共查询到20条相似文献,搜索用时 296 毫秒
1.
Haihong Zong Claire C. Bastie Jun Xu Reinhard Fassler Kevin P. Campbell Irwin J. Kurland Jeffrey E. Pessin 《The Journal of biological chemistry》2009,284(7):4679-4688
Integrin receptor plays key roles in mediating both inside-out and
outside-in signaling between cells and the extracellular matrix. We have
observed that the tissue-specific loss of the integrin β1 subunit in
striated muscle results in a near complete loss of integrin β1 subunit
protein expression concomitant with a loss of talin and to a lesser extent, a
reduction in F-actin content. Muscle-specific integrin β1-deficient mice
had no significant difference in food intake, weight gain, fasting glucose,
and insulin levels with their littermate controls. However, dynamic analysis
of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a
44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose
clearance, respectively. The whole body insulin resistance resulted from a
specific inhibition of skeletal muscle glucose uptake and glycogen synthesis
without any significant effect on the insulin suppression of hepatic glucose
output or insulin-stimulated glucose uptake in adipose tissue. The reduction
in skeletal muscle insulin responsiveness occurred without any change in GLUT4
protein expression levels but was associated with an impairment of the
insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not
threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation
occurred concomitantly with a decrease in integrin-linked kinase expression
but with no change in the mTOR·Rictor·LST8 complex (mTORC2).
These data demonstrate an in vivo crucial role of integrin β1
signaling events in mediating cross-talk to that of insulin action.Integrin receptors are a large family of integral membrane proteins
composed of a single α and β subunit assembled into a heterodimeric
complex. There are 19 α and 8 β mammalian subunit isoforms that
combine to form 25 distinct α,β heterodimeric receptors
(1-5).
These receptors play multiple critical roles in conveying extracellular
signals to intracellular responses (outside-in signaling) as well as altering
extracellular matrix interactions based upon intracellular changes (inside-out
signaling). Despite the large overall number of integrin receptor complexes,
skeletal muscle integrin receptors are limited to seven α subunit
subtypes (α1, α3, α4, α5, α6, α7, and
αν subunits), all associated with the β1 integrin subunit
(6,
7).Several studies have suggested an important cross-talk between
extracellular matrix and insulin signaling. For example, engagement of β1
subunit containing integrin receptors was observed to increase
insulin-stimulated insulin receptor substrate
(IRS)2
phosphorylation, IRS-associated phosphatidylinositol 3-kinase, and activation
of protein kinase B/Akt
(8-11).
Integrin receptor regulation of focal adhesion kinase was reported to modulate
insulin stimulation of glycogen synthesis, glucose transport, and cytoskeleton
organization in cultured hepatocytes and myoblasts
(12,
13). Similarly, the
integrin-linked kinase (ILK) was suggested to function as one of several
potential upstream kinases that phosphorylate and activate Akt
(14-18).
In this regard small interfering RNA gene silencing of ILK in fibroblasts and
conditional ILK gene knockouts in macrophages resulted in a near complete
inhibition of insulin-stimulated Akt serine 473 (Ser-473) phosphorylation
concomitant with an inhibition of Akt activity and phosphorylation of Akt
downstream targets (19).
However, a complex composed of mTOR·Rictor·LST8 (termed mTORC2)
has been identified in several other studies as the Akt Ser-473 kinase
(20,
21). In addition to Ser-473,
Akt protein kinase activation also requires phosphorylation on threonine 308
Thr-30 by phosphoinositide-dependent protein kinase, PDK1
(22-24).In vivo, skeletal muscle is the primary tissue responsible for
postprandial (insulin-stimulated) glucose disposal that results from the
activation of signaling pathways leading to the translocation of the
insulin-responsive glucose transporter, GLUT4, from intracellular sites to the
cell surface membranes (25,
26). Dysregulation of any step
of this process in skeletal muscle results in a state of insulin resistance,
thereby predisposing an individual for the development of diabetes
(27-33).
Although studies described above have utilized a variety of tissue culture
cell systems to address the potential involvement of integrin receptor
signaling in insulin action, to date there has not been any investigation of
integrin function on insulin action or glucose homeostasis in vivo.
To address this issue, we have taken advantage of Cre-LoxP technology to
inactivate the β1 integrin receptor subunit gene in striated muscle. We
have observed that muscle creatine kinase-specific integrin β1 knock-out
(MCKItgβ1 KO) mice display a reduction of insulin-stimulated glucose
infusion rate and glucose clearance. The impairment of insulin-stimulated
skeletal muscle glucose uptake and glycogen synthesis resulted from a decrease
in Akt Ser-473 phosphorylation concomitant with a marked reduction in ILK
expression. Together, these data demonstrate an important cross-talk between
integrin receptor function and insulin action and suggests that ILK may
function as an Akt Ser-473 kinase in skeletal muscle. 相似文献
2.
Guorong Li Coralia Luna Jianming Qiu David L. Epstein Pedro Gonzalez 《The Journal of biological chemistry》2010,285(8):5461-5471
MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H2O2. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells. 相似文献
3.
4.
Yuya Sato Tomoya Isaji Michiko Tajiri Shumi Yoshida-Yamamoto Tsuyoshi Yoshinaka Toshiaki Somehara Tomohiko Fukuda Yoshinao Wada Jianguo Gu 《The Journal of biological chemistry》2009,284(18):11873-11881
Recently we reported that N-glycans on the β-propeller domain
of the integrin α5 subunit (S-3,4,5) are essential for α5β1
heterodimerization, expression, and cell adhesion. Herein to further
investigate which N-glycosylation site is the most important for the
biological function and regulation, we characterized the S-3,4,5 mutants in
detail. We found that site-4 is a key site that can be specifically modified
by N-acetylglucosaminyltransferase III (GnT-III). The introduction of
bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell
adhesion and migration on fibronectin, whereas overexpression of
N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration.
The phenomenon is similar to previous observations that the functions of the
wild-type α5 subunit were positively and negatively regulated by GnT-V
and GnT-III, respectively, suggesting that the α5 subunit could be
duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified
the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by
erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in
cell adhesion was consistently observed in the S-4,5 mutant but not in the
S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a
substantial decrease in erythroagglutinating phytohemagglutinin lectin
staining and suppression of cell spread induced by GnT-III compared with that
of either the site-3 single mutant or wild-type α5. These results, taken
together, strongly suggest that N-glycosylation of site-4 on the
α5 subunit is the most important site for its biological functions. To
our knowledge, this is the first demonstration that site-specific modification
of N-glycans by a glycosyltransferase results in functional
regulation.Glycosylation is a crucial post-translational modification of most secreted
and cell surface proteins (1).
Glycosylation is involved in a variety of physiological and pathological
events, including cell growth, migration, differentiation, and tumor invasion.
It is well known that glycans play important roles in cell-cell communication,
intracellular signal transduction, protein folding, and stability
(2,
3).Integrins comprise a family of receptors that are important for cell
adhesion. The major function of integrins is to connect cells to the
extracellular matrix, activate intracellular signaling pathways, and regulate
cytoskeletal formation (4).
Integrin α5β1 is well known as a fibronectin
(FN)3 receptor. The
interaction between integrin α5 and FN is essential for cell migration,
cell survival, and development
(5–8).
In addition, integrins are N-glycan carrier proteins. For example,
α5β1 integrin contains 14 and 12 putative N-glycosylation
sites on the α5 and β1 subunits, respectively. Several studies
suggest that N-glycosylation is essential for functional integrin
α5β1. When human fibroblasts were cultured in the presence of
1-deoxymannojirimycin, which prevents N-linked oligosaccharide
processing, immature α5β1 integrin appeared on the cell surface,
and FN-dependent adhesion was greatly reduced
(9). Treatment of purified
integrin α5β1 with N-glycosidase F, which cleaves between
the innermost N-acetylglucosamine (GlcNAc) and asparagine
N-glycan residues of N-linked glycoproteins, prevented the
inherent association between subunits and blocked α5β1 binding to
FN (10).A growing body of evidence indicates that the presence of the appropriate
oligosaccharide can modulate integrin activation.
N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the addition
of GlcNAc to mannose that is β1,4-linked to an underlying
N-acetylglucosamine, producing what is known as a
“bisecting” GlcNAc linkage as shown in
Fig. 1B. GnT-III is
generally regarded as a key glycosyltransferase in N-glycan
biosynthetic pathways and contributes to inhibition of metastasis. The
introduction of a bisecting GlcNAc catalyzed by GnT-III suppresses additional
processing and elongation of N-glycans. These reactions, which are
catalyzed in vitro by other glycosyltransferases, such as
N-acetylglucosaminyltransferase V (GnT-V), which catalyzes the
formation of β1,6 GlcNAc branching structures
(Fig. 1B) and plays
important roles in tumor metastasis, do not proceed because the enzymes cannot
utilize the bisected N-glycans as a substrate. Introduction of the
bisecting GlcNAc to integrin α5 by overexpression of GnT-III resulted in
decreased in ligand binding and down-regulation of cell adhesion and migration
(11–13).
Contrary to the functions of GnT-III, overexpression of GnT-V promoted
integrin α5β1-mediated cell migration on FN
(14). These observations
clearly demonstrate that the alteration of N-glycan structure
affected the biological functions of integrin α5β1. Similarly
characterization of the carbohydrate moieties in integrin α3β1 from
non-metastatic and metastatic human melanoma cell lines showed that expression
of β1,6 GlcNAc branched structures was higher in metastatic cells
compared with non-metastatic cells, confirming the notion that the β1,6
GlcNAc branched structure confers invasive and metastatic properties to cancer
cells. In fact, Partridge et al.
(15) reported that
GnT-V-modified N-glycans containing
poly-N-acetyllactosamine, the preferred ligand for galectin-3, on
surface receptors oppose their constitutive endocytosis, promoting
intracellular signaling and consequently cell migration and tumor
metastasis.Open in a separate windowFIGURE 1.Potential N-glycosylation sites on the α5 subunit and its
modification by GnT-III and GnT-V. A, schematic diagram of
potential N-glycosylation sites on the α5 subunit. Putative
N-glycosylation sites are indicated by triangles, and point
mutations are indicated by crosses (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q).
B, illustration of the reaction catalyzed by GnT-III and GnT-V.
Square, GlcNAc; circle, mannose. TM, transmembrane
domain.In addition, sialylation on the non-reducing terminus of N-glycans
of α5β1 integrin plays an important role in cell adhesion. Colon
adenocarcinomas express elevated levels of α2,6 sialylation and
increased activity of ST6GalI sialyltransferase. Elevated ST6GalI positively
correlated with metastasis and poor survival. Therefore, ST6GalI-mediated
hypersialylation likely plays a role in colorectal tumor invasion
(16,
17). In fact, oncogenic
ras up-regulated ST6GalI and, in turn, increased sialylation of
β1 integrin adhesion receptors in colon epithelial cells
(18). However, this is not
always the case. The expression of hyposialylated integrin α5β1 was
induced by phorbol esterstimulated differentiation in myeloid cells in which
the expression of the ST6GalI was down-regulated by the treatment, increasing
FN binding (19). A similar
phenomenon was also observed in hematopoietic or other epithelial cells. In
these cells, the increased sialylation of the β1 integrin subunit was
correlated with reduced adhesiveness and metastatic potential
(20–22).
In contrast, the enzymatic removal of α2,8-linked oligosialic acids from
the α5 integrin subunit inhibited cell adhesion to FN
(23). Collectively these
findings suggest that the interaction of integrin α5β1 with FN is
dependent on its N-glycosylation and the processing status of
N-glycans.Because integrin α5β1 contains multipotential
N-glycosylation sites, it is important to determine the sites that
are crucial for its biological function and regulation. Recently we found that
N-glycans on the β-propeller domain (sites 3, 4, and 5) of the
integrin α5 subunit are essential for α5β1
heterodimerization, cell surface expression, and biological function
(24). In this study, to
further investigate the underlying molecular mechanism of GnT-III-regulated
biological functions, we characterized the N-glycans on the α5
subunit in detail using genetic and biochemical approaches and found that
site-4 is a key site that can be specifically modified by GnT-III. 相似文献
5.
Yuya Sato Toshihiko Uemura Keisuke Morimitsu Ryoko Sato-Nishiuchi Ri-ichiroh Manabe Junichi Takagi Masashi Yamada Kiyotoshi Sekiguchi 《The Journal of biological chemistry》2009,284(21):14524-14536
Integrin α8β1 interacts with a variety of Arg-Gly-Asp
(RGD)-containing ligands in the extracellular matrix. Here, we examined the
binding activities of α8β1 integrin toward a panel of
RGD-containing ligands. Integrin α8β1 bound specifically to
nephronectin with an apparent dissociation constant of 0.28 ± 0.01
nm, but showed only marginal affinities for fibronectin and other
RGD-containing ligands. The high-affinity binding to α8β1 integrin
was fully reproduced with a recombinant nephronectin fragment derived from the
RGD-containing central “linker” segment. A series of deletion
mutants of the recombinant fragment identified the LFEIFEIER sequence on the
C-terminal side of the RGD motif as an auxiliary site required for
high-affinity binding to α8β1 integrin. Alanine scanning
mutagenesis within the LFEIFEIER sequence defined the EIE sequence as a
critical motif ensuring the high-affinity integrin-ligand interaction.
Although a synthetic LFEIFEIER peptide failed to inhibit the binding of
α8β1 integrin to nephronectin, a longer peptide containing both the
RGD motif and the LFEIFEIER sequence was strongly inhibitory, and was
∼2,000-fold more potent than a peptide containing only the RGD motif.
Furthermore, trans-complementation assays using recombinant fragments
containing either the RGD motif or LFEIFEIER sequence revealed a clear
synergism in the binding to α8β1 integrin. Taken together, these
results indicate that the specific high-affinity binding of nephronectin to
α8β1 integrin is achieved by bipartite interaction of the integrin
with the RGD motif and LFEIFEIER sequence, with the latter serving as a
synergy site that greatly potentiates the RGD-driven integrin-ligand
interaction but has only marginal activity to secure the interaction by
itself.Integrins are a family of adhesion receptors that interact with a variety
of extracellular ligands, typically cell-adhesive proteins in the
extracellular matrix
(ECM).2 They play
mandatory roles in embryonic development and the maintenance of tissue
architectures by providing essential links between cells and the ECM
(1). Integrins are composed of
two non-covalently associated subunits, termed α and β. In mammals,
18 α and 8 β subunits have been identified, and combinations of
these subunits give rise to at least 24 distinct integrin heterodimers. Based
on their ligand-binding specificities, ECM-binding integrins are classified
into three groups, namely laminin-, collagen- and RGD-binding integrins
(2,
3), of which the RGD-binding
integrins have been most extensively investigated. The RGD-binding integrins
include α5β1, α8β1, αIIbβ3, and
αV-containing integrins, and have been shown to interact with a variety
of ECM ligands, such as fibronectin and vitronectin, with distinct binding
specificities.The α8 integrin subunit was originally identified in chick nerves
(4). Integrin α8β1
is expressed in the metanephric mesenchyme and plays a crucial role in
epithelial-mesenchymal interactions during the early stages of kidney
morphogenesis. Disruption of the α8 gene in mice was found to be
associated with severe defects in kidney morphogenesis
(5) and stereocilia development
(6). To date, α8β1
integrin has been shown to bind to fibronectin, vitronectin, osteopontin,
latency-associated peptide of transforming growth factor-β1, tenascin-W,
and nephronectin (also named POEM)
(7–13),
among which nephronectin is believed to be an α8β1 integrin ligand
involved in kidney development
(10).Nephronectin is one of the basement membrane proteins whose expression and
localization patterns are restricted in a tissue-specific and developmentally
regulated manner (10,
11). Nephronectin consists of
five epidermal growth factor-like repeats, a linker segment containing the RGD
cell-adhesive motif (designated RGD-linker) and a meprin-A5 protein-receptor
protein-tyrosine phosphatase μ (MAM) domain (see
Fig. 3A). Although the
physiological functions of nephronectin remain only poorly understood, it is
thought to play a role in epithelial-mesenchymal interactions through binding
to α8β1 integrin, thereby transmitting signals from the epithelium
to the mesenchyme across the basement membrane
(10). Recently, mice deficient
in nephronectin expression were produced by homologous recombination
(14). These
nephronectin-deficient mice frequently displayed kidney agenesis, a phenotype
reminiscent of α8 integrin knock-out mice
(14), despite the fact that
other RGD-containing ligands, including fibronectin and osteopontin, were
expressed in the embryonic kidneys
(9,
15). The failure of the other
RGD-containing ligands to compensate for the deficiency of nephronectin in the
developing kidneys suggests that nephronectin is an indispensable
α8β1 ligand that plays a mandatory role in epithelial-mesenchymal
interactions during kidney development.Open in a separate windowFIGURE 3.Binding activities of α8β1 integrin to nephronectin and its
fragments. A, schematic diagrams of full-length nephronectin
(NN) and its fragments. RGD-linker and RGD-linker
(GST), the central RGD-containing linker segments expressed in
mammalian and bacterial expression systems, respectively; PRGDV, a
short RGD-containing peptide modeled after nephronectin and expressed as a GST
fusion protein (see Fig.
4A for the peptide sequence). The arrowheads
indicate the positions of the RGD motif. B, purified recombinant
proteins were analyzed by SDS-PAGE in 7–15% gradient (left and
center panels) and 12% (right panels) gels, followed by
Coomassie Brilliant Blue (CBB) staining, immunoblotting with an
anti-FLAG mAb, or lectin blotting with PNA. The quantities of proteins loaded
were: 0.5 μg (for Coomassie Brilliant Blue staining) and 0.1 μg (for
blotting with anti-FLAG and PNA) in the left and center
panels;1 μg in the right panel. C, recombinant proteins (10
nm) were coated on microtiter plates and assessed for their binding
activities toward α8β1 integrin (10 nm) in the presence
of 1 mm Mn2+. The backgrounds were subtracted as
described in the legend to Fig.
2. The results represent the mean ± S.D. of triplicate
determinations. D, titration curves of α8β1 integrin bound
to full-length nephronectin (NN, closed squares), the RGD-linker
segments expressed in 293F cells (RGD-linker, closed triangles) and
E. coli (RGD-linker (GST), open
triangles), the MAM domain (MAM, closed diamonds), and the PRGDV
peptide expressed as a GST fusion protein in E. coli (PRGDV
(GST), open circles). The assays were performed as described
in the legend to Fig.
2B. The results represent the means of duplicate
determinations.Although ligand recognition by RGD-binding integrins is primarily
determined by the RGD motif in the ligands, it is the residues outside the RGD
motif that define the binding specificities and affinities toward individual
integrins (16,
17). For example,
α5β1 integrin specifically binds to fibronectin among the many
RGD-containing ligands, and requires not only the RGD motif in the 10th type
III repeat but also the so-called “synergy site” within the
preceding 9th type III repeat for fibronectin recognition
(18). Recently, DiCara et
al. (19) demonstrated
that the high-affinity binding of αVβ6 integrin to its natural
ligands, e.g. foot-and-mouth disease virus, requires the RGD motif
immediately followed by a Leu-Xaa-Xaa-Leu/Ile sequence, which forms a helix to
align the two conserved hydrophobic residues along the length of the helix.
Given the presence of many naturally occurring RGD-containing ligands, it is
conceivable that the specificities of the RGD-binding integrins are dictated
by the sequences flanking the RGD motif or those in neighboring domains that
come into close proximity with the RGD motif in the intact ligand proteins.
However, the preferences of α8β1 integrin for RGD-containing
ligands and how it secures its high-affinity binding toward its preferred
ligands remain unknown.In the present study, we investigated the binding specificities of
α8β1 integrin toward a panel of RGD-containing cell-adhesive
proteins. Our data reveal that nephronectin is a preferred ligand for
α8β1 integrin, and that a LFEIFEIER sequence on the C-terminal side
of its RGD motif serves as a synergy site to ensure the specific high-affinity
binding of nephronectin to α8β1 integrin. 相似文献
6.
Norihisa Nishimichi Fumiko Higashikawa Hiromi H. Kinoh Yoshiko Tateishi Haruo Matsuda Yasuyuki Yokosaki 《The Journal of biological chemistry》2009,284(22):14769-14776
Osteopontin (OPN) is a cytokine and ligand for multiple members of the
integrin family. OPN undergoes the in vivo polymerization catalyzed
by cross-linking enzyme transglutaminase 2, which consequently increases the
bioactivity through enhanced interaction with integrins. The integrin
α9β1, highly expressed on neutrophils, binds to the sequence
SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence
appears to be cryptic in intact OPN because α9β1 does not recognize
intact OPN. Because transglutaminase 2-catalyzed polymers change their
physical and chemical properties, we hypothesized that the SVVYGLR site might
also be exposed on polymeric OPN. As expected, α9β1 turned into a
receptor for polymeric OPN, a result obtained by cell adhesion and migration
assays with α9-transfected cells and by detection of direct binding of
recombinant soluble α9β1 with colorimetry and surface plasmon
resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a
ligand for α9β1, has been reported to attract neutrophils, we next
examined migration of neutrophils to polymeric OPN using time-lapse
microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which
was clearly inhibited by anti-α9β1 antibody. Unexpectedly,
mutagenesis studies showed that α9β1 bound to polymeric OPN
independently of the SVVYGLR sequence, and further, SVVYGLR sequence of
polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize
polymeric OPN. These results demonstrate that polymerization of OPN generates
a novel α9β1-binding site and that the interaction of this site
with the α9β1 integrin is critical to the neutrophil chemotaxis
induced by polymeric OPN.Acidic phosphorylated secreted glycoprotein osteopontin
(OPN),4 known as a
cytokine, has multiple functions, including roles in tissue remodeling,
fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis
(1–3).
OPN is also an integrin ligand. At least nine integrins can function as OPN
receptors. α5β1, α8β1, αvβ1, αvβ3,
αvβ5 (1), and
αvβ6 (4) recognize
the linear tripeptide RGD, and α9β1, α4β1, and
α4β7 recognize the sequence, SVVYGLR
(5), adjacent to RGD but only
after OPN has been cleaved by the protease, thrombin
(Fig. 1).Open in a separate windowFIGURE 1.Schematic diagram of OPN. Two integrin-binding sites
(boxed), a thrombin cleavage site (arrow), and a putative
transglutamination site (circled) are shown. The term
thrombin-cleaved nOPN is defined as in the figure.The overlap of receptors for OPN does not necessarily mean that these
integrins play redundant roles in cellular responses to OPN because the
patterns of integrin expression and utilization vary widely among cell types.
In addition, interactions of different integrins with a single ligand can
exert distinct effects on cell behavior in a single cell type. For example, we
have previously reported that signals by ligation of αvβ3,
αvβ6, or α9β1 to a single ligand, tenascin-C,
differently affected cell adhesion, spreading, and proliferation of the colon
cancer cell line, SW480 (6).
Furthermore, intact OPN or thrombin- or matrix metalloproteinase-cleaved OPN
interact with distinct subsets of integrins and exhibit distinct effects on
cell behavior (4,
7,
8). Collectively, some of the
functional diversity of OPN could be attributed to this multiplicity of
receptors and responses. We have recently shown that polymerization of OPN
results in enhanced biological activity
(9). We thus set out to
determine whether polymerized OPN exerts its effects through unique
interactions with integrins.OPN is polymerized by transglutaminase 2 (TG2, EC 2.3.2.13)
(10) that catalyzes formation
of isopeptide cross-links between glutamine and lysine residues in substrate
proteins (11) including OPN.
Polymeric OPN has been identified in vivo in bone
(12) and calcified aorta
(13). We have previously
reported that upon polymerization, OPN displays increased integrin binding
accompanied by enhanced cell adhesion, spreading, migration, and focal contact
formation (9). However, very
little is known about how polymeric OPN induces its biological effects.Integrin α9β1, highly expressed on neutrophils
(14), does not act as a
receptor for intact OPN but does bind to an N-terminal fragment of OPN (nOPN)
that is generated by thrombin cleavage
(15) through the new
C-terminal sequence, SVVYGLR. Protein polymerization can expose otherwise
cryptic domains (16), so we
hypothesized that the SVVYGLR site might be exposed upon polymerization and
serve as a binding site for α9β1. In the present study, we
demonstrate that α9β1 is indeed a receptor for polymeric OPN and
that neutrophil migration induced by polymeric OPN is largely mediated by this
interaction. However, mutational analysis and antibody studies demonstrate
that this interaction does not involve the SVVYGLR site, suggesting the
presence of de novo binding site in polymeric OPN. 相似文献
7.
8.
Haipeng Cheng Kulandaivelu S. Vetrivel Renaldo C. Drisdel Xavier Meckler Ping Gong Jae Yoon Leem Tong Li Meghan Carter Ying Chen Phuong Nguyen Takeshi Iwatsubo Taisuke Tomita Philip C. Wong William N. Green Maria Z. Kounnas Gopal Thinakaran 《The Journal of biological chemistry》2009,284(3):1373-1384
Proteolytic processing of amyloid precursor protein (APP) by β- and
γ-secretases generates β-amyloid (Aβ) peptides, which
accumulate in the brains of individuals affected by Alzheimer disease.
Detergent-resistant membrane microdomains (DRM) rich in cholesterol and
sphingolipid, termed lipid rafts, have been implicated in Aβ production.
Previously, we and others reported that the four integral subunits of the
γ-secretase associate with DRM. In this study we investigated the
mechanisms underlying DRM association of γ-secretase subunits. We report
that in cultured cells and in brain the γ-secretase subunits nicastrin
and APH-1 undergo S-palmitoylation, the post-translational covalent
attachment of the long chain fatty acid palmitate common in lipid
raft-associated proteins. By mutagenesis we show that nicastrin is
S-palmitoylated at Cys689, and APH-1 is
S-palmitoylated at Cys182 and Cys245.
S-Palmitoylation-defective nicastrin and APH-1 form stable
γ-secretase complexes when expressed in knock-out fibroblasts lacking
wild type subunits, suggesting that S-palmitoylation is not essential
for γ-secretase assembly. Nevertheless, fractionation studies show that
S-palmitoylation contributes to DRM association of nicastrin and
APH-1. Moreover, pulse-chase analyses reveal that S-palmitoylation is
important for nascent polypeptide stability of both proteins. Co-expression of
S-palmitoylation-deficient nicastrin and APH-1 in cultured cells
neither affects Aβ40, Aβ42, and AICD production, nor intramembrane
processing of Notch and N-cadherin. Our findings suggest that
S-palmitoylation plays a role in stability and raft localization of
nicastrin and APH-1, but does not directly modulate γ-secretase
processing of APP and other substrates.Alzheimer disease is the most common among neurodegenerative diseases that
cause dementia. This debilitating disorder is pathologically characterized by
the cerebral deposition of 39–42 amino acid peptides termed Aβ,
which are generated by proteolytic processing of amyloid precursor protein
(APP)2 by β- and
γ-secretases (1,
2). The β-site APP
cleavage enzyme 1 cleaves full-length APP within its luminal domain to
generate a secreted ectodomain leaving behind a C-terminal fragment
(β-CTF). γ-Secretase cleaves β-CTF within the transmembrane
domain to release Aβ and APP intracellular
C-terminal domain (AICD). γ-Secretase is a
multiprotein complex, comprising at least four subunits: presenilins (PS1 and
PS2), nicastrin, APH-1, and PEN-2 for its activity
(3). PS1 is synthesized as a
42–43-kDa polypeptide and undergoes highly regulated endoproteolytic
processing within the large cytoplasmic loop domain connecting putative
transmembrane segments 6 and 7 to generate stable N-terminal (NTF) and
C-terminal fragments (CTF) by an uncharacterized proteolytic activity
(4). This endoproteolytic event
has been identified as the activation step in the process of PS1 maturation as
it assembles with other γ-secretase subunits
(3). Nicastrin is a heavily
glycosylated type I membrane protein with a large ectodomain that has been
proposed to function in substrate recognition and binding
(5), but this putative function
has not been confirmed by others
(6). APH-1 is a
seven-transmembrane protein encoded by two human or three rodent genes that
are alternatively spliced (7).
Although PS1 (or PS2), nicastrin, APH-1, and PEN-2 are sufficient for
γ-secretase processing of APP, a type I membrane protein, termed p23
(also referred toTMP21), was recently identified as a γ-secretase
component that modulates γ-secretase activity and regulates secretory
trafficking of APP (8,
9).A growing number of type I integral membrane proteins has been identified
as γ-secretase substrates within the last few years, including Notch1
homologues, Notch ligands, Delta and Jagged, cell adhesion receptors N- and
E-cadherins, low density lipoprotein receptor-related protein, ErbB-4, netrin
receptor DCC, and others (10).
Mounting evidence suggests that APP processing occurs within cholesterol- and
sphingolipid-enriched lipid rafts, which are biochemically defined as
detergentresistant membrane microdomains (DRM)
(11,
12). Previously we reported
that each of the γ-secretase subunits localizes in lipid rafts in
post-Golgi and endosome membranes enriched in syntaxin 6
(13). Moreover, loss of
γ-secretase activity by gene deletion or exposure to γ-secretase
inhibitors results in the accumulation of APP CTFs in lipid rafts indicating
that cleavage of APP CTFs likely occurs in raft microdomains
(14). In contrast, CTFs
derived from Notch1, Jagged2, N-cadherin, and DCC are processed by
γ-secretase in non-raft membranes
(14). The mechanisms
underlying association of γ-secretase subunits with lipid rafts need
further clarification to elucidate spatial segregation of amyloidogenic
processing of APP in membrane microdomains.Post-translational S-palmitoylation is increasingly recognized as
a potential mechanism for regulating raft association, stability,
intracellular trafficking, and function of several cytosolic and transmembrane
proteins
(15–17).
S-palmitoylation refers to the addition of 16-carbon palmitoyl moiety
to certain cysteine residues through thioester linkage. Cysteines close to
transmembrane domains or membrane-associated domains in non-integral membrane
proteins are preferred S-palmitoylation sites, although no conserved
motif has been identified
(18). Palmitoylation modifies
numerous neuronal proteins, including postsynaptic density protein PSD-95
(19),
a-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid receptors
(20), nicotinic α7
receptors (21), neuronal
t-SNAREs SNAP-25, synaptobrevin 2 and synaptogagmin
(22,
23), neuronal
growth-associated protein GAP-43
(24), protein kinase CLICK-III
(CL3)/CaMKIγ (25),
β-secretase (26), and
Huntingtin (27). Although
palmitoylation can occur in vitro without the involvement of an
enzyme, a family of palmitoyltransferases that specifically catalyze
S-palmitoylation has been identified
(28,
29).In this study, we have identified S-palmitoylation of
γ-secretase subunits nicastrin and APH-1, and characterized its role on
DRM association, protein stability, and γ-secretase enzyme activities.
We show that nicastrin is S-palmitoylated at Cys689, and
APH-1 at Cys182 and Cys245. Mutagenesis of
palmitoylation sites results in increased degradation of nascent nicastrin and
APH-1 polypeptides and reduced association with DRM. Nevertheless, in cultured
cells overexpression of S-palmitoylation-deficient nicastrin and
APH-1 does not modulate γ-secretase processing of APP or other
substrates. 相似文献
9.
10.
Johann Schredelseker Anamika Dayal Thorsten Schwerte Clara Franzini-Armstrong Manfred Grabner 《The Journal of biological chemistry》2009,284(2):1242-1251
The paralyzed zebrafish strain relaxed carries a null mutation for
the skeletal muscle dihydropyridine receptor (DHPR) β1a
subunit. Lack of β1a results in (i) reduced membrane
expression of the pore forming DHPR α1S subunit, (ii)
elimination of α1S charge movement, and (iii) impediment of
arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine
receptor (RyR1), a structural prerequisite for skeletal muscle-type
excitation-contraction (EC) coupling. In this study we used relaxed
larvae and isolated myotubes as expression systems to discriminate specific
functions of β1a from rather general functions of β
isoforms. Zebrafish and mammalian β1a subunits quantitatively
restored α1S triad targeting and charge movement as well as
intracellular Ca2+ release, allowed arrangement of DHPRs in
tetrads, and most strikingly recovered a fully motile phenotype in
relaxed larvae. Interestingly, the cardiac/neuronal
β2a as the phylogenetically closest, and the ancestral
housefly βM as the most distant isoform to β1a
also completely recovered α1S triad expression and charge
movement. However, both revealed drastically impaired intracellular
Ca2+ transients and very limited tetrad formation compared with
β1a. Consequently, larval motility was either only partially
restored (β2a-injected larvae) or not restored at all
(βM). Thus, our results indicate that triad expression and
facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common
features of all tested β subunits, whereas the efficient arrangement of
DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the
β1a isoform. Consequently, we postulate a model that presents
β1a as an allosteric modifier of α1S
conformation enabling skeletal muscle-type EC coupling.Excitation-contraction
(EC)3 coupling in
skeletal muscle is critically dependent on the close interaction of two
distinct Ca2+ channels. Membrane depolarizations of the myotube are
sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the
sarcolemma, leading to a rearrangement of charged amino acids (charge
movement) in the transmembrane segments S4 of the pore-forming DHPR
α1S subunit
(1,
2). This conformational change
induces via protein-protein interaction
(3,
4) the opening of the
sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca2+
influx through the DHPR (5).
The release of Ca2+ from the sarcoplasmic reticulum via RyR1
consequently induces muscle contraction. The protein-protein interaction
mechanism between DHPR and RyR1 requires correct ultrastructural targeting of
both channels. In Ca2+ release units (triads and peripheral
couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled
to every other RyR1 and hence are geometrically arranged following the
RyR-specific orthogonal arrays
(6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of
the voltage-sensing and pore-forming α1S subunit and
auxiliary subunits β1a, α2δ-1, and
γ1 (7). While
gene knock-out of the DHPR γ1 subunit
(8,
9) and small interfering RNA
knockdown of the DHPR α2δ-1 subunit
(10-12)
have indicated that neither subunit is essential for coupling of the DHPR with
RyR1, the lack of the α1S or of the intracellular
β1a subunit is incompatible with EC coupling and accordingly
null model mice die perinatally due to asphyxia
(13,
14). β subunits of
voltage-gated Ca2+ channels were repeatedly shown to be responsible
for the facilitation of α1 membrane insertion and to be
potent modulators of α1 current kinetics and voltage
dependence (15,
16). Whether the loss of EC
coupling in β1-null mice was caused by decreased DHPR membrane
expression or by the lack of a putative specific contribution of the β
subunit to the skeletal muscle EC coupling apparatus
(17,
18) was not clearly resolved.
Recently, other β-functions were identified in skeletal muscle using the
β1-null mutant zebrafish relaxed
(19,
20). Like the
β1-knock-out mouse
(14) zebrafish
relaxed is characterized by complete paralysis of skeletal muscle
(21,
22). While
β1-knock-out mouse pups die immediately after birth due to
respiratory paralysis (14),
larvae of relaxed are able to survive for several days because of
oxygen and metabolite diffusion via the skin
(23). Using highly
differentiated myotubes that are easy to isolate from these larvae, the lack
of EC coupling could be described by quantitative immunocytochemistry as a
moderate ∼50% reduction of α1S membrane expression
although α1S charge movement was nearly absent, and, most
strikingly, as the complete lack of the arrangement of DHPRs in tetrads
(19). Thus, in skeletal muscle
the β subunit enables EC coupling by (i) enhancing α1S
membrane targeting, (ii) facilitating α1S charge movement,
and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal
muscle β1a and which ones are rather general properties of
Ca2+ channel β subunits. Previous reconstitution studies made
in the β1-null mouse system
(24,
25) using different β
subunit constructs (26) did
not allow differentiation between β-induced enhancement of non-functional
α1S membrane expression and the facilitation of
α1S charge movement, due to the lack of information on
α1S triad expression levels. Furthermore, the β-induced
arrangement of DHPRs in tetrads was not detected as no ultrastructural
information was obtained.In the present study, we established zebrafish mutant relaxed as
an expression system to test different β subunits for their ability to
restore skeletal muscle EC coupling. Using isolated myotubes for in
vitro experiments (19,
27) and complete larvae for
in vivo expression studies
(28-31)
and freeze-fracture electron microscopy, a clear differentiation between the
major functional roles of β subunits was feasible in the zebrafish
system. The cloned zebrafish β1a and a mammalian (rabbit)
β1a were shown to completely restore all parameters of EC
coupling when expressed in relaxed myotubes and larvae. However, the
phylogenetically closest β subunit to β1a, the
cardiac/neuronal isoform β2a from rat, as well as the
ancestral βM isoform from the housefly (Musca
domestica), could recover functional α1S membrane
insertion, but led to very restricted tetrad formation when compared with
β1a, and thus to impaired DHPR-RyR1 coupling. This impairment
caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional
α1S membrane expression is a common function of all the
tested β subunits, from β1a to even the most distant
βM, whereas the effective formation of tetrads and thus proper
skeletal muscle EC coupling is an exclusive function of the skeletal muscle
β1a subunit. In context with previous studies, our results
suggest a model according to which β1a acts as an allosteric
modifier of α1S conformation. Only in the presence of
β1a, the α1S subunit is properly folded to
allow RyR1 anchoring and thus skeletal muscle-type EC coupling. 相似文献
11.
Karen Vanhoorelbeke Simon F. De Meyer Inge Pareyn Chantal Melchior Sebastien Plan?on Christiane Margue Olivier Pradier Pierre Fondu Nelly Kieffer Timothy A. Springer Hans Deckmyn 《The Journal of biological chemistry》2009,284(22):14914-14920
Three heterozygous mutations were identified in the genes encoding platelet
integrin receptor αIIbβ3 in a patient with an ill defined platelet
disorder: one in the β3 gene (S527F) and two in the αIIb gene
(R512W and L841M). Five stable Chinese hamster ovary cell lines were
constructed expressing recombinant αIIbβ3 receptors bearing the
individual R512W, L841M, or S527F mutation; both the R512W and L841M
mutations; or all three mutations. All receptors were expressed on the cell
surface, and mutations R512W and L841M had no effect on integrin function.
Interestingly, the β3 S527F mutation produced a constitutively active
receptor. Indeed, both fibrinogen and the ligand-mimetic antibody PAC-1 bound
to non-activated αIIbβ3 receptors carrying the S527F mutation,
indicating that the conformation of this receptor was altered and corresponded
to the high affinity ligand binding state. In addition, the conformational
change induced by S527F was evident from basal anti-ligand-induced binding
site antibody binding to the receptor. A molecular model bearing this mutation
was constructed based on the crystal structure of αIIbβ3 and
revealed that the S527F mutation, situated in the third integrin epidermal
growth factor-like (I-EGF3) domain, hindered the αIIbβ3 receptor
from adopting a wild type-like bent conformation. Movement of I-EGF3 into a
cleft in the bent conformation may be hampered both by steric hindrance
between Phe527 in β3 and the calf-1 domain in αIIb and
by decreased flexibility between I-EGF2 and I-EGF3.The platelet receptor αIIbβ3 belongs to the family of integrin
receptors that consist of noncovalently linked α/β-heterodimers.
They are cell-surface receptors that play a role in cell-cell and cell-matrix
interactions. Under resting conditions, integrin receptors adopt the low
affinity conformation and do not interact with their ligands. Inside-out
signaling turns the receptor into a high affinity conformation capable of
ligand binding. Ligand binding itself induces additional conformational
changes resulting in exposure of neoantigenic sites called ligand-induced
binding sites (LIBS)3
and generates in turn outside-in signaling, which triggers a range of
downstream signals (1,
2).Integrin αIIbβ3 is expressed on platelets and megakaryocytes. In
flowing blood under resting conditions, αIIbβ3 does not interact
with its ligand fibrinogen. When a blood vessel is damaged, platelets adhere
at sites of vascular injury and become activated. As a consequence,
αIIbβ3 adopts the high affinity conformation and binds fibrinogen.
This results in platelet aggregation and thrombus formation, which eventually
will stop the bleeding (3).The topology of integrins comprises an extracellular, globular, N-terminal
ligand-binding head domain (the β-propeller domain in the αIIb
chain and the βI domain in the β3 chain) standing on two long legs
or stalks (consisting of thigh, calf-1, and calf-2 domains in the αIIb
chain and hybrid, plexin/semaphorin/integrin (PSI), four integrin endothelial
growth factor-like (I-EGF), and β-tail domains in the β3 chain),
followed by transmembrane and cytoplasmic domains
(1,
2). X-ray crystal structures of
the extracellular domain of non-activated αVβ3 revealed that the
legs are severely bent, putting the head domain next to the membrane-proximal
portions of the legs (4,
5). The bending occurs between
I-EGF1 and I-EGF2 in the β-subunit and between the thigh and calf-1
domains in the α-subunit. This bent conformation represents the low
affinity state of the receptor. The high affinity state of the receptor is
induced by activation and is associated with a large-scale conformational
rearrangement in which the integrin extends with a switchblade-like motion
(2). Recently, the crystal
structure of the entire extracellular domain of αIIbβ3 in its low
affinity conformation was resolved and revealed that this integrin also adopts
the bent conformation under resting conditions
(6). Structural rearrangements
in αIIbβ3 between the bent and extended conformations are similar
to what has been reported for other integrins
(7).We report here that the S527F mutation in the I-EGF3 region of the β3
polypeptide chain of the αIIbβ3 receptor induces a constitutively
active receptor adopting an extended high affinity conformation. This was
evidenced by spontaneous PAC-1, fibrinogen, and anti-LIBS antibody binding.
These data were further corroborated by modeling the replacement of
Ser527 with Phe in the crystal structure of the extracellular
domain of αIIbβ3. In this model, the S527F mutation decreases the
flexibility of I-EGF3 and appears to prevent movement of the lower β-leg
into the cleft between the upper β-leg and the lower α-leg. As a
consequence, formation of the bent conformation of the non-activated receptor
is hampered. 相似文献
12.
Control of TANK-binding Kinase 1-mediated Signaling by the
��134.5 Protein of Herpes Simplex Virus
1
Dustin Verpooten Yijie Ma Songwang Hou Zhipeng Yan Bin He 《The Journal of biological chemistry》2009,284(2):1097-1105
TANK-binding kinase 1 (TBK1) is a key component of Toll-like
receptor-dependent and -independent signaling pathways. In response to
microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and
cytokine expression. Here we show that TBK1 is a novel target of the
γ134.5 protein, a virulence factor whose expression is
regulated in a temporal fashion. Remarkably, the γ134.5
protein is required to inhibit IRF3 phosphorylation, nuclear translocation,
and the induction of antiviral genes in infected cells. When expressed in
mammalian cells, the γ134.5 protein forms complexes with TBK1
and disrupts the interaction of TBK1 and IRF3, which prevents the induction of
interferon and interferon-stimulated gene promoters. Down-regulation of TBK1
requires the amino-terminal domain. In addition, unlike wild type virus, a
herpes simplex virus mutant lacking γ134.5 replicates
efficiently in TBK1-/- cells but not in TBK1+/+ cells.
Addition of exogenous interferon restores the antiviral activity in both
TBK1-/- and TBK+/+ cells. Hence, control of
TBK1-mediated cell signaling by the γ134.5 protein
contributes to herpes simplex virus infection. These results reveal that TBK1
plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1
(HSV-1)3 is a large
DNA virus that establishes latent or lytic infection, in which the virus
triggers innate immune responses. In HSV-infected cells, a number of antiviral
mechanisms operate in a cell type- and time-dependent manner
(1). In response to
double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor
TIR domain-containing adaptor inducing IFN-β and stimulates cytokine
expression (2,
3). In the cytoplasm, RNA
helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma
differentiation associated gene 5) recognize intracellular viral
5′-triphosphate RNA or dsRNA
(2,
4). Furthermore, a
DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded
DNA in the cytoplasm and induces cytokine expression
(5). There is also evidence
that viral entry induces antiviral programs independent of TLR and RIG-I
pathways (6). While recognizing
distinct viral components, these innate immune pathways relay signals to the
two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB
kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate
IRF3 (interferon regulatory factor 3), as well as the closely related IRF7,
which translocates to the nucleus and induces antiviral genes, such as
interferon-α/β and ISG56 (interferon-stimulated gene 56)
(7,
8). TBK1 is constitutively
expressed, whereas IKKi is engaged as an inducible gene product of innate
immune signaling (9,
10). IRF3 activation is
attenuated in TBK1-deficient but not in IKKi-deficient cells
(11,
12). Its activation is
completely abolished in double-deficient cells
(12), suggesting a partially
redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates
the STAT-signaling pathway
(13). TBK1/IKKi interacts with
several proteins, such as TRAF family member-associated NF-κB activator
(TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor
(SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and
secretory protein 5 (Sec5) in host cells
(5,
14–18).
These interactions are thought to regulate TBK1/IKKi, which delineates innate
as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the
induction of antiviral immunity. When treated with UV or cycloheximide, HSV
induces an array of antiviral genes in human lung fibroblasts
(19,
20). Furthermore, an HSV
mutant, with deletion in immediate early protein ICP0, induces ISG56
expression (21). Accordingly,
expression of ICP0 inhibits the induction of antiviral programs mediated by
IRF3 or IRF7
(21–23).
However, although ICP0 negatively regulates IFN-β expression, it is not
essential for this effect
(24). In HSV-infected human
macrophages or dendritic cells, an immediate early protein ICP27 is required
to suppress cytokine induction involving IRF3
(25). In this context, it is
notable that an HSV mutant, lacking a leaky late gene γ134.5,
replicates efficiently in cells devoid of IFN-α/β genes
(26). Additionally, the
γ134.5 null mutant induces differential cytokine expression
as compared with wild type virus
(27). Thus, HSV modulation of
cytokine expression is a complex process that involves multiple viral
components. Currently, the molecular mechanism governing this event is
unclear. In this study, we show that HSV γ134.5 targets TBK1
and inhibits antiviral signaling. The data herein reveal a previously
unrecognized mechanism by which γ134.5 facilitates HSV
replication. 相似文献
13.
Kazuyuki Kitatani Kely Sheldon Vinodh Rajagopalan Viviana Anelli Russell W. Jenkins Ying Sun Gregory A. Grabowski Lina M. Obeid Yusuf A. Hannun 《The Journal of biological chemistry》2009,284(19):12972-12978
Activation of protein kinase C (PKC) promotes the salvage pathway of
ceramide formation, and acid sphingomyelinase has been implicated, in part, in
providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007)
J. Biol. Chem. 282, 11549–11561). In the present study, we
examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes
glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated
formation of ceramide from recycled sphingosine. Glucosylceramide levels
declined after treatment of MCF-7 cells with a potent PKC activator, phorbol
12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs
significantly attenuated acid glucocerebrosidase activity and decreased
PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced
degradation of glucosylceramide and generation of sphingosine, the source for
ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased
ceramide levels. These observations indicate that GBA1 activation can generate
the source (sphingosine) for PMA-induced formation of ceramide through the
salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in
the formation of ceramide was determined. By attenuating expression of
PKCδ, cells failed to trigger PMA-induced alterations in levels of
ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is
suggested to stimulate the degradation of both sphingomyelin and
glucosylceramide leading to the salvage pathway of ceramide formation.
Collectively, GBA1 is identified as a novel source of regulated formation of
ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which
are emerging as bioactive lipid mediators thought to play crucial roles in
cellular responses (1,
2). Ceramide, a central
sphingolipid, serves as the main precursor for various sphingolipids,
including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of
formation of ceramide has been demonstrated through the action of three major
pathways: the de novo pathway
(3,
4), the sphingomyelinase
pathway (5), and the salvage
pathway
(6–8).
The latter plays an important role in constitutive sphingolipid turnover by
salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that
serve as sphingolipid backbones for ceramide and dihydroceramide as well as
all complex sphingolipids (Fig.
1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and
inhibition of PMA induction of ceramide by fumonisin B1. A, the
scheme of the sphingosine salvage pathway of ceramide formation. B,
previously published data as to effects of fumonisin B1 on ceramide mass
profiles (23) are re-plotted
as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or
without 100 μm fumonisin B1 for 2 h followed by treatment with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Results are expressed as sum of
increased mass of ceramide species. Dotted or open columns
represents C16-ceramide or sum of other ceramide species
(C14-ceramide, C18-ceramide, C18:1-ceramide,
C20-ceramide, C24-ceramide, and
C24:1-ceramide), respectively. The data represent mean ±
S.E. of three to five values.Metabolically, ceramide is also formed from degradation of
glycosphingolipids (Fig.
1A) usually in acidic compartments, the lysosomes and/or
late endosomes (9). The
stepwise hydrolysis of complex glycosphingolipids eventually results in the
formation of glucosylceramide, which in turn is converted to ceramide by the
action of acid β-glucosidase 1
(GBA1)2
(9,
10). Severe defects in GBA1
activity cause Gaucher disease, which is associated with aberrant accumulation
of the lipid substrates
(10–14).
On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also
form ceramide (15,
16). Either process results in
the generation of lysosomal ceramide that can then be deacylated by acid
ceramidase (17), releasing
sphingosine that may escape the lysosome
(18). The released sphingosine
may become a substrate for either sphingosine kinases or ceramide synthases,
forming sphingosine 1-phosphate or ceramide, respectively
(3,
19–21).In a related line of investigation, our studies
(20,
22,
23) have begun to implicate
protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage
pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol
ester (PMA) was shown to stimulate the salvage pathway resulting in increases
in ceramide. All the induced ceramide was inhibited by pretreatment with a
ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating
acute activation of the de novo pathway and establishing a role for
ceramide synthesis (20,
23). Moreover, labeling
studies also implicated the salvage pathway because PMA induced turnover of
steady state-labeled sphingolipids but did not affect de novo labeled
ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream
molecule for the activation of acid sphingomyelinase in the salvage pathway
(22). Interestingly, the
PKCδ isoform induced the phosphorylation of acid sphingomyelinase at
serine 508, leading to its activation and consequent formation of ceramide.
The activation of acid sphingomyelinase appeared to contribute to ∼50% of
the salvage pathway-induced increase in ceramide
(28) (also, see
Fig. 4C). This raised
the possibility that distinct routes of ceramide metabolism may account for
the remainder of ceramide generation. In this study, we investigated
glucocerebrosidase GBA1 as a candidate for one of the other routes accounting
for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide
after PMA treatment. A, MCF-7 cells were transfected with 5
nm siRNAs of each of four individual sequences (SCR, GBA1-a,
GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA
for 1 h. Lipids were extracted, and then the levels of the
C16-ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to nine values. B, MCF-7 cells were transfected with 5
nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with
100 nm PMA for 1 h. Lipids were extracted, and then the levels of
individual ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. The data represent mean ± S.E.
of three to five values. C14-Cer,
C14-ceramide; C16-Cer,
C16-ceramide; C18-Cer;
C18-ceramide; C18:1-Cer,
C18:1-ceramide; C20-Cer,
C20-ceramide; C20-Cer,
C24-ceramide; C24:1-Cer,
C24:1-ceramide. C, MCF-7 cells were transfected with 5
nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a
(GBA1) for 48 h following stimulation with (PMA) or without
(Control) 100 nm PMA for 1 h. Lipids were extracted, and
then the levels of ceramide species were determined by high-performance liquid
chromatography-tandem mass spectrometry. Levels of C16-ceramide are
shown. The data represent mean ± S.E. of four to five values.
Significant changes from SCR-transfected cells treated with PMA are shown in
A–C (*, p < 0.02; **,
p < 0.05; ***, p < 0.01). 相似文献
14.
Aurelia Raducanu Ernst B. Hunziker Inga Drosse Attila Asz��di 《The Journal of biological chemistry》2009,284(35):23780-23792
The lack of β1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of β1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the β1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to β1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. β1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of β1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.Chondrocytes of the articular cartilage (AC)2 secrete a unique set of extracellular matrix (ECM) molecules that assemble into interactive associates composed of collagens, proteoglycans (PGs), and non-collagenous glycoproteins (1). The fibrillar collagen meshwork supplies cartilage with its tensile strength, whereas the hydrated glycosaminoglycan (GAG) chains of PGs (mainly aggrecan) generate an osmotic swelling pressure that resists compressive forces. In diarthrodial joints, the molecular composition and the physical properties of the cartilage are principal determinants for the shock-absorbing function of articular surfaces upon mechanical loading. During the development of osteoarthritis (OA), an imbalance between anabolic and catabolic processes increases the proteolysis of PGs and collagens (2, 3), which eventually leads to the mechanical weakening of the AC and culminates in its progressive destruction. Physiological and pathological remodeling of the AC ECM is primarily attributed to the activities of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin-like repeat (ADAMTS) proteases (4, 5) and is controlled by the communication between the cells and their environment.An increasing amount of evidence suggests that interactions between chondrocytes and the ECM through the integrin family of heterodimeric (αβ) transmembrane receptors play a central role in cartilage function (6). Integrins connect the pericellular matrix to cytoskeletal and intracellular signaling complexes and modulate various cellular functions, including survival, proliferation, differentiation, and matrix assembly and metabolism (7, 8). Chondrocytes express several integrin receptors for cartilage matrix ligands, such as α1β1, α2β1, and α10β1 (for collagen II); α5β1, αvβ3, and αvβ5 (for fibronectin); and α6β1 (for laminin) (6, 9). We have previously demonstrated that β1fl/fl-Col2a1cre+ mice, in which the floxed β1 integrin gene (β1fl/fl) was deleted using the chondrocyte-specific Col2a1cre transgene, display severe chondrodysplasia and a high mortality rate at birth (10). Homozygous mutant mice exhibit multiple growth plate abnormalities during endochondral bone formation, characterized by defects in chondrocyte adhesion, shape, proliferation, cytokinesis, and actin organization. In addition, the cartilage matrix shows a sparse, distorted collagen network. Similar, but milder abnormalities were found in mice lacking the collagen-binding integrin α10β1 or integrin-linked kinase in cartilage (11, 12).Although these works have identified β1 integrins as essential regulators of growth plate development, the role of integrins in joint morphogenesis, adult joint function, and pathology is incompletely understood. In the embryonic mouse limb culture system, administration of β1 and α5 blocking antibodies or RGD peptides induced ectopic joint formation between proliferating and hypertrophic chondrocytes of the growth plate, suggesting that α5β1 integrin controls the decision between cartilage differentiation and joint formation during development (13). In adult joints, increased immunostaining of β1 integrin was reported in osteoarthritic monkey cartilage compared with normal cartilage (14) and in human OA samples at minimally damaged locations compared with areas with more severe lesions (15). In another study, the neoexpression of α2, α4, and β2 integrins was observed in osteoarthritic human femoral head cartilage (16). In vitro experiments have suggested that signaling through the fibronectin (FN) receptor α5β1 integrin is pivotal to prevent cell death of normal and osteoarthritic human articular chondrocytes (17). FN fragments (FN-fs) present in synovial fluid and cartilage of OA patients have been implicated in cartilage breakdown (18–21). Human AC chondrocytes treated with the central, 110–120-kDa cell-binding FN-f but not with intact FN were shown to increase MMP-13 synthesis through the stimulation of α5β1 integrin and the subsequent activation of the proline-rich tyrosine kinase-2 and mitogen-activated protein kinases (MAPKs) ERK-1/2, JNK, and p38 (22, 23). Similarly, an adhesion-blocking antibody against α2β1 integrin induced the phosphorylation of MAPKs in human AC chondrocytes (22). Treatment of cultured rabbit synovial fibroblasts with central FN-fs or activating antibodies against α5β1 integrin elevated MMP-1 and MMP-3 expression (24). Although these experiments suggest that blocking integrin signaling through α2β1/α5β1 in response to degradation fragments may attenuate OA, mice lacking α1β1 integrin are prone to osteoarthritis (25). Knee joints of α1-null mice display precocious PG loss, cartilage erosion associated with increased MMP-2 and MMP-3 expression, and synovial hyperplasia.To further explore the role of β1 integrins in joint biology, here we report the deletion of the floxed β1 integrin gene in embryonic limb bud mesenchymal cells using the Prx1cre transgene (26). β1fl/fl-Prx1cre+ mice were born alive with short limbs due to the lack of β1 integrin heterodimers on chondrocytes. We found that β1 integrin deficiency in knee joints leads to multiple abnormalities of the AC and the synovium, but it is not associated with accelerated AC destruction, perturbed AC metabolism, and MAPK signaling. Our data suggest that β1 integrins are required for the proper structural organization of the AC by anchoring chondrocytes to the ECM, but signaling through β1 integrins is less important for normal cartilage homeostasis. 相似文献
15.
Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
16.
Yun Liu Yun-wu Zhang Xin Wang Han Zhang Xiaoqing You Francesca-Fang Liao Huaxi Xu 《The Journal of biological chemistry》2009,284(18):12145-12152
Excessive accumulation of β-amyloid peptides in the brain is a major
cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived
from β-amyloid precursor protein (APP) through sequential cleavages by
β- and γ-secretases, whose enzymatic activities are tightly
controlled by subcellular localization. Delineation of how intracellular
trafficking of these secretases and APP is regulated is important for
understanding Alzheimer disease pathogenesis. Although APP trafficking is
regulated by multiple factors including presenilin 1 (PS1), a major component
of the γ-secretase complex, and phospholipase D1 (PLD1), a
phospholipid-modifying enzyme, regulation of intracellular trafficking of
PS1/γ-secretase and β-secretase is less clear. Here we demonstrate
that APP can reciprocally regulate PS1 trafficking; APP deficiency results in
faster transport of PS1 from the trans-Golgi network to the cell
surface and increased steady state levels of PS1 at the cell surface, which
can be reversed by restoring APP levels. Restoration of APP in APP-deficient
cells also reduces steady state levels of other γ-secretase components
(nicastrin, APH-1, and PEN-2) and the cleavage of Notch by
PS1/γ-secretase that is more highly correlated with cell surface levels
of PS1 than with APP overexpression levels, supporting the notion that Notch
is mainly cleaved at the cell surface. In contrast, intracellular trafficking
of β-secretase (BACE1) is not regulated by APP. Moreover, we find that
PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell
surface accumulation of PS1 in an APP-independent manner. Our results clearly
elucidate a physiological function of APP in regulating protein trafficking
and suggest that intracellular trafficking of PS1/γ-secretase is
regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease
(AD)4 is the formation
of senile plaques in the brains of patients. The major components of those
plaques are β-amyloid peptides (Aβ), whose accumulation triggers a
cascade of neurodegenerative steps ending in formation of senile plaques and
intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible
brain regions (1,
2). Aβ is proteolytically
derived from the β-amyloid precursor protein (APP) through sequential
cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl
protease (3,
4), and by γ-secretase, a
high molecular weight complex consisting of at least four components:
presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and
presenilin enhancer-2 (PEN-2)
(5,
6). APP is a type I
transmembrane protein belonging to a protein family that includes APP-like
protein 1 (APLP1) and 2 (APLP2) in mammals
(7,
8). Full-length APP is
synthesized in the endoplasmic reticulum (ER) and transported through the
Golgi apparatus. Most secreted Aβ peptides are generated within the
trans-Golgi network (TGN), also the major site of steady state APP in
neurons
(9–11).
APP can be transported to the cell surface in TGN-derived secretory vesicles
if not proteolyzed to Aβ or an intermediate metabolite. At the cell
surface APP is either cleaved by α-secretase to produce soluble
sAPPα (12) or
reinternalized for endosomal/lysosomal degradation
(13,
14). Aβ may also be
generated in endosomal/lysosomal compartments
(15,
16). In contrast to neurotoxic
Aβ peptides, sAPPα possesses neuroprotective potential
(17,
18). Thus, the subcellular
distribution of APP and proteases that process it directly affect the ratio of
sAPPα to Aβ, making delineation of the mechanisms responsible for
regulating trafficking of all of these proteins relevant to AD
pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the
two mammalian PS gene homologues, PS1 and PS2, PS1
encodes the major form (PS1) in active γ-secretase
(19,
20). Nascent PSs undergo
endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a
carboxyl-terminal fragment (CTF) to form a functional PS heterodimer
(21). Based on observations
that PSs possess two highly conserved aspartate residues indispensable for
γ-secretase activity and that specific transition state analogue
γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers
(5,
22), PSs are believed to be
the catalytic component of the γ-secretase complex. PS assembles with
three other components, NCT, APH-1, and PEN-2, to form the functional
γ-secretase (5,
6). Strong evidence suggests
that PS1/γ-secretase resides principally in the ER, early Golgi, TGN,
endocytic and intermediate compartments, most of which (except the TGN) are
not major subcellular sites for APP
(23,
24). In addition to generating
Aβ and cleaving APP to release the APP intracellular domain,
PS1/γ-secretase cleaves other substrates such as Notch
(25), cadherin
(26), ErbB4
(27), and CD44
(28), releasing their
respective intracellular domains. Interestingly, PS1/γ-secretase
cleavage of different substrates seems to occur at different subcellular
compartments; APP is mainly cleaved at the TGN and early endosome domains,
whereas Notch is predominantly cleaved at the cell surface
(9,
11,
29). Thus, perturbing
intracellular trafficking of PS1/γ-secretase may alter interactions
between PS1/γ-secretase and APP, contributing to either abnormal Aβ
generation and AD pathogenesis or decreased access of PS1/γ-secretase to
APP such that Aβ production is reduced. However, mechanisms regulating
PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates
intracellular trafficking of several membrane proteins, including other
γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate
APP (reviewed in Ref. 30).
Intracellular APP trafficking is highly regulated and requires other factors
such as mint family members and SorLA
(2). Moreover, we recently
found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that
regulates membrane trafficking events, can interact with PS1, and can regulate
budding of APP-containing vesicles from the TGN and delivery of APP to the
cell surface (31,
32). Interestingly, Kamal
et al. (33)
identified an axonal membrane compartment that contains APP, BACE1, and PS1
and showed that fast anterograde axonal transport of this compartment is
mediated by APP and kinesin-I, implying a traffic-regulating role for APP.
Increased APP expression is also shown to decrease retrograde axonal transport
of nerve growth factor (34).
However, whether APP indeed regulates intracellular trafficking of proteins
including BACE1 and PS1/γ-secretase requires further validation. In the
present study we demonstrate that intracellular trafficking of PS1, as well as
that of other γ-secretase components, but not BACE1, is regulated by
APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase
complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In
addition, we find that PLD1 also regulates intracellular trafficking of PS1
through a different mechanism and more potently than APP. 相似文献
17.
Yongmei Pu Susan H. Garfield Noemi Kedei Peter M. Blumberg 《The Journal of biological chemistry》2009,284(2):1302-1312
Classic and novel protein kinase C (PKC) isozymes contain two zinc finger
motifs, designated “C1a” and “C1b” domains, which
constitute the recognition modules for the second messenger diacylglycerol
(DAG) or the phorbol esters. However, the individual contributions of these
tandem C1 domains to PKC function and, reciprocally, the influence of protein
context on their function remain uncertain. In the present study, we prepared
PKCδ constructs in which the individual C1a and C1b domains were
deleted, swapped, or substituted for one another to explore these issues. As
isolated fragments, both the δC1a and δC1b domains potently bound
phorbol esters, but the binding of [3H]phorbol 12,13-dibutyrate
([3H]PDBu) by the δC1a domain depended much more on the
presence of phosphatidylserine than did that of the δC1b domain. In
intact PKCδ, the δC1b domain played the dominant role in
[3H]PDBu binding, membrane translocation, and down-regulation. A
contribution from the δC1a domain was nonetheless evident, as shown by
retention of [3H]PDBu binding at reduced affinity, by increased
[3H]PDBu affinity upon expression of a second δC1a domain
substituting for the δC1b domain, and by loss of persistent plasma
membrane translocation for PKCδ expressing only the δC1b domain,
but its contribution was less than predicted from the activity of the isolated
domain. Switching the position of the δC1b domain to the normal position
of the δC1a domain (or vice versa) had no apparent effect on the
response to phorbol esters, suggesting that the specific position of the C1
domain within PKCδ was not the primary determinant of its activity.One of the essential steps for protein kinase C
(PKC)2 activation is
its translocation from the cytosol to the membranes. For conventional
(α, βI, βII, and γ) and novel (δ, ε, η,
and θ) PKCs, this translocation is driven by interaction with the
lipophilic second messenger sn-1,2-diacylglycerol (DAG), generated
from phosphatidylinositol 4,5-bisphosphate upon the activation of
receptor-coupled phospholipase C or indirectly from phosphatidylcholine via
phospholipase D (1). A pair of
zinc finger structures in the regulatory domain of the PKCs, the
“C1” domains, are responsible for the recognition of the DAG
signal. The DAG-C1 domain-membrane interaction is coupled to a conformational
change in PKC, both causing the release of the pseudosubstrate domain from the
catalytic site to activate the enzyme and triggering the translocation to the
membrane (2). By regulating
access to substrates, PKC translocation complements the intrinsic enzymatic
specificity of PKC to determine its substrate profile.The C1 domain is a highly conserved cysteine-rich motif (∼50 amino
acids), which was first identified in PKC as the interaction site for DAG or
phorbol esters (3). It
possesses a globular structure with a hydrophilic binding cleft at one end
surrounded by hydrophobic residues. Binding of DAG or phorbol esters to the C1
domain caps the hydrophilic cleft and forms a continuous hydrophobic surface
favoring the interaction or penetration of the C1 domain into the membrane
(4). In addition to the novel
and classic PKCs, six other families of proteins have also been identified,
some of whose members possess DAG/phorbol ester-responsive C1 domains. These
are the protein kinase D (5),
the chimaerin (6), the munc-13
(7), the RasGRP (guanyl
nucleotide exchange factors for Ras and Rap1)
(8), the DAG kinase
(9), and the recently
characterized MRCK (myotonic dystrophy kinase-related
Cdc42-binding kinase) families
(10). Of these C1
domain-containing proteins, the PKCs have been studied most extensively and
are important therapeutic targets
(11). Among the drug
candidates in clinical trials that target PKC, a number such as bryostatin 1
and PEP005 are directed at the C1 domains of PKC rather than at its catalytic
site.Both the classic and novel PKCs contain in their N-terminal regulatory
region tandem C1 domains, C1a and C1b, which bind DAG/phorbol ester
(12). Multiple studies have
sought to define the respective roles of these two C1 domains in PKC
regulation, but the issue remains unclear. Initial in vitro binding
measurements with conventional PKCs suggested that 1 mol of phorbol ester
bound per mole of PKC
(13-15).
On the other hand, Stubbs et al., using a fluorescent phorbol ester
analog, reported that PKCα bound two ligands per PKC
(16). Further, site-directed
mutagenesis of the C1a and C1b domains of intact PKCα indicated that the
C1a and C1b domains played equivalent roles for membrane translocation in
response to phorbol 12-myristate 13-acetate (PMA) and (-)octylindolactam V
(17). Likewise, deletion
studies indicated that the C1a and C1b domains of PKCγ bound PDBu
equally with high potency (3,
18). Using a functional assay
with PKCα expression in yeast, Shieh et al.
(19) deleted individual C1
domains and reported that C1a and C1b were both functional and equivalent upon
stimulation by PMA, with either deletion causing a similar reduction in
potency of response, whereas for mezerein the response depended essentially on
the C1a domain, with much weaker response if only the C1b domain was present.
Using isolated C1 domains, Irie et al.
(20) suggested that the C1a
domain of PKCα but not those of PKCβ or PKCγ bound
[3H]PDBu preferentially; different ligands showed a generally
similar pattern but with different extents of selectivity. Using synthesized
dimeric bisphorbols, Newton''s group reported
(21) that, although both C1
domains of PKCβII are oriented for potential membrane interaction, only
one C1 domain bound ligand in a physiological context.In the case of novel PKCs, many studies have been performed on PKCδ
to study the equivalency of the twin C1 domains. The P11G point mutation of
the C1a domain, which caused a 300-fold loss of binding potency in the
isolated domain (22), had
little effect on the phorbol ester-dependent translocation of PKCδ in
NIH3T3 cells, whereas the same mutation of the C1b caused a 20-fold shift in
phorbol ester potency for inducing translocation, suggesting a major role of
the C1b domain for phorbol ester binding
(23). A secondary role for the
C1a domain was suggested, however, because mutation in the C1a domain as well
as the C1b domain caused a further 7-fold shift in potency. Using the same
mutations in the C1a and C1b domains, Bögi et al.
(24) found that the binding
selectivity for the C1a and C1b domains of PKCδ appeared to be
ligand-dependent. Whereas PMA and the indole alkaloids indolactam and
octylindolactam were selectively dependent on the C1b domain, selectivity was
not observed for mezerein, the 12-deoxyphorbol 13-monoesters prostratin and
12-deoxyphorbol 13-phenylacetate, and the macrocyclic lactone bryostatin 1
(24). In in vitro
studies using isolated C1a and C1b domains of PKCδ, Cho''s group
(25) described that the two C1
domains had opposite affinities for DAG and phorbol ester; i.e. the
C1a domain showed high affinity for DAG and the C1b domain showed high
affinity for phorbol ester. No such difference in selectivity was observed by
Irie et al. (20).PKC has emerged as a promising therapeutic target both for cancer and for
other conditions, such as diabetic retinopathy or macular degeneration
(26-30).
Kinase inhibitors represent one promising approach for targeting PKC, and
enzastaurin, an inhibitor with moderate selectivity for PKCβ relative to
other PKC isoforms (but still with activity on some other non-PKC kinases) is
currently in multiple clinical trials. An alternative strategy for drug
development has been to target the regulatory C1 domains of PKC. Strong proof
of principle for this approach is provided by multiple natural products,
e.g. bryostatin 1 and PEP005, which are likewise in clinical trials
and which are directed at the C1 domains. A potential advantage of this
approach is the lesser number of homologous targets, <30 DAG-sensitive C1
domains compared with over 500 kinases, as well as further opportunities for
specificity provided by the diversity of lipid environments, which form a
half-site for ligand binding to the C1 domain. Because different PKC isoforms
may induce antagonistic activities, inhibition of one isoform may be
functionally equivalent to activation of an antagonistic isoform
(31).Along with the benzolactams
(20,
32), the DAG lactones have
provided a powerful synthetic platform for manipulating ligand: C1 domain
interactions (31). For
example, the DAG lactone derivative 130C037 displayed marked selectivity among
the recombinant C1a and C1b domains of PKCα and PKCδ as well as
substantial selectivity for RasGRP relative to PKCα
(33). Likewise, we have shown
that a modified DAG lactone (dioxolanones) can afford an additional point of
contact in ligand binding to the C1b domain of PKCδ
(34). Such studies provide
clear examples that ligand-C1 domain interactions can be manipulated to yield
novel patterns of recognition. Further selectivity might be gained with
bivalent compounds, exploiting the spacing and individual characteristics of
the C1a and C1b domains (35).
A better understanding of the differential roles of the two C1 domains in PKC
regulation is critical for the rational development of such compounds. In this
study, by molecularly manipulating the C1a or C1b domains in intact
PKCδ, we find that both the C1a and C1b domains play important roles in
PKCδ regulation. The C1b domain is predominant for ligand binding and
for membrane translocation of the whole PKCδ molecule. The C1a domain of
intact PKCδ plays only a secondary role in ligand binding but stabilizes
the PKCδ molecule at the plasma membrane for downstream signaling. In
addition, we show that the effect of the individual C1 domains of PKCδ
does not critically depend on their position within the regulatory domain. 相似文献
18.
Nelli Mnatsakanyan Arathianand M. Krishnakumar Toshiharu Suzuki Joachim Weber 《The Journal of biological chemistry》2009,284(17):11336-11345
ATP synthase uses a unique rotational mechanism to convert chemical energy
into mechanical energy and back into chemical energy. The helix-turn-helix
motif, termed “DELSEED-loop,” in the C-terminal domain of the
β subunit was suggested to be involved in coupling between catalysis and
rotation. Here, the role of the DELSEED-loop was investigated by functional
analysis of mutants of Bacillus PS3 ATP synthase that had 3–7
amino acids within the loop deleted. All mutants were able to catalyze ATP
hydrolysis, some at rates several times higher than the wild-type enzyme. In
most cases ATP hydrolysis in membrane vesicles generated a transmembrane
proton gradient, indicating that hydrolysis occurred via the normal rotational
mechanism. Except for two mutants that showed low activity and low abundance
in the membrane preparations, the deletion mutants were able to catalyze ATP
synthesis. In general, the mutants seemed less well coupled than the wild-type
enzyme, to a varying degree. Arrhenius analysis demonstrated that in the
mutants fewer bonds had to be rearranged during the rate-limiting catalytic
step; the extent of this effect was dependent on the size of the deletion. The
results support the idea of a significant involvement of the DELSEED-loop in
mechanochemical coupling in ATP synthase. In addition, for two deletion
mutants it was possible to prepare an
α3β3γ subcomplex and measure nucleotide
binding to the catalytic sites. Interestingly, both mutants showed a severely
reduced affinity for MgATP at the high affinity site.F1F0-ATP synthase catalyzes the final step of
oxidative phosphorylation and photophosphorylation, the synthesis of ATP from
ADP and inorganic phosphate. F1F0-ATP synthase consists
of the membrane-embedded F0 subcomplex, with, in most bacteria, a
subunit composition of ab2c10, and the peripheral
F1 subcomplex, with a subunit composition of
α3β3γδε. The energy
necessary for ATP synthesis is derived from an electrochemical transmembrane
proton (or, in some organisms, a sodium ion) gradient. Proton flow down the
gradient through F0 is coupled to ATP synthesis on F1 by
a unique rotary mechanism. The protons flow through (half) channels at the
interface of the a and c subunits, which drives rotation of the ring of c
subunits. The c10 ring, together with F1 subunits
γ and ε, forms the rotor. Rotation of γ leads to
conformational changes in the catalytic nucleotide binding sites on the β
subunits, where ADP and Pi are bound. The conformational changes
result in the formation and release of ATP. Thus, ATP synthase converts
electrochemical energy, the proton gradient, into mechanical energy in the
form of subunit rotation and back into chemical energy as ATP. In bacteria,
under certain physiological conditions, the process runs in reverse. ATP is
hydrolyzed to generate a transmembrane proton gradient, which the bacterium
requires for such functions as nutrient import and locomotion (for reviews,
see Refs.
1–6).F1 (or F1-ATPase) has three catalytic nucleotide
binding sites located on the β subunits at the interface to the adjacent
α subunit. The catalytic sites have pronounced differences in their
nucleotide binding affinity. During rotational catalysis, the sites switch
their affinities in a synchronized manner; the position of γ determines
which catalytic site is the high affinity site
(Kd1 in the nanomolar range), which site is the
medium affinity site (Kd2 ≈ 1
μm), and which site is the low affinity site
(Kd3 ≈ 30–100 μm; see
Refs. 7 and
8). In the original crystal
structure of bovine mitochondrial F1
(9), one of the three catalytic
sites, was filled with the ATP analog
AMP-PNP,2 a second was
filled with ADP (plus azide) (see Ref.
10), and the third site was
empty. Hence, the β subunits are referred to as βTP,
βDP, and βE. The occupied β subunits,
βTP and βDP, were in a closed conformation,
and the empty βE subunit was in an open conformation. The main
difference between these two conformations is found in the C-terminal domain.
Here, the “DELSEED-loop,” a helix-turn-helix structure containing
the conserved DELSEED motif, is in an “up” position when the
catalytic site on the respective β subunit is filled with nucleotide and
in a “down” position when the site is empty
(Fig. 1A). When all
three catalytic sites are occupied by nucleotide, the previously open
βE subunit assumes an intermediate, half-closed
(βHC) conformation. It cannot close completely because of
steric clashes with γ
(11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the
βTP and βE subunits with theγ
subunit.β subunits are shown in yellow andγ in
blue. The DELSEED-loop (shown in orange, with the DELSEED
motif itself in green)of βTP interacts with the
C-terminal helixγ and the short helix that runs nearly perpendicular to
the rotation axis. The DELSEED-loop of βE makes contact with
the convex portion of γ, formed mainly by the N-terminal helix. A
nucleotide molecule (shown in stick representation) occupies the catalytic
site of βTP, and the subunit is in the closed conformation.
The catalytic site on βE is empty, and the subunit is in the
open conformation. This figure is based on Protein Data Bank file 1e79
(32). B, deletions in
the βDELSEED-loop. The loop was “mutated” in silico
to represent the PS3 ATP synthase. The 3–4-residue segments that are
removed in the deletion mutants are color-coded as follows:
380LQDI383, pink;
384IAIL387, green;
388GMDE391, yellow;
392LSD394, cyan;
395EDKL398, orange;
399VVHR402, blue. Residues that are the most
involved in contacts with γ are labeled. All figures were generated
using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with
the γ subunit. In some cases, these contacts consist of hydrogen bonds
or salt bridges between the negatively charged residues of the DELSEED motif
and positively charged residues on γ. The interactions of the
DELSEED-loop with γ, its movement during catalysis, the conservation of
the DELSEED motif (see 12–14).
Thus, the finding that an AALSAAA mutant in the
α3β3γ complex of ATP synthase from the
thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges
to γ are removed simultaneously, could drive rotation of γ with
the same torque as the wild-type enzyme
(14) came as a surprise. On
the other hand, it seems possible that it is the bulk of the DELSEED-loop,
more so than individual interactions, that drives rotation of γ.
According to a model favored by several authors
(6,
15,
16) (see also Refs.
17–19),
binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site
on βE and the subsequent closure of this site, accompanied by
its conversion into the high affinity site, are responsible for driving the
large (80–90°) rotation substep during ATP hydrolysis, with the
DELSEED-loop acting as a “pushrod.” A recent molecular dynamics
(20) study supports this model
and implicates mainly the region around several hydrophobic residues upstream
of the DELSEED motif (specifically βI386 and
βL387)3 as being
responsible for making contact with γ during the large rotation
substep.