles in regulating the
phosphorylation states of MAPs; hence, the dynamic properties of
microtubules.The selective loss of dopaminergic (DA) neurons in substantia nigra is the
pathological hallmark of Parkinson disease and directly contributes to its
locomotor symptoms. Nigral DA neurons project to striatal target areas with
very long axons, which rely on microtubules to transport dopamine vesicles
over long distances. Our previous studies have shown that these dopaminergic
neurons are particularly vulnerable to microtubule-depolymerizing agents
including rotenone (7), an
environmental toxin that causes PD-like symptoms and pathologies in animal
models (8). Microtubule
depolymerization disrupts vesicular transport, which significantly elevates
oxidative stress due to increased oxidation of cytosolic dopamine leaked from
vesicles (7). On the other hand
our previous studies have shown that parkin, a protein-ubiquitin E3 ligase
linked to Parkinson disease, strongly binds to microtubules
(9) through redundant, high
affinity interactions mediated by three independent domains
(10). In addition, parkin
increases the ubiquitination and degradation of both α- and
β-tubulin (9), whose
complex folding reactions are prone to produce misfolded intermediates
(11). These results suggest
that parkin plays an important role in maintaining the stability and normal
functions of microtubules, which are critically involved in the survival of
nigral DA neurons (12).In the present study we examined the impact of parkin on MAP kinase
activation induced by microtubule depolymerization. Our results showed that
MAP kinases, including JNK, ERK, and p38, were activated by
microtubule-depolymerizing agents such as colchicine and nocodazole. This
effect was greatly attenuated by overexpression of wild-type parkin or any one
of its three microtubule binding domains. The ability of parkin to suppress
microtubule depolymerization and the ensuing MAP kinase activation was
abrogated in PD patients with parkin mutations such as exon 4 deletion, which
produced a truncated protein lacking any microtubule-binding domain. This
mutation also prevented parkin from protecting dopaminergic neurons against
the selective toxicity of microtubule-depolymerizing toxins such as rotenone
or colchicine. Blocking MAP kinase activation by small interfering RNA (siRNA)
of MAP kinase kinases significantly reduced the selective toxicity of rotenone
or colchicine, whereas overexpression of MAP kinases produced toxicities that
were significantly reduced by parkin. Together, the results suggest that
parkin protects midbrain dopaminergic neurons against
microtubule-depolymerizing PD toxins by stabilizing microtubules to rein in
MAP kinase activation. 相似文献
7.
Ivano Bertini Marco Fragai Claudio Luchinat Maxime Melikian Efstratios Mylonas Niko Sarti Dmitri I. Svergun 《The Journal of biological chemistry》2009,284(19):12821-12828
The presence of extensive reciprocal conformational freedom between the
catalytic and the hemopexin-like domains of full-length matrix
metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray
scattering experiments. This finding is discussed in relation to the
essentiality of the hemopexin-like domain for the collagenolytic activity of
MMP-1. The conformational freedom experienced by the present system, having
the shortest linker between the two domains, when compared with similar
findings on MMP-12 and MMP-9 having longer and the longest linker within the
family, respectively, suggests this type of conformational freedom to be a
general property of all MMPs.Matrix metalloproteinases
(MMP)2 are
extracellular hydrolytic enzymes involved in a variety of processes including
connective tissue cleavage and remodeling
(1–3).
All 23 members of the family are able to cleave simple peptides derived from
connective tissue components such as collagen, gelatin, elastin, etc. A subset
of MMPs is able to hydrolyze more resistant polymeric substrates, such as
cross-linked elastin, and partially degraded collagen forms, such as gelatin
and type IV collagens (4).
Intact triple helical type I–III collagen is only attacked by
collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14
(5–12).
Although the detailed mechanism of cleavage of single chain peptides by MMP
has been largely elucidated
(13–19),
little is known about the process of hydrolysis of triple helical collagen. In
fact, triple helical collagen cannot be accommodated in the substrate-binding
groove of the catalytic site of MMPs
(9).All MMPs (but MMP-7) in their active form are constituted by a catalytic
domain (CAT) and a hemopexin-like domain (HPX)
(20–22).
The CAT domain contains two zinc ions and one to three calcium ions. One zinc
ion is at the catalytic site and is responsible for the activity, whereas the
other metal ions have structural roles. The isolated CAT domains retain full
catalytic activity toward simple peptides and single chain polymeric
substrates such as elastin, whereas hydrolysis of triple helical collagen also
requires the presence of the HPX domain
(9,
23–25).
It has been shown that the isolated CAT domain regains a small fraction of the
activity of the full-length (FL) protein when high amounts of either
inactivated full-length proteins or isolated HPX domains are added to the
assay solution (9). Finally, it
has been shown that the presence of the HPX domain alone alters the CD
spectrum of triple helical collagen in a way that suggests its partial
unwinding (26,
27). It is tempting to
speculate that full-length collagenases attack collagen by first locally
unwinding the triple helical structure with the help of the HPX domain and
then cleaving the resulting, exposed, single filaments
(9,
28).Until 2007, three-dimensional structures of full-length MMPs had been
reported only for collagenase MMP-1
(29–31)
and gelatinase MMP-2 (32). The
structures of the two proteins are very similar and show a compact arrangement
of the two domains, which are connected by a short linker (14 and 20 amino
acids, respectively). It is difficult to envisage that rigid and compact
molecules of this type can interact with triple helical collagen in a way that
can lead to first unwinding and then cleavage of individual filaments. It has
been recently suggested that such concerted action could occur much more
easily if the two domains could enjoy at least a partial conformational
independence (9). Slight
differences in the reciprocal orientation of the CAT and HPX domains of MMP-1
in the presence (29) and
absence (30,
31) of the prodomain were
indeed taken as a hint that the two domains could experience relative mobility
(29).Two recent solution studies have shown that conformational independence is
indeed occurring in gelatinase MMP-9
(33) and elastase MMP-12
(34), whereas the x-ray
structure of the latter (34)
is only slightly less compact than those of MMP-1
(29–31)
and MMP-2 (32). Among MMPs,
MMP-9 features an exceptionally long linker (68 amino acid)
(33,
35), which in fact constitutes
a small domain by itself (the O-glycosylated domain)
(33), and therefore, this
inspiring observation can hardly be taken as evidence that conformational
freedom is a general characteristic of the two-domain MMPs. MMP-12 features a
much more normal 16-amino acid linker, thereby making more probable a general
functional role for this conformational freedom
(34). However, both MMP-9 and
MMP-12 retain their full catalytic activity against their substrates even when
deprived of the HPX domain (9).
Therefore, the question remains of whether conformational freedom is also a
required characteristic for those MMPs that are only active as full-length
proteins, i.e. collagenases. Interestingly, the three collagenases
(MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all
MMPs. Demonstrating or negating the presence of conformational freedom in one
of these collagenases would therefore constitute a significant step forward to
formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution
(34) have shown that a
combination of NMR relaxation studies and small angle x-ray scattering (SAXS)
is enough to show the presence and the extent of the relative conformational
freedom of the two domains of MMPs. Here we apply the same strategy to
full-length MMP-1 and show that sizable conformational freedom is indeed
experienced even by this prototypical collagenase, although somewhat less
pronounced than that observed for MMP-12. 相似文献
8.
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
9.
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
10.
11.
12.
John W. Hardin Francis E. Reyes Robert T. Batey 《The Journal of biological chemistry》2009,284(22):15317-15324
In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are
responsible for 2′-O-methylation of tRNAs and rRNAs. The
archaeal box C/D small RNP complex requires a small RNA component (sRNA)
possessing Watson-Crick complementarity to the target RNA along with three
proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from
S-adenosylmethionine to the target RNA is performed by fibrillarin,
which by itself has no affinity for the sRNA-target duplex. Instead, it is
targeted to the site of methylation through association with Nop5p, which in
turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a
bridge between the targeting and catalytic functions of the box C/D small RNP
complex, we have employed alanine scanning to evaluate the interaction between
the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA
complex. From these data, we were able to construct an isolated RNA-binding
domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography
and binds to the L7Ae box C/D RNA complex with near wild type affinity. These
data demonstrate that the Nop-RBD is an autonomously folding and functional
module important for protein assembly in a number of complexes centered on the
L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full
functionality in the cell (1).
Currently there are over 100 known RNA modification types ranging from small
functional group substitutions to the addition of large multi-cyclic ring
structures (2). Transfer RNA,
one of many functional RNAs targeted for modification
(3-6),
possesses the greatest modification type diversity, many of which are
important for proper biological function
(7). Ribosomal RNA, on the
other hand, contains predominantly two types of modified nucleotides:
pseudouridine and 2′-O-methylribose
(8). The crystal structures of
the ribosome suggest that these modifications are important for proper folding
(9,
10) and structural
stabilization (11) in
vivo as evidenced by their strong tendency to localize to regions
associated with function (8,
12,
13). These roles have been
verified biochemically in a number of cases
(14), whereas newly emerging
functional modifications are continually being investigated.Box C/D ribonucleoprotein
(RNP)3 complexes serve
as RNA-guided site-specific 2′-O-methyltransferases in both
archaea and eukaryotes (15,
16) where they are referred to
as small RNP complexes and small nucleolar RNPs, respectively. Target RNA
pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl
group of the nucleotide five bases upstream of either the D or D′ box
motif of the sRNA (Fig. 1,
star) (17,
18). In archaea, the internal
C′ and D′ motifs generally conform to a box C/D consensus sequence
(19), and each sRNA contains
two guide regions ∼12 nucleotides in length
(20). The bipartite
architecture of the RNP potentially enables the complex to methylate two
distinct RNA targets (21) and
has been shown to be essential for site-specific methylation
(22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components
of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D
sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D,
C′, and D′ boxes are labeled. The target RNA binds the sRNA
through Watson-Crick pairing and is methylated by fibrillarin at the fifth
nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three
proteins for activity (23):
the ribosomal protein L7Ae
(24,
25), fibrillarin, and the
Nop56/Nop58 homolog Nop5p (Fig.
1). L7Ae binds to both box C/D and the C′/D′ motifs
(26), which respectively
comprise kink-turn (27) or
k-loop structures (28), to
initiate the assembly of the RNP
(29,
30). Fibrillarin performs the
methyl group transfer from the cofactor S-adenosylmethionine to the
target RNA
(31-33).
For this to occur, the active site of fibrillarin must be positioned precisely
over the specific 2′-hydroxyl group to be methylated. Although
fibrillarin methylates this functional group in the context of a Watson-Crick
base-paired helix (guide/target), it has little to no binding affinity for
double-stranded RNA or for the L7Ae-sRNA complex
(22,
26,
33,
34). Nop5p serves as an
intermediary protein bringing fibrillarin to the complex through its
association with both the L7Ae-sRNA complex and fibrillarin
(22). Along with its role as
an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses
other functions not yet fully understood. For example, Nop5p self-dimerizes
through a coiled-coil domain
(35) that in most archaea and
eukaryotic homologs includes a small insertion sequence of unknown function
(36,
37). However, dimerization and
fibrillarin binding have been shown to be mutually exclusive in
Methanocaldococcus jannaschii Nop5p, potentially because of the
presence of this insertion sequence
(36). Thus, whether Nop5p is a
monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate
its interaction with a L7Ae box C/D RNA complex because both the
fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal
structures (29,
35,
38). Individual residues on
the surface of a monomeric form of Nop5p (referred to as mNop5p)
(22) were mutated to alanine,
and the effect on binding affinity for a L7Ae box C/D motif RNA complex was
assessed through the use of electrophoretic mobility shift assays. These data
reveal that residues important for binding cluster within the highly conserved
NOP domain (39,
40). To demonstrate that this
domain is solely responsible for the affinity of Nop5p for the preassembled
L7Ae box C/D RNA complex, we expressed and purified it in isolation from the
full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA
complex with nearly wild type affinity, demonstrating that the Nop-RBD is
truly an autonomously folding and functional module. Comparison of our data
with the crystal structure of the homologous spliceosomal hPrp31-15.5K
protein-U4 snRNA complex (41)
suggests the adoption of a similar mode of binding, further supporting a
crucial role for the NOP domain in RNP complex assembly. 相似文献
13.
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.
George Minasov Sivaraman Padavattan Ludmilla Shuvalova Joseph S. Brunzelle Darcie J. Miller Arnaud Basl�� Claudia Massa Frank R. Collart Tilman Schirmer Wayne F. Anderson 《The Journal of biological chemistry》2009,284(19):13174-13184
Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is
involved in the regulation of cell surface-associated traits and the
persistence of infections. Omnipresent GGDEF and EAL domains, which occur in
various combinations with regulatory domains, catalyze c-di-GMP synthesis and
degradation, respectively. The crystal structure of full-length YkuI from
Bacillus subtilis, composed of an EAL domain and a C-terminal
PAS-like domain, has been determined in its native form and in complex with
c-di-GMP and Ca2+. The EAL domain exhibits a triose-phosphate
isomerase-barrel fold with one antiparallel β-strand. The complex with
c-di-GMP-Ca2+ defines the active site of the putative
phosphodiesterase located at the C-terminal end of the β-barrel. The EAL
motif is part of the active site with Glu-33 of the motif being involved in
cation coordination. The structure of the complex allows the proposal of a
phosphodiesterase mechanism, in which the divalent cation and the general base
Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on
the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its
pocket-like structure could accommodate a yet unknown ligand. YkuI forms a
tight dimer via EAL-EAL and trans EAL-PAS-like domain association.
The possible regulatory significance of the EAL-EAL interface and a mechanism
for signal transduction between sensory and catalytic domains of
c-di-GMP-specific phosphodiesterases are discussed.The dinucleotide cyclic di-GMP (c-di-GMP) was discovered about 20 years ago
when it was found to regulate the activity of cellulase synthase in
Acetobacter xylinum
(1). However, its prominent
role as a global second messenger has been realized only upon the recent
recognition of the omnipresence of genes coding for domains that catalyze
c-di-GMP biosynthesis and degradation in eubacteria
(2). GGDEF domains catalyze the
condensation of two GTP molecules to the cyclic 2-fold symmetric dinucleotide
(diguanylate cyclase activity
(3-6)),
whereas EAL domains are involved in its degradation to yield the linear
dinucleotide pGpG (phosphodiesterase
(PDE)4 A activity)
(3,
7-9).
Recently, also HD-GYP domains have been implicated in c-di-GMP-specific PDE
activity (10). All the domains
have been named according to their sequence signature motifs. They are
typically found in combinations with various other, mostly sensory or
regulatory, domains. It is thought that the balance between antagonistic
diguanylate cyclase and PDE-A activities determines the cellular level of
c-di-GMP and, thus, affects a variety of physiological processes in
bacteria.It has been shown that, in general, c-di-GMP regulates cell
surface-associated traits and community behavior such as biofilm formation
(for reviews see Refs.
11-12),
and its relevance to the virulence of pathogenic bacteria has been
demonstrated (11,
13,
14). In particular, the
dinucleotide has been proposed to orchestrate the switch between acute and
persistent phase of infection.The best characterized diguanylate cyclase is PleD from Caulobacter
crescentus with a Rec-Rec-GGDEF domain architecture (Rec indicates
response regulator receiver domain). The structure of its GGDEF domain
revealed a single GTP-binding site and suggested that dimerization is the
prerequisite for enzymatic activity
(4). This has been corroborated
recently by crystallography showing directly that
modification of the first Rec
domain, mimicking phosphorylation by the cognate kinase, induces formation of
a tightly packed dimer (15).
Additionally, an upper limit of c-di-GMP levels in the cell seems to be
ensured by potent allosteric product inhibition of the PleD cyclase
(4,
15,
16). Recently, the crystal
structure of another diguanylate cyclase, WspR from Pseudomonas
aeruginosa with a Rec-GGDEF domain architecture, has been determined
(17), which showed a
tetrameric quaternary structure and active and feedback inhibition sites that
are very similar to those in PleD.For EAL domains, it has been demonstrated that genetic knock-out results in
phenotypes that are in line with the paradigm that an elevated cellular
c-di-GMP concentration corresponds to a sessile and a low concentration to a
motile bacterial life style
(13,
18,
19). Only recently,
EAL-mediated PDE-A activity has been measured in vitro
(7-9,
20-22).The Bacillus subtilis YkuI protein was targeted for structure
determination by the Midwest Center for Structural Genomics as a member of the
large sequence family that contains EAL (Pfam number PF00563) domains. Here we
report the crystal structure of YkuI showing the fold of the N-terminal EAL
domain and the C-terminal PAS-like domain. Co-crystallization with c-di-GMP
revealed the substrate binding mode and allows the proposal of a catalytic
mechanism. The PAS-like domain most probably has regulatory function, which is
discussed. Recently, another EAL structure has been deposited in the Protein
Data Bank by the Midwest Center for Structural Genomics, the EAL domain of a
GGDEF-EAL protein from Thiobacillus denitrificans (tdEAL; PDB code
2r6o). Comparison of the two structures suggests a possible regulatory
mechanism. 相似文献
19.
20.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献