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1.
Michael S. Friedman Sivan M. Oyserman Kurt D. Hankenson 《The Journal of biological chemistry》2009,284(21):14117-14125
Wnt11 signals through both canonical (β-catenin) and non-canonical
pathways and is up-regulated during osteoblast differentiation and fracture
healing. In these studies, we evaluated the role of Wnt11 during
osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases
β-catenin accumulation and promotes bone morphogenetic protein
(BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11
dramatically increases expression of the osteoblast-associated genes
Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating
endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also
increases expression of Rspo2 (R-spondin 2), a secreted factor known
to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing
Dmp1, Phex, and Bsp expression and promotes bone
morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates
Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor
(Tcf)/β-catenin signaling with dominant negative Tcf blocks
Wnt11-mediated expression of Dmp1, Phex, and Rspo2
and decreases mineralization. However, dominant negative Tcf fails to block
the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11
signals through β-catenin, activating Rspo2 expression, which is
then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and
maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b
enhances osteoblast differentiation
(1). Canonical Wnt signaling
through β-catenin has also been shown to enhance the chondroinductive and
osteoinductive properties of
BMP22
(2,
3). During BMP2-induced
osteoblast differentiation of mesenchymal stem cell lines, cross-talk between
BMP and Wnt pathways converges through the interaction of Smad4 with
β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and
homeostasis. During limb development, expression of Wnt3a in the apical
ectodermal ridge of limb buds maintains cells in a highly proliferative and
undifferentiated state (4,
5). Disruption of canonical Wnt
signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and
digit-patterning defects (6).
Wnt signaling is also involved in the maintenance of post-natal bone mass.
Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass,
whereas loss of Lrp5 function is associated with decreased bone mass and
osteoporosis pseudoglioma syndrome
(7,
8). Mice with increased Wnt10b
expression have increased trabecular bone, whereas Wnt10b-deficient mice have
reduced trabecular bone (9).
Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased
trabecular bone accrual throughout adulthood
(10).Although canonical Wnt signaling regulates osteoblastogenesis and bone
formation, the profile of endogenous Wnts that play a role in osteoblast
differentiation and maturation is not well described. During development,
Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum
(11). Wnt11 expression is
increased during glucocorticoid-induced osteogenesis
(12), indicating a potential
role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates
both β-catenin-dependent as well as β-catenin-independent signaling
pathways (13). Targeted
disruption of Wnt11 results in late embryonic/early post-natal death because
of cardiac dysfunction (14).
Although these mice have no reported skeletal developmental abnormalities,
early lethality obfuscates a detailed examination of post-natal skeletal
modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of
R-spondin 2 (Rspo2) in the apical ectodermal ridge
(11,
15). R-spondins are a novel
family of proteins that share structural features, including two conserved
cysteinerich furin-like domains and a thrombospondin type I repeat
(16). The four R-spondin
family members can activate canonical Wnt signaling
(15,
17–19).
Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling.
Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting
Dkk-mediated receptor internalization
(20). Rspo1 was also shown to
potentiate Wnt3a-mediated osteoblast differentiation
(21). Rspo2 knock-out
mice, which die at birth, have limb patterning defects associated with altered
β-catenin signaling
(22–24).
However, the role of Rspo2 in osteoblast differentiation and maturation
remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts
activates β-catenin and augments BMP-induced osteoblast maturation and
mineralization. Wnt11 increases the expression of Rspo2.
Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced
osteoblast maturation and mineralization. Although antagonism of
Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2
rescues this block, and knockdown of Rspo2 shows that it is required for
Wnt11-mediated osteoblast maturation and mineralization. These studies
identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and
mineralization. 相似文献
2.
3.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
4.
5.
6.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
7.
8.
9.
Kaliyamurthi Venkatachalam Balachandar Venkatesan Anthony J. Valente Peter C. Melby Sailesh Nandish Jane E. B. Reusch Robert A. Clark Bysani Chandrasekar 《The Journal of biological chemistry》2009,284(21):14414-14427
WNT1-inducible signaling pathway protein-1 (WISP1), a member of the
CYR61/CTGF/Nov family of growth factors, can mediate cell growth,
transformation, and survival. Previously we demonstrated that WISP1 is
up-regulated in post-infarct heart, stimulates cardiac fibroblast
proliferation, and is induced by the proinflammatory cytokine tumor necrosis
factor-α (TNF-α). Here we investigated (i) the localization of
TNF-α and WISP1 in post-infarct heart, (ii) the mechanism of
TNF-α-mediated WISP1 induction in primary human cardiac fibroblasts
(CF), (iii) the role of WISP1 in TNF-α-mediated CF proliferation and
collagen production, and (iv) the effects of WISP1 on TNF-α-mediated
cardiomyocyte death. TNF-α and WISP1 expressions were increased in the
border zones and non-ischemic remote regions of the post-ischemic heart. In
CF, TNF-α potently induced WISP1 expression in cyclic AMP response
element-binding protein (CREB)-dependent manner. TNF-α induced CREB
phosphorylation in vitro and DNA binding and reporter gene activities
in vivo. TNF-α induced CREB activation via ERK1/2, and
inhibition of ERK1/2 and CREB blunted TNF-α-mediated WISP1 induction.
Most importantly, WISP1 knockdown attenuated TNF-α stimulated collagen
production and CF proliferation. Furthermore, WISP1 attenuated
TNF-α-mediated cardiomyocyte death, thus demonstrating pro-mitogenic and
pro-survival effects for WISP1 in myocardial constituent cells. Our results
suggest that a TNF-α/WISP1 signaling pathway may contribute to
post-infarct cardiac remodeling, a condition characterized by fibrosis and
progressive cardiomyocyte loss.Acute myocardial infarction
(MI)2 is a major cause
of morbidity and mortality worldwide. After acute MI, the two principal
determinants of patient outcome are myocardial infarct size and the extent of
left ventricular (LV) remodeling
(1–3).
Infarct size is determined during the acute phase immediately post-MI. LV
remodeling on the other hand is a continuous and maladaptive process
characterized by progressive left ventricular hypertrophy, fibrosis,
ventricular dilatation, and by the gradual deterioration of cardiac
performance, leading ultimately to congestive heart failure
(1–3).Numerically, fibroblasts are the major cell type in the heart, but they
constitute a smaller total volume compared with cardiomyocytes
(4). Fibroblasts are associated
primarily with modulation of extracellular matrix and tissue healing/repair
(4–6).
Fibroblasts secrete collagens, fibrillins, fibronectin, laminin, and matrix
metalloproteinases and, thus, are responsible for the maintenance of
connective tissue homeostasis
(4–7).
We and others have shown that primary cardiac fibroblasts also produce various
cytokines, chemokines, and growth factors
(4–6,
8,
9), which tightly regulate the
physiological function of the cells. Under pathological conditions, however,
where the expression of cytokines and growth factors is significantly altered,
the fibroblasts can undergo differentiation, migration, and proliferation,
resulting in pathological fibrosis because of excessive accumulation of
collagens and other ECM proteins
(4–6).The six members of the CCN (CYR61/CTGF/Nov) family of
growth factors (Cyr61 (cysteine-rich 61, CCN1), CTGF (connective tissue growth
factor, CCN2), Nov (nephroblastoma-overexpressed, CCN3), WISP1 (WNT1-inducible
signaling pathway protein-1, CCN4), WISP2 (CCN5), and WISP3 (CCN6)) are
involved in a number of cellular processes, including adhesion, migration, and
proliferation
(10–12).
Although their roles in angiogenesis and oncogenesis are well characterized,
with the exception of CTGF, their precise contribution to myocardial
remodeling is unknown. Recently we demonstrated that whereas WISP1 is
expressed in the heart at low basal levels, permanent occlusion of the left
anterior descending coronary artery significantly up-regulates its expression
in the non-ischemic myocardium
(13). Furthermore, we have
found that WISP1 exerts both pro-hypertrophic and pro-mitogenic effects in
vitro, stimulating Akt-dependent cardiomyocyte growth, as well as
collagen synthesis and fibroblast proliferation
(13). Because cardiomyocyte
hypertrophy and fibroblast proliferation play central roles in remodeling and
WISP1 regulates these two critical processes, it is plausible that WISP1 also
mediates cardiac remodeling after myocardial ischemia, infarction, and
inflammation. However, the mechanisms involved in the induction and regulation
of WISP1 under these conditions have not been well characterized.The proinflammatory cytokine tumor necrosis factor (TNF)-α is
expressed at low basal levels in the normal myocardium but is significantly
up-regulated after infection, inflammation, and injury
(14,
15). Although low levels are
considered cytoprotective (16,
17), aberrant expression of
TNF-α during myocardial ischemic injury and inflammation induces
cardiomyocyte death, hypertrophy of surviving cardiomyocytes, contractile
dysfunction, fibroblast proliferation, fibrosis, and adverse remodeling
(14–17).
TNF-α exerts its biological effects via two cell surface receptors,
TNFR1 (55 kDa) and TNFR2 (75 kDa)
(18). TNFR1, which is more
abundantly expressed in the heart, appears to be the main signaling receptor
for TNF-α and is implicated in transmitting its deleterious effects
(18). On the other hand,
signaling through TNFR2 appears to exert a protective effect in the heart
(18,
19). Of note, all myocardial
constituent cells, including fibroblasts, express both TNFR1 and TNFR2 and,
therefore, are targets of TNF-α.We have recently demonstrated that TNF-α induces WISP1 expression in
cardiomyocytes (13). However,
neither its localization in post-infarct myocardium, its role in
TNF-α-mediated cardiac fibroblast (CF) proliferation and collagen
production, nor its effects on TNF-α-mediated cardiomyocyte death are
known. Our results show that both TNF-α and WISP1 are localized in the
border zone and the non-infarct remote region in post-infarct myocardium.
Furthermore, TNF-α induces WISP1 expression in CF via a
TNFR2/MEK1/ERK1/2/CREB pathway and stimulates fibroblast collagen production
in part via WISP1. In addition, WISP1 exerts pro-survival effects in
cardiomyocytes, antagonizing TNF-α-mediated cardiomyocyte death.
Collectively, these results indicate that WISP1 exerts pro-mitogenic and
pro-survival effects in cardiac cell type-specific manner, and
TNF-α/WISP1 signaling may be an important contributing mechanism in
post-infarct cardiac remodeling. 相似文献
10.
11.
12.
13.
14.
15.
16.
17.
18.
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
19.
Kelly J. Inglis David Chereau Elizabeth F. Brigham San-San Chiou Susanne Sch?bel Normand L. Frigon Mei Yu Russell J. Caccavello Seth Nelson Ruth Motter Sarah Wright David Chian Pamela Santiago Ferdie Soriano Carla Ramos Kyle Powell Jason M. Goldstein Michael Babcock Ted Yednock Frederique Bard Guriqbal S. Basi Hing Sham Tamie J. Chilcote Lisa McConlogue Irene Griswold-Prenner John P. Anderson 《The Journal of biological chemistry》2009,284(5):2598-2602
Several neurological diseases, including Parkinson disease and dementia
with Lewy bodies, are characterized by the accumulation of α-synuclein
phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for
this phosphorylation have been the subject of intense investigation. Here we
submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible
kinase or SNK) is a principle contributor to α-synuclein phosphorylation
at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at
Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases
inhibited α-synuclein phosphorylation both in primary cortical cell
cultures and in mouse brain in vivo. Finally, specific knockdown of
PLK2 expression by transduction with short hairpin RNA constructs or by
knock-out of the plk2 gene reduced p-Ser-129 levels. These results
indicate that PLK2 plays a critical role in α-synuclein phosphorylation
in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson
disease (PD)4 and the
related disorder, dementia with Lewy bodies (DLB), is suggested by its
association with Lewy bodies and Lewy neurites, the inclusions that
characterize these diseases
(1–3),
and demonstrated by the existence of mutations that cause syndromes mimicking
sporadic PD and DLB
(4–6).
Furthermore, three separate mutations cause early onset forms of PD and DLB.
It is particularly telling that duplications or triplications of the gene
(7–9),
which increase levels of α-synuclein with no alteration in sequence,
also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine
residues, at Ser-87 and Ser-129
(10), although to date only
the Ser-129 phosphorylation has been identified in the central nervous system
(11,
12). Phosphorylation at
tyrosine residues has been observed by some investigators
(13,
14) but not by others
(10–12).
Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the
majority of synuclein in Lewy bodies contains this modification
(15). In addition, p-Ser-129
was found to be the most extensive and consistent modification in a survey of
synuclein in Lewy bodies (11).
Results have been mixed from studies investigating the function of
phosphorylation using S129A and S129D mutations to respectively block and
mimic the modification. Although the phosphorylation mimic was associated with
pathology in studies in Drosophila
(16) and in transgenic mouse
models (17,
18), studies using
adeno-associated virus vectors to overexpress α-synuclein in rat
substantia nigra found an exacerbation of pathology with the S129A mutation,
whereas the S129D mutation was benign, if not protective
(19). Interpretation of these
studies is complicated by a recent study showing that the S129D and S129A
mutations themselves have effects on the aggregation properties of
α-synuclein independent of their effects on phosphorylation, with the
S129A mutation stimulating fibril formation
(20). Clearly, determination
of the role of p-Ser-129 phosphorylation would be helped by identification of
the responsible kinase. In addition, identification will provide a
pathologically relevant way to increase phosphorylation in a cell or animal
model.Several kinases have been proposed to phosphorylate α-synuclein,
including casein kinases 1 and 2
(10,
12,
21) and members of the
G-protein-coupled receptor kinase family
(22). In this report, we offer
evidence that a member of the polo-like kinase (PLK) family, PLK2 (or
serum-inducible kinase, SNK), functions as an α-synuclein kinase. The
ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is
established by overexpression in cell culture and by in vitro
reaction with the purified kinase. We show that PLK2 phosphorylates
α-synuclein in cells, including primary neuronal cultures, using a
series of kinase inhibitors as well as inhibition of expression with RNA
interference. In addition, inhibitor and knock-out studies in mouse brain
support a role for PLK2 as an α-synuclein kinase in vivo. 相似文献
20.
Elena Sotillo Judit Garriga Amol Padgaonkar Alison Kurimchak Jeanette Gowen Cook Xavier Gra?a 《The Journal of biological chemistry》2009,284(21):14126-14135
We have previously shown that SV40 small t antigen (st) cooperates with
deregulated cyclin E to activate CDK2 and bypass quiescence in normal human
fibroblasts (NHF). Here we show that st expression in serum-starved and
density-arrested NHF specifically induces up-regulation and loading of CDC6
onto chromatin. Coexpression of cyclin E results in further accumulation of
CDC6 onto chromatin concomitantly with phosphorylation of CDK2 on Thr-160 and
CDC6 on Ser-54. Investigation of the mechanism leading to CDC6 accumulation
and chromatin loading indicates that st is a potent inducer of cdc6
mRNA expression and increases CDC6 protein stability. We also show that CDC6
expression in quiescent NHF efficiently promotes cyclin E loading onto
chromatin, but it is not sufficient to activate CDK2. Moreover, we show that
CDC6 expression is linked to phosphorylation of the activating T loop of CDK2
in serum-starved NHF stimulated with mitogens or ectopically expressing cyclin
E and st. Our data suggest a model where the combination of st and deregulated
cyclin E result in cooperative and coordinated activation of both an essential
origin licensing factor, CDC6, and an activity required for origin firing,
CDK2, resulting in progression from quiescence to S phase.Upon mitogenic stimulation mammalian G1
CDKs4 trigger passage
through the restriction point and the transition into DNA replication. In
particular, cyclin E/CDK2 is activated in mid to late G1 and phosphorylates a
variety of substrates that play critical roles in these processes. CDK2
cooperates with D-type cyclin/CDKs to inactivate E2F/pocket protein repressor
complexes inducing the expression of DNA synthesis factors and other cell
cycle regulators (reviewed in Refs.
1 and
2). CDK2 also phosphorylates
DNA replication factors facilitating prereplication complex assembly and
origin firing and plays additional roles in centrosome duplication and histone
synthesis (reviewed in Ref. 1).
In particular, it has been proposed that CDK2 phosphorylates the essential
origin licensing factor CDC6 promoting its stabilization prior to inactivation
of the APCCdh1 ubiquitin ligase
(3). This is thought to ensure
that CDC6 accumulation precedes accumulation of other APC substrates that
inhibit origin licensing. Moreover, CDK2-independent cyclin E functions have
also been reported to be important for prereplication complex assembly in
cells in transit from G0 into G1
(4,
5). In keeping with its role as
positive regulator of major G1 transitions, deregulation of the cyclin E via
gene amplification or defective protein turnover is commonly seen in primary
tumors and is associated with poor prognosis
(6–8).
In normal fibroblasts, ectopic expression of cyclin E has been associated with
shortening of the G1 phase of the cell cycle
(9,
10), and with induction of DNA
damage (reviewed in Ref. 8).
Cyclin E deregulation in certain human tumor cell lines and immortalized rat
fibroblasts is associated with mitogen-independent cell cycle entry and
progression through the cell cycle
(11). However, when cyclin E
is ectopically expressed in quiescent normal human fibroblasts (NHF), cells
remain in G0 (12).We have recently reported that coexpression of SV40 small t antigen (st) in
quiescent NHF with deregulated cyclin E expression is sufficient to trigger
mitogen-independent cell cycle progression, proliferation beyond cell
confluence, and foci formation. The bypass of quiescence induced by
the expression of st and cyclin E is dependent on CDK2 activation
(12). Thus, contrary to what
is seen in normal murine cells
(13), CDK2 activity appears
essential for cell cycle progression when it is oncogenically driven by cyclin
E and st expression (12).
Because st is known to target pathways uniquely required for the
transformation of human cells
(14,
15), tumor cells with altered
pathways that mimic st/cyclin E expression could predictably be sensitive to
selective inhibition of CDK2 activity.Given the critical role of CDK2 activity in cyclin E and st cooperation in
inducing cell proliferation and transformation of NHF, we sought to determine
the factors and mechanisms by which st modulates CDK2 activation. In this
report we have identified the CDC6 replication licensing factor as a cellular
target of st. We also uncover CDC6 as a participant in the events leading to
chromatin association of cyclin E and CDK2 and in phosphorylation of CDK2 on
its activating T loop both in response to mitogenic stimulation, as well as
expression of cyclin E and st in NHF. 相似文献