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1.
Obidimma C. Ezezika Noah S. Younger Jia Lu Donald A. Kaiser Zachary A. Corbin Bradley J. Nolen David R. Kovar Thomas D. Pollard 《The Journal of biological chemistry》2009,284(4):2088-2097
Expression of human profilin-I does not complement the
temperature-sensitive cdc3-124 mutation of the single profilin gene
in fission yeast Schizosaccharomyces pombe, resulting in death from
cytokinesis defects. Human profilin-I and S. pombe profilin have
similar affinities for actin monomers, the FH1 domain of fission yeast formin
Cdc12p and poly-l-proline (Lu, J., and Pollard, T. D. (2001)
Mol. Biol. Cell 12, 1161–1175), but human profilin-I does not
stimulate actin filament elongation by formin Cdc12p like S. pombe
profilin. Two crystal structures of S. pombe profilin and homology
models of S. pombe profilin bound to actin show how the two profilins
bind to identical surfaces on animal and yeast actins even though 75% of the
residues on the profilin side of the interaction differ in the two profilins.
Overexpression of human profilin-I in fission yeast expressing native profilin
also causes cytokinesis defects incompatible with viability. Human profilin-I
with the R88E mutation has no detectable affinity for actin and does not have
this dominant overexpression phenotype. The Y6D mutation reduces the affinity
of human profilin-I for poly-l-proline by 1000-fold, but
overexpression of Y6D profilin in fission yeast is lethal. The most likely
hypotheses to explain the incompatibility of human profilin-I with Cdc12p are
differences in interactions with the proline-rich sequences in the FH1 domain
of Cdc12p and wider “wings” that interact with actin.The small protein profilin not only helps to maintain a cytoplasmic pool of
actin monomers ready to elongate actin filament barbed ends
(2), but it also binds to type
II poly-l-proline helices
(3,
4). The actin
(5) and
poly-l-proline
(6–8)
binding sites are on opposite sides of the profilin molecule, so profilin can
link actin to proline-rich targets. Viability of fission yeast depends
independently on profilin binding to both actin and poly-l-proline,
although cells survive >10-fold reductions in affinity for either ligand
(1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p
(9,
10) and profilin
(11) to assemble actin
filaments for the cytokinetic contractile ring. Formins are multidomain
proteins that nucleate and assemble unbranched actin filaments
(12). Formin FH2 domains form
homodimers that can associate processively with the barbed ends of growing
actin filaments (13,
14). FH2 dimers slow the
elongation of barbed ends
(15). Most formin proteins
have an FH1 domain linked to the FH2 domain. Binding profilin-actin to
multiple polyproline sites in an FH1 domain concentrates actin near the barbed
end of an actin filament associated with a formin FH2 homodimer. Actin
transfers very rapidly from the FH1 domains onto the filament end
(16) allowing profilin to
stimulate elongation of the filament
(15,
17).We tested the ability of human (Homo sapiens,
Hs)7 profilin-I to
complement the temperature-sensitive cdc3-124 mutation
(11) in the single fission
yeast profilin gene with the aim of using yeast to characterize human profilin
mutations. The failure of expression of Hs profilin-I to complement the
cdc3-124 mutation prompted us to compare human and fission yeast
profilins more carefully. We report here a surprising incompatibility of Hs
profilin-I with fission yeast formin Cdc12p, a crystal structure of fission
yeast profilin, which allowed a detailed comparison with Hs profilin, and
mutations that revealed how overexpression of Hs profilin-I compromises the
viability of wild-type fission yeast. 相似文献
2.
Benjamin E. L. Lauffer Stanford Chen Cristina Melero Tanja Kortemme Mark von Zastrow Gabriel A. Vargas 《The Journal of biological chemistry》2009,284(4):2448-2458
Many G protein-coupled receptors (GPCRs) recycle after agonist-induced
endocytosis by a sequence-dependent mechanism, which is distinct from default
membrane flow and remains poorly understood. Efficient recycling of the
β2-adrenergic receptor (β2AR) requires a C-terminal PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin
cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth
factor-regulated substrate). The PDZbd is thought to link receptors to actin
through a series of protein interaction modules present in NHERF/EBP50
(Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein
of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not
known, however, if such actin connectivity is sufficient to recapitulate the
natural features of sequence-dependent recycling. We addressed this question
using a receptor fusion approach based on the sufficiency of the PDZbd to
promote recycling when fused to a distinct GPCR, the δ-opioid receptor,
which normally recycles inefficiently in HEK293 cells. Modular domains
mediating actin connectivity promoted receptor recycling with similarly high
efficiency as the PDZbd itself, and recycling promoted by all of the domains
was actin-dependent. Regulation of receptor recycling by Hrs, however, was
conferred only by the PDZbd and not by downstream interaction modules. These
results suggest that actin connectivity is sufficient to mimic the core
recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors
(GPCRs)2 comprise the
largest family of transmembrane signaling receptors expressed in animals and
transduce a wide variety of physiological and pharmacological information.
While these receptors share a common 7-transmembrane-spanning topology,
structural differences between individual GPCR family members confer diverse
functional and regulatory properties
(1-4).
A fundamental mechanism of GPCR regulation involves agonist-induced
endocytosis of receptors via clathrin-coated pits
(4). Regulated endocytosis can
have multiple functional consequences, which are determined in part by the
specificity with which internalized receptors traffic via divergent downstream
membrane pathways
(5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed
by the δ-opioid receptor (δOR), contributes to proteolytic
down-regulation of receptor number and produces a prolonged attenuation of
subsequent cellular responsiveness to agonist
(8,
9). Trafficking of internalized
GPCRs via a rapid recycling pathway, a major route traversed by the
β2-adrenergic receptor (β2AR), restores the complement of functional
receptors present on the cell surface and promotes rapid recovery of cellular
signaling responsiveness (6,
10,
11). When co-expressed in the
same cells, the δOR and β2AR are efficiently sorted between these
divergent downstream membrane pathways, highlighting the occurrence of
specific molecular sorting of GPCRs after endocytosis
(12).Recycling of various integral membrane proteins can occur by default,
essentially by bulk membrane flow in the absence of lysosomal sorting
determinants (13). There is
increasing evidence that various GPCRs, such as the β2AR, require
distinct cytoplasmic determinants to recycle efficiently
(14). In addition to requiring
a cytoplasmic sorting determinant, sequence-dependent recycling of the
β2AR differs from default recycling in its dependence on an intact actin
cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs
(hepatocyte growth factor receptor substrate)
(11,
14). Compared with the present
knowledge regarding protein complexes that mediate sorting of GPCRs to
lysosomes (15,
16), however, relatively
little is known about the biochemical basis of sequence-directed recycling or
its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called
PDZbd), and PDZ-mediated protein association(s) with this sequence appear to
be primarily responsible for its endocytic sorting activity
(17-20).
Fusion of this sequence to the cytoplasmic tail of the δOR effectively
re-routes endocytic trafficking of engineered receptors from lysosomal to
recycling pathways, establishing the sufficiency of the PDZbd to function as a
transplantable sorting determinant
(18). The β2AR-derived
PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ
proteins (21,
22). A well-established
biochemical function of NHERF/EBP50 family proteins is to associate integral
membrane proteins with actin-associated cytoskeletal elements. This is
achieved through a series of protein-interaction modules linking NHERF/EBP50
family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to
actin filaments
(23-26).
Such indirect actin connectivity is known to mediate other effects on plasma
membrane organization and function
(23), however, and NHERF/EBP50
family proteins can bind to additional proteins potentially important for
endocytic trafficking of receptors
(23,
25). Thus it remains unclear
if actin connectivity is itself sufficient to promote sequence-directed
recycling of GPCRs and, if so, if such connectivity recapitulates the normal
cellular regulation of sequence-dependent recycling. In the present study, we
took advantage of the modular nature of protein connectivity proposed to
mediate β2AR recycling
(24,
26), and extended the opioid
receptor fusion strategy used successfully for identifying diverse recycling
sequences in GPCRs
(27-29),
to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can
be effectively bypassed by linking receptors to ERM family proteins in the
absence of the PDZbd itself. Further, we establish that the protein
connectivity network can be further simplified by fusing receptors to an
interaction module that binds directly to actin filaments. We found that
bypassing the PDZ-mediated interaction using either domain is sufficient to
mimic the ability of the PDZbd to promote efficient, actin-dependent recycling
of receptors. Hrs-dependent regulation, however, which is characteristic of
sequence-dependent recycling of wild-type receptors, was recapitulated only by
the fused PDZbd and not by the proposed downstream interaction modules. These
results support a relatively simple architecture of protein connectivity that
is sufficient to mimic the core recycling activity of the β2AR-derived
PDZbd, but not its characteristic cellular regulation. Given that an
increasing number of GPCRs have been shown to bind PDZ proteins that typically
link directly or indirectly to cytoskeletal elements
(17,
27,
30-32),
the present results also suggest that actin connectivity may represent a
common biochemical principle underlying sequence-dependent recycling of
various GPCRs. 相似文献
3.
The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.Bni1 and Bnr1 are the two formin isoforms expressed in Saccharomyces cerevisiae (1, 2). These proteins, as other isoforms in the formin family, are large multidomain proteins (3, 4). Several regulatory domains, including one for binding the G-protein rho, are located at the N-terminal half of the protein (4–7). FH1, FH2, and Bud6 binding domains are located in the C-terminal half of the protein (8). The formin homology 1 (FH1)2 domain contains several sequential poly-l-proline motifs, and it interacts with the profilin/actin complex to recruit actin monomers and regulate the insertion of actin monomers at the barbed end of actin (9–11). The fomin homology domain 2 (FH2) forms a donut-shaped homodimer, which wraps around actin dimers at the barbed end of actin filaments (12, 13). One important function of formin is to facilitate actin polymerization by stabilizing actin dimers or trimers under polymerization conditions and then to processively associate with the barbed end of the elongating filament to control actin filament elongation kinetics (13–18).A major unsolved protein in the study of formins is the elucidation of the individual functions of different isoforms and their regulation. In vivo, these two budding yeast formins have distinct cellular locations and dynamics (1, 2, 19, 20). Bni1 concentrates at the budding site before the daughter cell buds from the mother cell, moves along with the tip of the daughter cell, and then travels back to the neck between daughter and mother cells at the end of segregation. Bnr1 localizes only at the neck of the budding cell in a very short period of time after bud emergence. Although a key cellular function of these two formins in yeast is to promote actin cable formation (8, 18), the roles of the individual formins in different cellular process is unclear because deleting either individual formin gene has limited impact on cell growth and deleting both genes together is lethal (21).Although each of the two formins can nucleate actin filament formation in vitro, the manner in which they affect polymerization is distinctly isoform-specific. Most of this mechanistic work in vitro has used formin fragments containing the FH1 and FH2 domains. Bni1 alone processively caps the barbed end of actin filaments partially inhibiting polymerization at this end (14, 16, 18). The profilin-actin complex, recruited to the actin barbed end through its binding to Bni1 FH1 domain, possibly raises the local actin concentration and appears to allow this inhibition to be overcome, thereby, accelerating barbed end polymerization. It has also been shown that this complex modifies the kinetics of actin dynamics at the barbed end (9, 11, 18, 22). Moreover, Bni1 participation leads only to the formation of single filaments (8). In comparison, the Bnr1 FH1-FH2 domain facilitates actin polymerization much more efficiently than does Bni1. Moseley and Goode (8) showed Bnr1 accelerates actin polymerization up to 10 times better than does Bni and produces actin filament bundles when the Bnr1/actin molar ratio is above 1:2. Finally, the regulation of Bni1 and Bnr1 by formin binding is different. For example, Bud 6/Aip3, a yeast cell polarity factor, binds to Bni1, but not Bnr1, and also stimulates its activity in vitro.For their studies, Moseley and Goode (8) utilized mammalian skeletal muscle actin instead of the S. cerevisiae actin with which the yeast formins are designed to function. It is entirely possible that the differences observed with the two formins are influenced quantitatively or qualitatively by the nature of the actin used in the study. This possibility must be seriously considered because although yeast and muscle actins are 87% identical in sequence, they display marked differences in their polymerization behavior (23). Yeast actin nucleates filaments better than muscle actin (24, 25). It appears to form shorter and more flexible filaments than does muscle actin (26, 27). Finally, the disposition of the Pi released during the hydrolysis of ATP that occurs during polymerization is different. Yeast actin releases its Pi concomitant with hydrolysis of the bound ATP whereas muscle actin retains the Pi for a significant amount of time following nucleotide hydrolysis (28, 29). This difference is significant because ADP-Pi F-actin has been shown to be more stable than ADP F-actin (30). Another example of this isoform dependence is the interaction of yeast Arp2/3 with yeast versus muscle actins (31). Yeast Arp2/3 complex accelerates polymerization of muscle actin only in the presence of a nucleation protein factor such as WASP. However, with yeast actin, no such auxiliary protein is required. In light of these actin behavioral differences, to better understand the functional differences of these two formins in vivo, we have studied the behavior of Bni 1 and Bnr 1 with WT and mutant yeast actins, and we have also explored the molecular basis underlying the Bnr 1-induced formation of actin nuclei from G-actin. 相似文献
4.
Ivana I. Knezevic Sanda A. Predescu Radu F. Neamu Matvey S. Gorovoy Nebojsa M. Knezevic Cordus Easington Asrar B. Malik Dan N. Predescu 《The Journal of biological chemistry》2009,284(8):5381-5394
It is known that platelet-activating factor (PAF) induces severe
endothelial barrier leakiness, but the signaling mechanisms remain unclear.
Here, using a wide range of biochemical and morphological approaches applied
in both mouse models and cultured endothelial cells, we addressed the
mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and
of increased endothelial permeability. The formation of interendothelial gaps
filled with filopodia and lamellipodia is the cellular event responsible for
the disruption of endothelial barrier. We observed that PAF ligation of its
receptor induced the activation of the Rho GTPase Rac1. Following PAF
exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were
found associated with a membrane fraction from which they
co-immunoprecipitated with PAF receptor. In the same time frame with
Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were
relocated from the IEJs, and formation of numerous interendothelial gaps was
recorded. Notably, the response was independent of myosin light chain
phosphorylation and thus distinct from other mediators, such as histamine and
thrombin. The changes in actin status are driven by the PAF-induced localized
actin polymerization as a consequence of Rac1 translocation and activation.
Tiam1 was required for the activation of Rac1, actin polymerization,
relocation of junctional associated proteins, and disruption of IEJs. Thus,
PAF-induced IEJ disruption and increased endothelial permeability requires the
activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic
target against increased vascular permeability associated with inflammatory
diseases.The endothelial barrier is made up of endothelial cells
(ECs)4 connected to
each other by interendothelial junctions (IEJs) consisting of protein
complexes organized as tight junctions (TJs) and adherens junctions (AJs). In
addition, the focal adhesion complex located at the basal plasma membrane
enables firm contact of ECs with the underlying basement membrane and also
contributes to the barrier function
(1-3).
The glycocalyx, the endothelial monolayer, and the basement membrane all
together constitute the vascular barrier.The structural integrity of the ECs along with their proper functionality
are the two most important factors controlling the tightness of the
endothelial barrier. Changes affecting these factors cause loss of barrier
restrictiveness and leakiness. Therefore, defining and understanding the
cellular and molecular mechanisms controlling these processes is of paramount
importance. Increased width of IEJs in response to permeability-increasing
mediators (4) regulates the
magnitude of transendothelial exchange of fluid and solutes. Disruption of
IEJs and the resultant barrier leakiness contribute to the genesis of diverse
pathological conditions, such as inflammation
(5), metastasis
(6,
7), and uncontrolled
angiogenesis (8,
9).Accumulated evidence demonstrated that IEJs changes are responsible for
increased or decreased vascular permeability, and the generally accepted
mechanism responsible for them was the myosin light chain (MLC)-mediated
contraction of ECs (5,
10). However, published
evidence showed that an increase in vascular permeability could be obtained
without a direct involvement of any contractile mechanism
(11-16).The main component of the vascular barrier, the ECs, has more than 10% of
their total protein represented by actin
(17), which under
physiological salt concentrations subsists as monomers (G-actin) and assembled
into filaments (F-actin). A large number of actin-interacting proteins may
modulate the assembly, disassembly, and organization of G-actin and of actin
filaments within a given cell type. Similar to the complexity of
actin-interacting proteins found in other cell types, the ECs utilize their
actin binding proteins to stabilize the endothelial monolayer in order to
efficiently function as a selective barrier
(11). In undisturbed ECs, the
actin microfilaments are organized as different networks with distinctive
functional and morphological characteristics: the peripheral filaments also
known as peripheral dense band (PDB), the cytoplasmic fibers identified as
stress fibers (SF), and the actin from the membrane cytoskeleton
(18). The peripheral web,
localized immediately under the membrane, is associated with (i) the luminal
plasmalemma (on the apical side), (ii) the IEJ complexes on the lateral
surfaces, and (iii) the focal adhesion complexes on the abluminal side (the
basal part) of polarized ECs. The SF reside inside the endothelial cytoplasm
and are believed to be directly connected with the plasmalemma proper on the
luminal as well as on the abluminal side of the cell. As described, the
endothelial actin cytoskeleton (specifically the SF) seems to be a stable
structure helping the cells to remain flat under flow
(19). It is also established
that the actin fibers participate in correct localization of different
junctional complexes while keeping them in place
(20). However, it was
suggested that the dynamic equilibrium between F- and G-actin might modulate
the tightness of endothelial barrier in response to different challenges
(13).Mediators effective at nanomolar concentrations or less that disrupt the
endothelial barrier and increase vascular permeability include C2 toxin of
Clostridium botulinum, vascular permeability factor, better known as
vascular endothelial growth factor, and PAF
(21). C2 toxin increases
endothelial permeability by ribosylating monomeric G-actin at Arg-177
(22). This results in the
impairment of actin polymerization
(23), followed by rounding of
ECs (16) and the disruption of
junctional integrity. Vascular permeability factor was shown to open IEJs by
redistribution of junctional proteins
(24,
25) and by interfering with
the equilibrium of actin pools
(26). PAF
(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocoline), a naturally
synthesized phospholipid is active at 10-10 m or less
(27). PAF is synthesized by
and acts on a variety of cell types, including platelets
(28), neutrophils
(29), monocytes
(30), and ECs
(31). PAF-mediated activation
of ECs induced cell migration
(32), angiogenesis
(7), and vascular
hyperpermeability (33)
secondary to disassembly of IEJs
(34). The effects of PAF on
the endothelium are initiated through a G protein-coupled receptor (PAF-R)
localized at the plasmalemma, in a large endosomal compartment inside the cell
(34), and also in the nuclear
membrane (35). In ECs, PAF-R
was shown to signal through Gαq and downstream activation of
phospholipase C isozymes (PLCβ3 and PLCγ1),
and via cSrc (32,
36). Studies have shown that
PAF challenge induced endothelial actin cytoskeletal rearrangement
(37) and marked vascular
leakiness (38); however, the
signaling pathways have not been elucidated.Therefore, in the present study, we carried out a systematic analysis of
PAF-induced morphological and biochemical changes of endothelial barrier
in vivo and in cultured ECs. We found that the opening of endothelial
barrier and the increased vascular leakiness induced by PAF are the result of
a shift in actin pools without involvement of EC contraction, followed by a
redistribution of tight junctional associated protein ZO-1 and adherens
junctional protein VE-cadherin. 相似文献
5.
Danielle M. Paul Edward P. Morris Robert W. Kensler John M. Squire 《The Journal of biological chemistry》2009,284(22):15007-15015
The troponin complex on the thin filament plays a crucial role in the
regulation of muscle contraction. However, the precise location of troponin
relative to actin and tropomyosin remains uncertain. We have developed a
method of reconstructing thin filaments using single particle analysis that
does not impose the helical symmetry of actin and is independent of a starting
model. We present a single particle three-dimensional reconstruction of the
thin filament. Atomic models of the F-actin filament were fitted into the
electron density maps and troponin and tropomyosin located. The structure
provides evidence that the globular head region of troponin labels the two
strands of actin with a 27.5-Å axial stagger. The density attributed to
troponin appears tapered with the widest point toward the barbed end. This
leads us to interpret the polarity of the troponin complex in the thin
filament as reversed with respect to the widely accepted model.Regulation of actin filament function is a fundamental biological process
with implications ranging from cell migration to muscle contraction. Skeletal
and cardiac muscle thin filaments consist of actin and the regulatory proteins
troponin and tropomyosin. Contraction is initiated by release of
Ca2+ into the sarcomere and the consequent binding of
Ca2+ to regulatory sites on troponin. Troponin is believed to
undergo a conformational change leading to an azimuthal movement of
tropomyosin, which allows myosin heads to interact with actin, hydrolyze ATP,
and generate force. The molecular basis by which troponin acts to regulate
muscle contraction is only partly understood. It is essential that the
structure of troponin in the thin filament at high and low Ca2+ is
determined to properly understand the mechanism of regulation.The basic structure of the thin filament was described by Ebashi in 1972
(1). In this structure each
tropomyosin molecule covers seven actin monomers, and there is a 27.5-Å
stagger between troponin molecules. The 7-Å tropomyosin structure
(2), the atomic model of
F-actin (3), and the troponin
“core domain” (4)
have recently been used to generate atomic models of the thin filament in low
and high Ca2+ states
(5). While the position of
troponin in these models was constrained by known distance measurements
between filament components, the exact arrangement of the complex on the
filament has not been determined a priori. Although recently
published crystal structures of partial troponin complexes
(4,
6) have provided valuable
insights into the arrangement of the globular head or core domain, the complex
in its entirety has not been crystallized.Troponin is believed to consist of a globular core domain with an extended
tail (7). The globular core
contains the Ca2+-binding subunit
(TnC),2 the inhibitory
subunit (TnI), and the C-terminal part (residues 156–262) of the
tropomyosin-binding subunit (TnT). The extended tail consists of the
N-terminal part of TnT (residues 1–155). A structural rearrangement
associated with Ca2+ dissociation from the troponin core has been
observed (4) such that the
helix connecting the two domains of TnC collapses, releasing the TnI
inhibitory segment. It is postulated that the TnI inhibitory segment then
becomes able to bind actin, in so doing biasing tropomyosin
(8). To understand properly how
Ca2+ binding to TnC leads to movement of tropomyosin, it is
necessary to determine a high resolution structure of troponin attached to the
thin filament, allowing unambiguous docking of the available crystal
structures and direct observation of any changes at a molecular level caused
by Ca2+ binding.Direct visualization of the thin filament is possible using electron
microscopy. Tropomyosin strands have been resolved in the low and high
Ca2+ states confirming the movement of tropomyosin and the steric
blocking model (9,
10). Until recently the actin
helical repeat has been imposed in the majority of reconstructions of the thin
filament causing artifacts. Helical averaging using the actin repeat spreads
troponin density over every actin monomer, which prevents the detailed
position and shape of the troponin complex from being found
(11). It is possible to avoid
this effect by applying a single particle approach. Individual filament images
are divided into segments and each segment treated as a particle.
Three-dimensional reconstruction may then be carried out by single particle
techniques of alignment, classification
(12,
13), Euler angle assignment
(14–16)
and exact filter back-projection
(17,
18).Two forms of single particle analysis have emerged: helical single particle
analysis (19), where the
determined helical symmetry is applied to the final reconstruction, and
non-helical single particle analysis, which treats the complex as a truly
asymmetric particle. Helical single particle analysis has been used to
successfully reconstruct a myosin containing invertebrate thick filament to a
resolution of 25 Å (20),
and non-helical single particle analysis has been applied to the vertebrate
skeletal muscle thick filament allowing azimuthal perturbations of the myosin
heads to be observed (21).Model-based single particle image processing methods have recently been
applied to the structural analysis of the vertebrate
(5,
22,
23) and the insect thin
filament (24). We have
deliberately avoided starting with a model and any potential model bias by
using a reference-free alignment procedure. The adaptation of conventional
procedures and their application to the structural study of the muscle thin
filament has been documented
(25). 相似文献
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.
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
8.
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
9.
Susan R. Wilson Christoph Peters Paul Saftig Dieter Br?mme 《The Journal of biological chemistry》2009,284(4):2584-2592
Cathepsin K is responsible for the degradation of type I collagen in
osteoclast-mediated bone resorption. Collagen fragments are known to be
biologically active in a number of cell types. Here, we investigate their
potential to regulate osteoclast activity. Mature murine osteoclasts were
seeded on type I collagen for actin ring assays or dentine discs for
resorption assays. Cells were treated with cathepsins K-, L-, or
MMP-1-predigested type I collagen or soluble bone fragments for 24 h. The
presence of actin rings was determined fluorescently by staining for actin. We
found that the percentage of osteoclasts displaying actin rings and the area
of resorbed dentine decreased significantly on addition of cathepsin
K-digested type I collagen or bone fragments, but not with cathepsin L or
MMP-1 digests. Counterintuitively, actin ring formation was found to decrease
in the presence of the cysteine proteinase inhibitor LHVS and in cathepsin
K-deficient osteoclasts. However, cathepsin L deficiency or the general MMP
inhibitor GM6001 had no effect on the presence of actin rings. Predigestion of
the collagen matrix with cathepsin K, but not by cathepsin L or MMP-1 resulted
in an increased actin ring presence in cathepsin K-deficient osteoclasts.
These studies suggest that cathepsin K interaction with type I collagen is
required for 1) the release of cryptic Arg-Gly-Asp motifs during the initial
attachment of osteoclasts and 2) termination of resorption via the creation of
autocrine signals originating from type I collagen degradation.Osteoclasts are monocyte-macrophage lineage-derived, large multinucleated
cells. They are the major bone resorbing cells, essential for bone turnover
and development. Active osteoclasts display characteristic membranes,
including the ruffled border, attachment zone, and the basolateral secretory
membrane. After attachment to bone, the ruffled border secretes enzymes and
protons enabling the solubilization and digestion of the bone matrix.
Osteoclasts express many proteases including cathepsins and matrix
metalloproteases
(MMPs)2 (for review
see Refs.
1-3).
However, it is the general consensus that cathepsin K (catK) is the major
bone-degrading enzyme
(4-7).Rapid cytoskeletal reorganization is essential for osteoclast function and
formation of the specialized membranes. Bone resorption occurs within the
sealing zone, which is formed by an actin ring structure. This can be
identified as a solid circular belt like formation and consists of an actin
filament core surrounded by actin-binding proteins such as talin,
α-actinin, and vinculin, which link matrix-recognizing integrins to the
cytoskeleton (8). The ruffled
border is contained within this structure. The actin ring is initiated by the
formation of podosomes, which represent dot-like actin structures of small
F-actin containing columns surrounded by proteins also found in focal adhesion
such as vinculin and paxillin
(9). It was previously thought
that the sealing zone was formed by the fusion of podosomes after the
osteoclast becomes activated
(10,
11), but it has since been
demonstrated that podosomes and the sealing zone are distinct structures
(12,
13). It should be noted that
bone resorption only occurs when the sealing zone is formed and the actin ring
is present (14).Osteoclasts bind and interact with the bone surface through specific
integrin receptors. The most abundant integrin present in osteoclasts is the
αvß3 receptor also known as the vitronectin receptor
(15,
16). This receptor attaches to
RGD sequence containing components of the bone matrix, e.g.
vitronectin, osteopontin, and type I collagen
(17-19).
This interaction enables the formation and regulation of the actin ring and
therefore osteoclast activity
(20-22).
It has previously been shown that soluble RGD containing peptides added to
cell supernatant are capable of inhibiting osteoclast binding and bone
resorption (18,
22-24).This study investigates the effect of collagen degradation fragments on
osteoclast activity. Soluble type I collagen and the bone powder of murine
long bones were subjected to digestion reactions by the cysteine proteases,
catK and catL, and the interstitial collagenase, MMP-1. The effect of these
degradation products on osteoclasts was investigated by monitoring actin ring
and resorption pit formation. We further investigated the role of cathepsins
using catK- and catL-deficient mice. Finally, we looked in more detail at the
effect of collagen, as a cell adhesion matrix, on osteoclast activity. 相似文献
10.
11.
Justin F. Shaffer Robert W. Kensler Samantha P. Harris 《The Journal of biological chemistry》2009,284(18):12318-12327
Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein
expressed in cardiac sarcomeres that is known to interact with myosin, titin,
and actin. cMyBP-C modulates actomyosin interactions in a
phosphorylation-dependent way, but it is unclear whether interactions with
myosin, titin, or actin are required for these effects. Here we show using
cosedimentation binding assays, that the 4 N-terminal domains of murine
cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation
constant (Kd) of ∼10 μm and a molar
binding ratio (Bmax) near 1.0, indicating 1:1 (mol/mol)
binding to actin. Electron microscopy and light scattering analyses show that
these domains cross-link F-actin filaments, implying multiple sites of
interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or
m-domain, reduced binding to actin (reduced Bmax) and
eliminated actin cross-linking. These results suggest that the N terminus of
cMyBP-C interacts with F-actin through multiple distinct binding sites and
that binding at one or more sites is reduced by phosphorylation. Reversible
interactions with actin could contribute to effects of cMyBP-C to increase
cross-bridge cycling.Cardiac myosin-binding protein C
(cMyBP-C)2
is a thick filament accessory protein that performs both structural and
regulatory functions within vertebrate sarcomeres. Both roles are likely to be
essential in deciphering how a growing number of mutations found in the
cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure
in a substantial number of the world''s population
(1,
2).Considerable progress has recently been made in determining the regulatory
functions of cMyBP-C and it is now apparent that cMyBP-C normally limits
cross-bridge cycling kinetics and is critical for cardiac function
(3-5).
Phosphorylation of cMyBP-C is essential for its regulatory effects because
elimination of phosphorylation sites (serine to alanine substitutions)
abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge
cycling kinetics and blunts cardiac responses to inotropic stimuli
(6). The substitutions further
impair cardiac function, reduce contractile reserve, and cause cardiac
hypertrophy in transgenic mice
(6,
7). By contrast, substitution
of aspartic acids at these sites to mimic constitutive phosphorylation is
benign or cardioprotective
(8).Although a role for cMyBP-C in modulating cross-bridge kinetics is
supported by several transgenic and knock-out mouse models
(6,
7,
9,
10), the precise mechanisms by
which cMyBP-C exerts these effects are not completely understood. For
instance, the unique regulatory motif or “m-domain” of cMyBP-C
binds to the S2 subfragment of myosin in vitro
(11) and binding is abolished
by PKA-mediated phosphorylation of the m-domain
(12). These observations have
led to the idea that (un)binding of the m-domain from myosin S2 mediates
PKA-induced increases in cross-bridge cycling kinetics. Consistent with this
idea, Calaghan and colleagues
(13) showed that S2 added to
transiently permeabilized myocytes increased their contractility, presumably
because added S2 displaced cMyBP-C from binding endogenous S2. However, other
reports indicate that cMyBP-C can influence actomyosin interactions through
mechanisms unrelated to S2 binding, because either purified cMyBP-C
(14) or recombinant N-terminal
domains of cMyBP-C (15)
affected acto-S1 filament sliding velocities and ATPase rates in the absence
of myosin S2. These results thus raise the possibility that interactions with
ligands other than myosin S2, such as actin or myosin S1, contribute to
effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge
cycling kinetics is supported by several studies that implicate the regulatory
m-domain or sequences near it in actin binding
(16-19).
cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and
consists of 11 repeating domains that bear homology to either Ig or
fibronectin-like folds. Domains are numbered sequentially from the N terminus
of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100
amino acids, is located between domains C1 and C2 and is phosphorylated on at
least 3 serine residues by PKA
(12). Although the precise
structure of the m-domain is not known, small angle x-ray scattering data
suggest that it is compact and folded in solution and is thus similar in size
and dimensions to the surrounding Ig domains
(20). Recombinant proteins
encompassing the m-domain and/or a combination of adjacent domains including
C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been
shown to bind actin (16,
18,
19).The purpose of the present study was to characterize binding interactions
of the N terminus of cMyBP-C with actin and to determine whether interactions
with actin are influenced by phosphorylation of the m-domain. Results
demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin
filaments with affinities similar to that reported for cMyBP-C binding to
myosin S2 (11). Furthermore,
actin binding was reduced by m-domain phosphorylation, suggesting that
reversible interactions of cMyBP-C with actin could contribute to modulation
of cross-bridge kinetics. 相似文献
12.
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
13.
14.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
15.
16.
Yvette R. Pittman Kimberly Kandl Marcus Lewis Louis Valente Terri Goss Kinzy 《The Journal of biological chemistry》2009,284(7):4739-4747
Eukaryotic translation elongation factor 1A (eEF1A) both shuttles
aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single
domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide
exchange factor eEF1Bα. We show that eEF1Bα has the ability to
disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα
F163, which binds in this domain, demonstrates effects on growth, eEF1A
binding, nucleotide exchange activity, and cell morphology. These phenotypes
can be partially restored by an intragenic W130A mutation. Furthermore, the
combination of F163A with the lethal K205A mutation restores viability by
drastically reducing eEF1Bα affinity for eEF1A. This also results in a
consistent increase in actin bundling and partially corrected morphology. The
consequences of the overlapping functions in this eEF1A domain and its unique
differences from the bacterial homologs provide a novel function for
eEF1Bα to balance the dual roles in actin bundling and protein
synthesis.The final step of gene expression takes place at the ribosome as mRNA is
translated into protein. In the yeast Saccharomyces cerevisiae,
elongation of the polypeptide chain requires the orchestrated action of three
soluble factors. The eukaryotic elongation factor 1
(eEF1)2 complex
delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating
ribosome (1). The eEF1A subunit
is a classic G-protein that acts as a “molecular switch” for the
active and inactive states based on whether GTP or GDP is bound, respectively
(2). Once an anticodon-codon
match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP
hydrolysis resulting in the release of inactive GDP-bound eEF1A from the
ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely
slow (3,
4), a guanine nucleotide
exchange factor (GEF) complex, eEF1B, is required
(5,
6). The yeast S.
cerevisiae eEF1B complex contains two subunits, the essential catalytic
subunit eEF1Bα (5) and
the non-essential subunit eEF1Bγ
(7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP:
Mg2+ and eEF1A:eEF1Bα C terminus:GDPNP
(8,
9) demonstrated a surprising
structural divergence from the bacterial EF-Tu-EF-Ts
(10) and mammalian
mitochondrial EF-Tumt-EF-Tsmt
(11). While the G-proteins
have a similar topology and consist of three well-defined domains, a striking
difference was observed in binding sites for their GEFs. The C terminus of
eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A,
creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts
and mammalian mitochondrial EF-Tsmt, make extensive contacts with
domain I and III of EF-Tu and EF-Tumt, respectively. The altered
binding interface of eEF1Bα to domain II of eEF1A is particularly
unexpected given the functions associated with domain II of eEF1A and EF-Tu.
The crystal structure of the EF-Tu:GDPNP:Phe-tRNAPhe complex
reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II
and tRNA to sustain stable contacts
(12). That eEF1A employs the
same aa-tRNA binding site is supported by genetic and biochemical data
(13-15).
Interestingly, eEF1Bα contacts many domain II eEF1A residues in the
region hypothesized to be involved in the binding of the aa-tRNA CCA end
(8). Because, the shared
binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is
significantly different between the eukaryotic and bacterial/mitochondrial
systems, eEF1Bα may play a unique function aside from guanine nucleotide
release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This
noncanonical function of eEF1A was initially observed in Dictyostelium
amoebae (16). It is
estimated that greater than 60% of Dictyostelium eEF1A is associated
with the actin cytoskeleton
(17). The eEF1A-actin
interaction is conserved among species from yeast to mammals, suggesting the
importance of eEF1A for cytoskeleton integrity. Using a unique genetic
approach, multiple eEF1A mutations were identified that altered cell growth
and morphology, and are deficient in bundling actin in vitro
(18,
19). Intriguingly, most
mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding
site. Previous studies have demonstrated that actin bundling by eEF1A is
significantly reduced in the presence of aa-tRNA while eEF1A bound to actin
filaments is not in complex with aa-tRNA
(20). Therefore, actin and
aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of
yeast eEF1A or actin-bundling deficient mutants do not affect translation
elongation (18,
19,
21), suggesting
eEF1A-dependent cytoskeletal organization is independent of its translation
elongation function (18,
20). Thus, while aa-tRNA
binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling
function associated with eEF1A domain II places greater importance on its
relationship with the “novel” binding interface between eEF1A
domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα
binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates
the equilibrium between actin and translation functions of eEF1A and is
perhaps the result of evolutionary selective pressure to balance the
eukaryotic-specific role of eEF1A in actin organization. Here, we present
kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the
importance of the interactions between domain II of eEF1A and eEF1Bα to
prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide
exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A
support that this complex formation is a determining factor for eEF1A-induced
actin organization. Interestingly, the F163A that reduces eEF1A affinity is an
intragenic suppressor of the lethal K205A eEF1Bα mutant that displays
increased affinity for eEF1A. This, along with a consistent change in the
actin bundling correlated with the affinity of eEF1Bα for eEF1A,
indicates that eEF1Bα is a balancer, directing eEF1A to translation
elongation and away from actin, and alterations in this balance result in
detrimental effects on cell growth and eEF1A function. 相似文献
17.
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
18.
Tushar K. Beuria Srinivas Mullapudi Eugenia Mileykovskaya Mahalakshmi Sadasivam William Dowhan William Margolin 《The Journal of biological chemistry》2009,284(21):14079-14086
Cytokinesis in bacteria depends upon the contractile Z ring, which is
composed of dynamic polymers of the tubulin homolog FtsZ as well as other
membrane-associated proteins such as FtsA, a homolog of actin that is required
for membrane attachment of the Z ring and its subsequent constriction. Here we
show that a previously characterized hypermorphic mutant FtsA (FtsA*)
partially disassembled FtsZ polymers in vitro. This effect was
strictly dependent on ATP or ADP binding to FtsA* and occurred at
substoichiometric levels relative to FtsZ, similar to cellular levels.
Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical
concentration for FtsZ assembly but was able to disassemble preformed FtsZ
polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination
of the inhibited FtsZ polymers revealed a transition from long, straight
polymers and polymer bundles to mainly short, curved protofilaments. These
results indicate that a bacterial actin, when activated by adenine
nucleotides, can modify the length distribution of bacterial tubulin polymers,
analogous to the effects of actin-depolymerizing factor/cofilin on
F-actin.Bacterial cell division requires a large number of proteins that colocalize
to form a putative protein machine at the cell membrane
(1). This machine, sometimes
called the divisome, recruits enzymes to synthesize the septum cell wall and
to initiate and coordinate the invagination of the cytoplasmic membrane (and
in Gram-negative bacteria, the outer membrane). The most widely conserved and
key protein for this process is FtsZ, a homolog of tubulin that forms a ring
structure called the Z ring, which marks the site of septum formation
(2,
3). Like tubulin, FtsZ
assembles into filaments with GTP but does not form microtubules
(4). The precise assembly state
and conformation of these FtsZ filaments at the division ring is not clear,
although recent electron tomography work suggests that the FtsZ ring consists
of multiple short filaments tethered to the membrane at discrete junctures
(5), which may represent points
along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One
of these, ZipA, is not well conserved but is an essential protein in E.
coli. ZipA binds to the C-terminal tail of FtsZ
(6–8),
and purified ZipA promotes bundling of FtsZ filaments in vitro
(9,
10). The other, FtsA, is also
essential in E. coli and is more widely conserved among bacterial
species. FtsA is a member of the HSP70/actin superfamily
(11,
12), and like ZipA, it
interacts with the C-terminal tail of FtsZ
(7,
13–15).
FtsA can self-associate (16,
17) and bind ATP
(12,
18), but reports of ATPase
activity vary, with Bacillus subtilis FtsA having high activity
(19) and Streptococcus
pneumoniae FtsA exhibiting no detectable activity
(20). There are no reports of
any other in vitro activities of FtsA, including effects on FtsZ
assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has
a number of key activities in the cell. It is required for recruitment of a
number of divisome proteins
(21,
22) and helps to tether the Z
ring to the membrane via a C-terminal membrane-targeting sequence
(23). FtsA, like ZipA and
other divisome proteins, is necessary to activate the contraction of the Z
ring (24,
25). In E. coli, the
FtsA:FtsZ ratio is crucial for proper cell division, with either too high or
too low a ratio inhibiting septum formation
(26,
27). This ratio is roughly
1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell
(28), which works out to
concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in
the protein can result in significant enhancement of divisome activity. For
example, the R286W mutation of FtsA, also called FtsA*, can substitute for the
native FtsA and divide the cell. However, this mutant FtsA causes E.
coli cells to divide at less than 80% of their normal length
(29) and allows efficient
division of E. coli cells in the absence of ZipA
(30), indicating that it has
gain-of-function activity. FtsA* and other hypermorphic mutations such as
E124A and I143L can also increase division activity in cells lacking other
essential divisome components
(31–33).
The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule,
allowing cell division to occur at higher ratios than with
WT2 FtsA. This may be
because the altered FtsA proteins self-associate more readily than WT FtsA,
which may cause different changes in FtsZ assembly state as compared with WT
FtsA (17,
34).In this study, we use an in vitro system with purified FtsZ and a
purified tagged version of FtsA* to elucidate the role of FtsA in activating
constriction of the Z ring in vivo. We show that FtsA*, at
physiological concentrations in the presence of ATP or ADP, has significant
effects on the assembly of FtsZ filaments. 相似文献
19.
20.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671