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1.
L. Andy Chen Jing Li Scott R. Silva Lindsey N. Jackson Yuning Zhou Hiroaki Watanabe Kirk L. Ives Mark R. Hellmich B. Mark Evers 《The Journal of biological chemistry》2009,284(4):2459-2471
The protein kinase D (PKD) family of serine/threonine kinases, which can be
activated by gastrointestinal hormones, consists of three distinct isoforms
that modulate a variety of cellular processes including intracellular protein
transport as well as constitutive and regulated secretion. Although
isoform-specific functions have been identified in a variety of cell lines,
the expression and function of PKD isoforms in normal, differentiated
secretory tissues is unknown. Here, we demonstrate that PKD isoforms are
differentially expressed in the exocrine and endocrine cells of the pancreas.
Specifically, PKD3 is the predominant isoform expressed in exocrine cells of
the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly
expressed in the pancreatic islets. Within isolated mouse pancreatic acinar
cells, PKD3 undergoes rapid membrane translocation, trans-activating
phosphorylation, and kinase activation after gastrointestinal hormone or
cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs
viaaCa2+-independent, diacylglycerol- and protein kinase
C-dependent mechanism. PKD phosphorylation can also be induced by physiologic
concentrations of secretagogues and by in vivo stimulation of the
pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK,
ribosomal S6 kinase) signaling and significantly enhances
cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a
novel distinction between the exocrine and endocrine cells of the pancreas and
further identify PKD3 as a signaling molecule that promotes hormone-stimulated
amylase secretion.Protein kinase D
(PKD),2 a
serine/threonine kinase family with a catalytic domain homologous to the
Ca2+/calmodulin-dependent kinase domain and two cysteine-rich
phorbol ester binding domains similar to those of protein kinase C (PKC), is a
physiologically important downstream mediator of diacylglycerol (DAG) signal
transduction (1,
2). The mammalian PKDs include
three members, PKD1, PKD2, and PKD3, which demonstrate different expression
patterns and functions depending on the cell type and external signal stimuli.
PKDs are ubiquitously expressed, but levels of individual isoforms vary with
developmental stage and cell type
(3). PKD proteins are reported
to localize in the cytosol, Golgi, nucleus, and vesicle structures
(4-9).
Activation of PKDs results in a dynamic translocation among subcellular
compartments (10,
11). Expression of multiple
isoforms in different cell types and in different subcellular localizations
suggests that individual PKD isoforms may serve specific functions. The
majority of findings demonstrating the diverse expression patterns and
functions of PKD have been described using established cell lines
(4-9,
12). However, little is known
about PKD isoform expression and function in normal differentiated cells and
tissues.Recent functional studies have shown that PKD isoforms differentially
regulate exocytic protein trafficking and cargo specificity
(9,
12-14).
Furthermore, PKD isoforms are differentially activated by oxidative stress
signaling via PKCδ-mediated tyrosine phosphorylation
(15). In each of these
studies, PKD3 was found to have a regulatory mechanism or cellular function
distinct from that of PKD1 and PKD2. Unlike the other two isoforms, PKD3 lacks
the N terminus hydrophobic domain or the C terminus PDZ binding motif and
contains divergent PH (pleckstrin homology) and C1 domains, which are
important for regulating its catalytic activity
(12,
16,
17). Current knowledge of the
physiologic function of PKD3 is limited. It has been demonstrated using
kinase-inactive mutants that PKD3 activity is required for basolateral
exocytosis in Madin-Darby canine kidney cells
(13). PKD3 has also been
implicated in the epigenetic control of chromatin by regulating class II
histone deacetylases in B lymphocytes
(18). Furthermore, PKD3 was
found to be a specific regulator of glucose transport in skeletal muscle cells
(19).The exocrine pancreas is highly specialized for the synthesis, storage, and
exocrine secretion of digestive enzymes and bicarbonate-rich fluid
(20). More than 90% of the
newly synthesized proteins in the pancreas is targeted to the secretory
pathway (21). In addition, the
pancreas contains a variety of endocrine cells localized to the islets which
secrete peptide hormones. Numerous steps in the secretory pathway are
modulated by DAG signaling, which promotes secretion by maintaining Golgi
function and/or activating DAG receptor kinases such as PKCs, which are
regulators of exocytic proteins
(1,
22-25).
PKD is also critical for DAG-mediated secretion, as it is recruited by DAG to
the trans-Golgi network, where it phosphorylates the lipid kinase
phosphatidylinositol 4-kinase to initiate the process of vesicle fission
(9,
26). Gastrointestinal (GI)
hormones such as cholecystokinin (CCK), gastrin, neurotensin (NT), and
bombesin (BBS)/gastrin-releasing peptide are potent regulatory peptides that
modulate pancreatic function
(27,
28). They are known to
activate PKDs to promote cell proliferation and survival in gut epithelial
cells
(29-32);
however, the role of PKDs in modulating the secretory actions of GI hormones
is unknown.Although the PKD isoforms have been reported to be expressed in secretory
tissues such as salivary glands, adrenal glands, intestinal mucosa, and the
pituitary (3,
5,
33), the role of PKD in the
process of regulated secretion remains poorly understood. Previously, we
demonstrated that PKD1 mediates NT peptide secretion from a pancreas-derived
neuroendocrine cell line, BON, and that PKD1 activation is regulated by PKC
and Rho/Rho kinase pathways
(4); PKD1 and PKD2 isoforms are
highly expressed in this endocrine cell line with little to no PKD3
expression, thus suggesting that PKD1/2 may be the predominant isoforms for
endocrine secretion. The distribution and role of PKD isoforms in the
pancreas, an organ with both exocrine and endocrine functions, is not known.
Interestingly, we demonstrate that in both human and mouse pancreas, PKD3 is
the predominant PKD isoform expressed in the exocrine acini, whereas PKD1 and
PKD2 are more highly expressed in endocrine islets. PKD3 is catalytically
activated by GI hormone stimulation of the pancreas, and its activation is
dependent on CCK1/2 receptor binding and on DAG/PKC activity. PKD3
overexpression in mouse pancreatic acinar cells significantly increased
CCK-mediated pancreatic amylase secretion, suggesting that PKD3, in concert
with other signaling molecules, contributes to stimulated amylase secretion.
Our findings reveal a distinct expression pattern in the exocrine and
endocrine cells of the mouse and human pancreas and identify PKD3 as a novel
DAG-activated mediator of the exocrine secretory process in response to GI
hormone signaling. 相似文献
2.
Cheuk-Lun Lee Poh-Choo Pang William S. B. Yeung Bérangère Tissot Maria Panico Terence T. H. Lao Ivan K. Chu Kai-Fai Lee Man-Kin Chung Kevin K. W. Lam Riitta Koistinen Hannu Koistinen Markku Sepp?l? Howard R. Morris Anne Dell Philip C. N. Chiu 《The Journal of biological chemistry》2009,284(22):15084-15096
Glycodelin is a human glycoprotein with four reported glycoforms, namely
glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S
(GdS). These glycoforms have the same protein core and appear to differ in
their N-glycosylation. The glycosylation of GdA is completely
different from that of GdS. GdA inhibits proliferation and induces cell death
of T cells. However, the glycosylation and immunomodulating activities of GdF
and GdC are not known. This study aimed to use ultra-high sensitivity mass
spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the
relationship between the immunological activity and glycosylation pattern
among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were
shown to contain an enormous diversity of bi-, tri-, and tetra-antennary
complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or
GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels
of fucose and sialic acid substitution. Interestingly, they all carried a
family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing
glycans, which were not identified in the earlier study because of less
sensitive methodologies used. Among the three glycodelins, GdA is the most
heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda
antennae. With the exception of the Sda epitope, the GdC N-glycome
appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase
activity, which may be responsible for transforming GdA/GdF to GdC, was
detected in cumulus cells. Both GdA and GdF inhibited the proliferation,
induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and
peripheral blood mononuclear cells. In contrast, no immunosuppressive effect
was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino
acid residues (1) with two
sites of N-linked glycosylation. There are four reported glycodelin
isoforms, namely glycodelin-A (amniotic fluid isoform,
GdA),4 glycodelin-F
(follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S
(seminal plasma, GdS)
(2–5).
Among the four glycodelin isoforms, only the N-glycan structures of
GdA and GdS have been previously determined. This was achieved using fast atom
bombardment mass spectrometry
(6,
7). The glycan structures of
GdA and GdS are completely different. In GdA, the Asn-28 site carries high
mannose, hybrid, and complex-type structures, whereas the second Asn-63 site
is exclusively occupied by complex-type glycans
(6). The major non-reducing
epitopes characterized in the complex-type glycans are
Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc),
NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc),
NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc),
Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and
GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood
group substance Lewis-x) (6).
Many of these oligosaccharides are rare in other human glycoproteins. GdS
glycans are unusually fucose-rich, and the major complex type glycan
structures are bi-antennary glycans with Lewis-x and Lewis-y antennae.
Glycosylation of GdS is highly site-specific. Asn-28 contains only high
mannose structures, whereas Asn-63 contains only complex type glycans. More
than 80% of the complex glycans have 3–5 fucose residues/glycan, and
none of the glycans is sialylated, which is unusual for a secreted human
glycoprotein (7). The glycan
structures of GdF and GdC are not known, although they differ in
lectin-binding properties and isoelectric point from the other two glycodelin
isoforms (5).Glycans are involved in various intracellular, intercellular, and
cell-matrix recognition events
(8,
9). Glycosylation determines
the biological activities of the glycodelin isoforms
(2,
10). For example, both GdA and
GdF inhibit the spermatozoa-zona pellucida binding
(11) via fucosyltransferase-5
(12), but only the latter
inhibits progesterone-induced acrosome reaction, thus preventing a premature
acrosome reaction of the spermatozoa. There is evidence that cumulus cells can
convert exogenous GdA and -F to GdC, the physicochemical properties of which
suggest that it is differently glycosylated compared with GdA/F
(5). Moreover, GdC stimulated
spermatozoa-zona pellucida binding in a dose-dependent manner, and it
effectively displaced sperm-bound GdA and -F
(4,
5). GdS suppresses capacitation
probably via its inhibitory activity on cholesterol efflux from spermatozoa
(13).Except for the effects on fertilization, GdA is involved in fetomaternal
defense. This glycodelin isoform suppresses proliferation and induces
apoptosis of T cells (2) and
inhibits natural killer cell
(14) and B-cell
(15) activities. Glycosylation
is involved in the binding of GdA to receptors on T cells
(16). The sialic acid of GdA
contributes to the apoptotic activity in T cells
(17,
18) and binding to CD45, a
potential GdA receptor (16).
The importance of glycosylation in glycodelin is further shown by the absence
of immunosuppressive activities in GdS with different glycosylation
(18). The immunomodulating
activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different
glycodelins to exhibit their binding activities and biological effects
(13,
19,
20). The present study aims to
identify the effect of all four glycodelin isoforms on lymphocyte viability,
cell death, and interleukin-2 (IL-2) secretion and to correlate these
bioactivities with their glycosylation patterns determined by mass
spectrometry. 相似文献
3.
4.
Benjamin E. L. Lauffer Stanford Chen Cristina Melero Tanja Kortemme Mark von Zastrow Gabriel A. Vargas 《The Journal of biological chemistry》2009,284(4):2448-2458
Many G protein-coupled receptors (GPCRs) recycle after agonist-induced
endocytosis by a sequence-dependent mechanism, which is distinct from default
membrane flow and remains poorly understood. Efficient recycling of the
β2-adrenergic receptor (β2AR) requires a C-terminal PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin
cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth
factor-regulated substrate). The PDZbd is thought to link receptors to actin
through a series of protein interaction modules present in NHERF/EBP50
(Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein
of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not
known, however, if such actin connectivity is sufficient to recapitulate the
natural features of sequence-dependent recycling. We addressed this question
using a receptor fusion approach based on the sufficiency of the PDZbd to
promote recycling when fused to a distinct GPCR, the δ-opioid receptor,
which normally recycles inefficiently in HEK293 cells. Modular domains
mediating actin connectivity promoted receptor recycling with similarly high
efficiency as the PDZbd itself, and recycling promoted by all of the domains
was actin-dependent. Regulation of receptor recycling by Hrs, however, was
conferred only by the PDZbd and not by downstream interaction modules. These
results suggest that actin connectivity is sufficient to mimic the core
recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors
(GPCRs)2 comprise the
largest family of transmembrane signaling receptors expressed in animals and
transduce a wide variety of physiological and pharmacological information.
While these receptors share a common 7-transmembrane-spanning topology,
structural differences between individual GPCR family members confer diverse
functional and regulatory properties
(1-4).
A fundamental mechanism of GPCR regulation involves agonist-induced
endocytosis of receptors via clathrin-coated pits
(4). Regulated endocytosis can
have multiple functional consequences, which are determined in part by the
specificity with which internalized receptors traffic via divergent downstream
membrane pathways
(5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed
by the δ-opioid receptor (δOR), contributes to proteolytic
down-regulation of receptor number and produces a prolonged attenuation of
subsequent cellular responsiveness to agonist
(8,
9). Trafficking of internalized
GPCRs via a rapid recycling pathway, a major route traversed by the
β2-adrenergic receptor (β2AR), restores the complement of functional
receptors present on the cell surface and promotes rapid recovery of cellular
signaling responsiveness (6,
10,
11). When co-expressed in the
same cells, the δOR and β2AR are efficiently sorted between these
divergent downstream membrane pathways, highlighting the occurrence of
specific molecular sorting of GPCRs after endocytosis
(12).Recycling of various integral membrane proteins can occur by default,
essentially by bulk membrane flow in the absence of lysosomal sorting
determinants (13). There is
increasing evidence that various GPCRs, such as the β2AR, require
distinct cytoplasmic determinants to recycle efficiently
(14). In addition to requiring
a cytoplasmic sorting determinant, sequence-dependent recycling of the
β2AR differs from default recycling in its dependence on an intact actin
cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs
(hepatocyte growth factor receptor substrate)
(11,
14). Compared with the present
knowledge regarding protein complexes that mediate sorting of GPCRs to
lysosomes (15,
16), however, relatively
little is known about the biochemical basis of sequence-directed recycling or
its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ
(PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called
PDZbd), and PDZ-mediated protein association(s) with this sequence appear to
be primarily responsible for its endocytic sorting activity
(17-20).
Fusion of this sequence to the cytoplasmic tail of the δOR effectively
re-routes endocytic trafficking of engineered receptors from lysosomal to
recycling pathways, establishing the sufficiency of the PDZbd to function as a
transplantable sorting determinant
(18). The β2AR-derived
PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ
proteins (21,
22). A well-established
biochemical function of NHERF/EBP50 family proteins is to associate integral
membrane proteins with actin-associated cytoskeletal elements. This is
achieved through a series of protein-interaction modules linking NHERF/EBP50
family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to
actin filaments
(23-26).
Such indirect actin connectivity is known to mediate other effects on plasma
membrane organization and function
(23), however, and NHERF/EBP50
family proteins can bind to additional proteins potentially important for
endocytic trafficking of receptors
(23,
25). Thus it remains unclear
if actin connectivity is itself sufficient to promote sequence-directed
recycling of GPCRs and, if so, if such connectivity recapitulates the normal
cellular regulation of sequence-dependent recycling. In the present study, we
took advantage of the modular nature of protein connectivity proposed to
mediate β2AR recycling
(24,
26), and extended the opioid
receptor fusion strategy used successfully for identifying diverse recycling
sequences in GPCRs
(27-29),
to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can
be effectively bypassed by linking receptors to ERM family proteins in the
absence of the PDZbd itself. Further, we establish that the protein
connectivity network can be further simplified by fusing receptors to an
interaction module that binds directly to actin filaments. We found that
bypassing the PDZ-mediated interaction using either domain is sufficient to
mimic the ability of the PDZbd to promote efficient, actin-dependent recycling
of receptors. Hrs-dependent regulation, however, which is characteristic of
sequence-dependent recycling of wild-type receptors, was recapitulated only by
the fused PDZbd and not by the proposed downstream interaction modules. These
results support a relatively simple architecture of protein connectivity that
is sufficient to mimic the core recycling activity of the β2AR-derived
PDZbd, but not its characteristic cellular regulation. Given that an
increasing number of GPCRs have been shown to bind PDZ proteins that typically
link directly or indirectly to cytoskeletal elements
(17,
27,
30-32),
the present results also suggest that actin connectivity may represent a
common biochemical principle underlying sequence-dependent recycling of
various GPCRs. 相似文献
5.
Kuen-Feng Chen Pei-Yen Yeh Chiun Hsu Chih-Hung Hsu Yen-Shen Lu Hsing-Pang Hsieh Pei-Jer Chen Ann-Lii Cheng 《The Journal of biological chemistry》2009,284(17):11121-11133
Hepatocellular carcinoma (HCC) is one of the most common and aggressive
human malignancies. Recombinant tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However,
many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we
showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in
HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib
and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis.
Comparing the molecular change in HCC cells treated with these agents, we
found that down-regulation of phospho-Akt (P-Akt) played a key role in
mediating TRAIL sensitization of bortezomib. The first evidence was that
bortezomib down-regulated P-Akt in a dose- and time-dependent manner in
TRAIL-treated HCC cells. Second, , a PI3K inhibitor, also sensitized
resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by
small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells.
Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells
abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a
protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in
bortezomib-treated cells, and PP2A knockdown by small interference RNA also
reduced apoptosis induced by the combination of TRAIL and bortezomib,
indicating that PP2A may be important in mediating the effect of bortezomib on
TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at
clinically achievable concentrations in hepatocellular carcinoma cells, and
this effect is mediated at least partly via inhibition of the PI3K/Akt
pathway.Hepatocellular carcinoma
(HCC) LY2940022 is currently
the fifth most common solid tumor worldwide and the fourth leading cause of
cancer-related death. To date, surgery is still the only curative treatment
but is only feasible in a small portion of patients
(1). Drug treatment is the
major therapy for patients with advanced stage disease. Unfortunately, the
response rate to traditional chemotherapy for HCC patients is unsatisfactory
(1). Novel pharmacological
therapy is urgently needed for patients with advanced HCC. In this regard, the
approval of sorafenib might open a new era of molecularly targeted therapy in
the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a
type II transmembrane protein and a member of the TNF family, is a promising
anti-tumor agent under clinical investigation
(2). TRAIL functions by
engaging its receptors expressed on the surface of target cells. Five
receptors specific for TRAIL have been identified, including DR4/TRAIL-R1,
DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4
and DR5 contain an effective death domain that is essential to formation of
death-inducing signaling complex (DISC), a critical step for TRAIL-induced
apoptosis. Notably, the trimerization of the death domains recruits an adaptor
molecule, Fas-associated protein with death domain (FADD), which subsequently
recruits and activates caspase-8. In type I cells, activation of caspase-8 is
sufficient to activate caspase-3 to induce apoptosis; however, in another type
of cells (type II), the intrinsic mitochondrial pathway is essential for
apoptosis characterized by cleavage of Bid and release of cytochrome
c from mitochondria, which subsequently activates caspase-9 and
caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal
cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms
responsible for the resistance include receptors and intracellular resistance.
Although the cell surface expression of DR4 or DR5 is absolutely required for
TRAIL-induced apoptosis, tumor cells expressing these death receptors are not
always sensitive to TRAIL due to intracellular mechanisms. For example, the
cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but
without protease activity, has been linked to TRAIL resistance in several
studies (4,
5). In addition, inactivation
of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL
in MMR-deficient tumors (6,
7), and reintroduction of Bax
into Bax-deficient cells restored TRAIL sensitivity
(8), indicating that the Bcl-2
family plays a critical role in intracellular mechanisms for resistance of
TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma
and mantle cell lymphoma, has been investigated intensively for many types of
cancer (9). Accumulating
studies indicate that the combination of bortezomib and TRAIL overcomes the
resistance to TRAIL in various types of cancer, including acute myeloid
leukemia (4), lymphoma
(10–13),
prostate
(14–17),
colon (15,
18,
19), bladder
(14,
16), renal cell carcinoma
(20), thyroid
(21), ovary
(22), non-small cell lung
(23,
24), sarcoma
(25), and HCC
(26,
27). Molecular targets
responsible for the sensitizing effect of bortezomib on TRAIL-induced cell
death include DR4 (14,
27), DR5
(14,
20,
22–23,
28), c-FLIP
(4,
11,
21–23,
29), NF-κB
(12,
24,
30), p21
(16,
21,
25), and p27
(25). In addition, Bcl-2
family also plays a role in the combinational effect of bortezomib and TRAIL,
including Bcl-2 (10,
21), Bax
(13,
22), Bak
(27), Bcl-xL
(21), Bik
(18), and Bim
(15).Recently, we have reported that Akt signaling is a major molecular
determinant in bortezomib-induced apoptosis in HCC cells
(31). In this study, we
demonstrated that bortezomib overcame TRAIL resistance in HCC cells through
inhibition of the PI3K/Akt pathway. 相似文献
6.
Isabel Molina-Ortiz Rub��n A. Bartolom�� Pablo Hern��ndez-Varas Georgina P. Colo Joaquin Teixid�� 《The Journal of biological chemistry》2009,284(22):15147-15157
Melanoma cells express the chemokine receptor CXCR4 that confers high
invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial
stages of the disease show reduction or loss of E-cadherin expression, but
recovery of its expression is frequently found at advanced phases. We
overexpressed E-cadherin in the highly invasive BRO lung metastatic cell
melanoma cell line to investigate whether it could influence CXCL12-promoted
cell invasion. Overexpression of E-cadherin led to defective invasion of
melanoma cells across Matrigel and type I collagen in response to CXCL12. A
decrease in individual cell migration directionality toward the chemokine and
reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent
inhibition of RhoA activation was responsible for the impairment in
chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore,
we show that p190RhoGAP and p120ctn associated predominantly on the plasma
membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn
contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association.
These results suggest that melanoma cells at advanced stages of the disease
could have reduced metastatic potency in response to chemotactic stimuli
compared with cells lacking E-cadherin, and the results indicate that
p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca2+-dependent adhesion molecules that
mediate cell-cell contacts and are expressed in most solid tissues providing a
tight control of morphogenesis
(1,
2). Classical cadherins, such
as epithelial (E) cadherin, are found in adherens junctions, forming core
protein complexes with β-catenin, α-catenin, and p120 catenin
(p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin,
whereas α-catenin associates with the complex through its binding to
β-catenin, providing a link with the actin cytoskeleton
(1,
2). E-cadherin is frequently
lost or down-regulated in many human tumors, coincident with morphological
epithelial to mesenchymal transition and acquisition of invasiveness
(3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis
starts, it is responsible for 80% of deaths from skin cancers
(7). Melanocytes express
E-cadherin
(8-10),
but melanoma cells at early radial growth phase show a large reduction in the
expression of this cadherin, and surprisingly, expression has been reported to
be partially recovered by vertical growth phase and metastatic melanoma cells
(9,
11,
12).Trafficking of cancer cells from primary tumor sites to intravasation into
blood circulation and later to extravasation to colonize distant organs
requires tightly regulated directional cues and cell migration and invasion
that are mediated by chemokines, growth factors, and adhesion molecules
(13). Solid tumor cells
express chemokine receptors that provide guidance of these cells to organs
where their chemokine ligands are expressed, constituting a homing model
resembling the one used by immune cells to exert their immune surveillance
functions (14). Most solid
cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called
SDF-1), which is expressed in lungs, bone marrow, and liver
(15). Expression of CXCR4 in
human melanoma has been detected in the vertical growth phase and on regional
lymph nodes, which correlated with poor prognosis and increased mortality
(16,
17). Previous in vivo
experiments have provided evidence supporting a crucial role for CXCR4 in the
metastasis of melanoma cells
(18).Rho GTPases control the dynamics of the actin cytoskeleton during cell
migration (19,
20). The activity of Rho
GTPases is tightly regulated by guanine-nucleotide exchange factors
(GEFs),4 which
stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating
proteins (GAPs), which promote GTP hydrolysis
(21,
22), whereas guanine
nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of
spontaneous activation (23).
Therefore, cell migration is finely regulated by the balance between GEF, GAP,
and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is
well documented (reviewed in Ref.
24), providing control of both
cell migration and growth. RhoA and RhoC are highly expressed in colon,
breast, and lung carcinoma
(25,
26), whereas overexpression of
RhoC in melanoma leads to enhancement of cell metastasis
(27). CXCL12 activates both
RhoA and Rac1 in melanoma cells, and both GTPases play key roles during
invasion toward this chemokine
(28,
29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and
metastasis, in this study we have addressed the question of whether changes in
E-cadherin expression on melanoma cells might affect cell invasiveness. We
show here that overexpression of E-cadherin leads to impaired melanoma cell
invasion to CXCL12, and we provide mechanistic characterization accounting for
the decrease in invasion. 相似文献
7.
Formin-homology (FH) 2 domains from formin proteins associate processively
with the barbed ends of actin filaments through many rounds of actin subunit
addition before dissociating completely. Interaction of the actin
monomer-binding protein profilin with the FH1 domain speeds processive barbed
end elongation by FH2 domains. In this study, we examined the energetic
requirements for fast processive elongation. In contrast to previous
proposals, direct microscopic observations of single molecules of the formin
Bni1p from Saccharomyces cerevisiae labeled with quantum dots showed
that profilin is not required for formin-mediated processive elongation of
growing barbed ends. ATP-actin subunits polymerized by Bni1p and profilin
release the γ-phosphate of ATP on average >2.5 min after becoming
incorporated into filaments. Therefore, the release of γ-phosphate from
actin does not drive processive elongation. We compared experimentally
observed rates of processive elongation by a number of different FH2 domains
to kinetic computer simulations and found that actin subunit addition alone
likely provides the energy for fast processive elongation of filaments
mediated by FH1FH2-formin and profilin. We also studied the role of FH2
structure in processive elongation. We found that the flexible linker joining
the two halves of the FH2 dimer has a strong influence on dissociation of
formins from barbed ends but only a weak effect on elongation rates. Because
formins are most vulnerable to dissociation during translocation along the
growing barbed end, we propose that the flexible linker influences the
lifetime of this translocative state.Formins are multidomain proteins that assemble unbranched actin filament
structures for diverse processes in eukaryotic cells (reviewed in Ref.
1). Formins stimulate
nucleation of actin filaments and, in the presence of the actin
monomer-binding protein profilin, speed elongation of the barbed ends of
filaments
(2-6).
The ability of formins to influence elongation depends on the ability of
single formin molecules to remain bound to a growing barbed end through
multiple rounds of actin subunit addition
(7,
8). To stay associated during
subunit addition, a formin molecule must translocate processively on the
barbed end as each actin subunit is added
(1,
9-12).
This processive elongation of a barbed end by a formin is terminated when the
formin dissociates stochastically from the growing end during translocation
(4,
10).The formin-homology
(FH)2 1 and
2 domains are the best conserved domains of formin proteins
(2,
13,
14). The FH2 domain is the
signature domain of formins, and in many cases, is sufficient for both
nucleation and processive elongation of barbed ends
(2-4,
7,
15). Head-to-tail homodimers
of FH2 domains (12,
16) encircle the barbed ends
of actin filaments (9). In
vitro, association of barbed ends with FH2 domains slows elongation by
limiting addition of free actin monomers. This “gating” behavior
is usually explained by a rapid equilibrium of the FH2-associated end between
an open state competent for actin monomer association and a closed state that
blocks monomer binding (4,
9,
17).Proline-rich FH1 domains located N-terminal to FH2 domains are required for
profilin to stimulate formin-mediated elongation. Individual tracks of
polyproline in FH1 domains bind 1:1 complexes of profilin-actin and transfer
the actin directly to the FH2-associated barbed end to increase processive
elongation rates
(4-6,
8,
10,
17).Rates of elongation and dissociation from growing barbed ends differ widely
for FH1FH2 fragments from different formin homologs
(4). We understand few aspects
of FH1FH2 domains that influence gating, elongation or dissociation. In this
study, we examined the source of energy for formin-mediated processive
elongation, and the influence of FH2 structure on elongation and dissociation
from growing ends. In contrast to previous proposals
(6,
18), we found that fast
processive elongation mediated by FH1FH2-formins is not driven by energy from
the release of the γ-phosphate from ATP-actin filaments. Instead, the
data show that the binding of an actin subunit to the barbed end provides the
energy for processive elongation. We found that in similar polymerizing
conditions, different natural FH2 domains dissociate from growing barbed ends
at substantially different rates. We further observed that the length of the
flexible linker between the subunits of a FH2 dimer influences dissociation
much more than elongation. 相似文献
8.
Ruben K. Dagda Salvatore J. Cherra III Scott M. Kulich Anurag Tandon David Park Charleen T. Chu 《The Journal of biological chemistry》2009,284(20):13843-13855
Mitochondrial dysregulation is strongly implicated in Parkinson disease.
Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial
parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is
neuroprotective, less is known about neuronal responses to loss of PINK1
function. We found that stable knockdown of PINK1 induced mitochondrial
fragmentation and autophagy in SH-SY5Y cells, which was reversed by the
reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1.
Moreover, stable or transient overexpression of wild-type PINK1 increased
mitochondrial interconnectivity and suppressed toxin-induced
autophagy/mitophagy. Mitochondrial oxidant production played an essential role
in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines.
Autophagy/mitophagy served a protective role in limiting cell death, and
overexpressing Parkin further enhanced this protective mitophagic response.
The dominant negative Drp1 mutant inhibited both fission and mitophagy in
PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins
Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting
oxidative stress, suggesting active involvement of autophagy in morphologic
remodeling of mitochondria for clearance. To summarize, loss of PINK1 function
elicits oxidative stress and mitochondrial turnover coordinated by the
autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may
cooperate through different mechanisms to maintain mitochondrial
homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects
∼1% of the population worldwide. The causes of sporadic cases are unknown,
although mitochondrial or oxidative toxins such as
1-methyl-4-phenylpyridinium, 6-hydroxydopamine
(6-OHDA),3 and
rotenone reproduce features of the disease in animal and cell culture models
(1). Abnormalities in
mitochondrial respiration and increased oxidative stress are observed in cells
and tissues from parkinsonian patients
(2,
3), which also exhibit
increased mitochondrial autophagy
(4). Furthermore, mutations in
parkinsonian genes affect oxidative stress response pathways and mitochondrial
homeostasis (5). Thus,
disruption of mitochondrial homeostasis represents a major factor implicated
in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD
encodes for PTEN-induced kinase 1 (PINK1)
(6,
7). PINK1 is a cytosolic and
mitochondrially localized 581-amino acid serine/threonine kinase that
possesses an N-terminal mitochondrial targeting sequence
(6,
8). The primary sequence also
includes a putative transmembrane domain important for orientation of the
PINK1 domain (8), a conserved
kinase domain homologous to calcium calmodulin kinases, and a C-terminal
domain that regulates autophosphorylation activity
(9,
10). Overexpression of
wild-type PINK1, but not its PD-associated mutants, protects against several
toxic insults in neuronal cells
(6,
11,
12). Mitochondrial targeting
is necessary for some (13) but
not all of the neuroprotective effects of PINK1
(14), implicating involvement
of cytoplasmic targets that modulate mitochondrial pathobiology
(8). PINK1 catalytic activity
is necessary for its neuroprotective role, because a kinase-deficient K219M
substitution in the ATP binding pocket of PINK1 abrogates its ability to
protect neurons (14). Although
PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated
mutations differentially destabilize the protein, resulting in loss of
neuroprotective activities
(13,
15).Recent studies indicate that PINK1 and Parkin interact genetically
(3,
16-18)
to prevent oxidative stress
(19,
20) and regulate mitochondrial
morphology (21). Primary cells
derived from PINK1 mutant patients exhibit mitochondrial fragmentation with
disorganized cristae, recapitulated by RNA interference studies in HeLa cells
(3).Mitochondria are degraded by macroautophagy, a process involving
sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs)
for delivery to lysosomes (22,
23). Interestingly,
mitochondrial fission accompanies autophagic neurodegeneration elicited by the
PD neurotoxin 6-OHDA (24,
25). Moreover, mitochondrial
fragmentation and increased autophagy are observed in neurodegenerative
diseases including Alzheimer and Parkinson diseases
(4,
26-28).
Although inclusion of mitochondria in autophagosomes was once believed to be a
random process, as observed during starvation, studies involving hypoxia,
mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic
substrates in facultative anaerobes support the concept of selective
mitochondrial autophagy (mitophagy)
(29,
30). In particular,
mitochondrially localized kinases may play an important role in models
involving oxidative mitochondrial injury
(25,
31,
32).Autophagy is involved in the clearance of protein aggregates
(33-35)
and normal regulation of axonal-synaptic morphology
(36). Chronic disruption of
lysosomal function results in accumulation of subtly impaired mitochondria
with decreased calcium buffering capacity
(37), implicating an important
role for autophagy in mitochondrial homeostasis
(37,
38). Recently, Parkin, which
complements the effects of PINK1 deficiency on mitochondrial morphology
(3), was found to promote
autophagy of depolarized mitochondria
(39). Conversely, Beclin
1-independent autophagy/mitophagy contributes to cell death elicited by the PD
toxins 1-methyl-4-phenylpyridinium and 6-OHDA
(25,
28,
31,
32), causing neurite
retraction in cells expressing a PD-linked mutation in leucine-rich repeat
kinase 2 (40). Whereas
properly regulated autophagy plays a homeostatic and neuroprotective role,
excessive or incomplete autophagy creates a condition of “autophagic
stress” that can contribute to neurodegeneration
(28).As mitochondrial fragmentation
(3) and increased mitochondrial
autophagy (4) have been
described in human cells or tissues of PD patients, we investigated whether or
not the engineered loss of PINK1 function could recapitulate these
observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous
PINK1 gave rise to mitochondrial fragmentation and increased autophagy and
mitophagy, whereas stable or transient overexpression of PINK1 had the
opposite effect. Autophagy/mitophagy was dependent upon increased
mitochondrial oxidant production and activation of fission. The data indicate
that PINK1 is important for the maintenance of mitochondrial networks,
suggesting that coordinated regulation of mitochondrial dynamics and autophagy
limits cell death associated with loss of PINK1 function. 相似文献
9.
Graham H. Diering John Church Masayuki Numata 《The Journal of biological chemistry》2009,284(20):13892-13903
NHE5 is a brain-enriched Na+/H+ exchanger that
dynamically shuttles between the plasma membrane and recycling endosomes,
serving as a mechanism that acutely controls the local pH environment. In the
current study we show that secretory carrier membrane proteins (SCAMPs), a
group of tetraspanning integral membrane proteins that reside in multiple
secretory and endocytic organelles, bind to NHE5 and co-localize predominantly
in the recycling endosomes. In vitro protein-protein interaction
assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic
extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5
increased cell-surface abundance as well as transporter activity of NHE5
across the plasma membrane. Expression of a deletion mutant lacking the
SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the
N-terminal extension, reduced the transporter activity. Although both Arf6 and
Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across
the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by
dominant-negative Arf6 but not by dominant-negative Rab11. Together, these
results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes
and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are
especially sensitive to perturbations of pH
(1). Many voltage- and
ligand-gated ion channels that control membrane excitability are sensitive to
changes in cellular pH
(1-3).
Neurotransmitter release and uptake are also influenced by cellular and
organellar pH (4,
5). Moreover, the intra- and
extracellular pH of both neurons and glia are modulated in a highly transient
and localized manner by neuronal activity
(6,
7). Thus, neurons and glia
require sophisticated mechanisms to finely tune ion and pH homeostasis to
maintain their normal functions.Na+/H+ exchangers
(NHEs)3 were
originally identified as a class of plasma membrane-bound ion transporters
that exchange extracellular Na+ for intracellular H+,
and thereby regulate cellular pH and volume. Since the discovery of NHE1 as
the first mammalian NHE (8),
eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have
been isolated in mammals (9,
10). NHE1-5 commonly exhibit
transporter activity across the plasma membrane, whereas NHE6-9 are mostly
found in organelle membranes and are believed to regulate organellar pH in
most cell types at steady state
(11). More recently, NHE10 was
identified in human and mouse osteoclasts
(12,
13). However, the cDNA
encoding NHE10 shares only a low degree of sequence similarity with other
known members of the NHE gene family, raising the possibility that
this sodium-proton exchanger may belong to a separate gene family distantly
related to NHE1-9 (see Ref.
9).NHE gene family members contain 12 putative transmembrane domains
at the N terminus followed by a C-terminal cytosolic extension that plays a
role in regulation of the transporter activity by protein-protein interactions
and phosphorylation. NHEs have been shown to regulate the pH environment of
synaptic nerve terminals and to regulate the release of neurotransmitters from
multiple neuronal populations
(14-16).
The importance of NHEs in brain function is further exemplified by the
findings that spontaneous or directed mutations of the ubiquitously expressed
NHE1 gene lead to the progression of epileptic seizures, ataxia, and
increased mortality in mice
(17,
18). The progression of the
disease phenotype is associated with loss of specific neuron populations and
increased neuronal excitability. However, NHE1-null mice appear to
develop normally until 2 weeks after birth when symptoms begin to appear.
Therefore, other mechanisms may compensate for the loss of NHE1
during early development and play a protective role in the surviving neurons
after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene
family whose mRNA is expressed almost exclusively in the brain
(19,
20), although more recent
studies have suggested that NHE5 might be functional in other cell
types such as sperm (21,
22) and osteosarcoma cells
(23). Curiously, mutations
found in several forms of congenital neurological disorders such as
spinocerebellar ataxia type 4
(24-26)
and autosomal dominant cerebellar ataxia
(27-29)
have been mapped to chromosome 16q22.1, a region containing NHE5.
However, much remains unknown as to the molecular regulation of NHE5 and its
role in brain function.Very few if any proteins work in isolation. Therefore identification and
characterization of binding proteins often reveal novel functions and
regulation mechanisms of the protein of interest. To begin to elucidate the
biological role of NHE5, we have started to explore NHE5-binding proteins.
Previously, β-arrestins, multifunctional scaffold proteins that play a
key role in desensitization of G-protein-coupled receptors, were shown to
directly bind to NHE5 and promote its endocytosis
(30). This study demonstrated
that NHE5 trafficking between endosomes and the plasma membrane is regulated
by protein-protein interactions with scaffold proteins. More recently, we
demonstrated that receptor for activated
C-kinase 1 (RACK1), a scaffold protein that links
signaling molecules such as activated protein kinase C, integrins, and Src
kinase (31), directly
interacts with and activates NHE5 via integrin-dependent and independent
pathways (32). These results
further indicate that NHE5 is partly associated with focal adhesions and that
its targeting to the specialized microdomain of the plasma membrane may be
regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily
conserved tetra-spanning integral membrane proteins. SCAMPs are found in
multiple organelles such as the Golgi apparatus, trans-Golgi network,
recycling endosomes, synaptic vesicles, and the plasma membrane
(33,
34) and have been shown to
play a role in exocytosis
(35-38)
and endocytosis (39).
Currently, five isoforms of SCAMP have been identified in mammals. The
extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats,
which may allow these isoforms to participate in clathrin coat assembly and
vesicle budding by binding to Eps15 homology (EH)-domain proteins
(40,
41). Further, SCAMP2 was shown
recently to bind to the small GTPase Arf6
(38), which is believed to
participate in traffic between the recycling endosomes and the cell surface
(42,
43). More recent studies have
suggested that SCAMPs bind to organellar membrane type NHE7
(44) and the serotonin
transporter SERT (45) and
facilitate targeting of these integral membrane proteins to specific
intracellular compartments. We show in the current study that SCAMP2 binds to
NHE5, facilitates the cell-surface targeting of NHE5, and elevates
Na+/H+ exchange activity at the plasma membrane, whereas
expression of a SCAMP2 deletion mutant lacking the N-terminal domain
containing the NPF repeats suppresses the effect. Further we show that this
activity of SCAMP2 requires an active form of a small GTPase Arf6, but not
Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal
compartment and control its cell-surface abundance via an Arf6-dependent
pathway. 相似文献
10.
Matthias Gralle Michelle Gralle Botelho Fred S. Wouters 《The Journal of biological chemistry》2009,284(22):15016-15025
The amyloid precursor protein (APP) is implied both in cell growth and
differentiation and in neurodegenerative processes in Alzheimer disease.
Regulated proteolysis of APP generates biologically active fragments such as
the neuroprotective secreted ectodomain sAPPα and the neurotoxic
β-amyloid peptide. Furthermore, it has been suggested that the intact
transmembrane APP plays a signaling role, which might be important for both
normal synaptic plasticity and neuronal dysfunction in dementia. To understand
APP signaling, we tracked single molecules of APP using quantum dots and
quantitated APP homodimerization using fluorescence lifetime imaging
microscopy for the detection of Förster resonance energy transfer in
living neuroblastoma cells. Using selective labeling with synthetic
fluorophores, we show that the dimerization of APP is considerably higher at
the plasma membrane than in intracellular membranes. Heparan sulfate
significantly contributes to the almost complete dimerization of APP at the
plasma membrane. Importantly, this technique for the first time structurally
defines the initiation of APP signaling by binding of a relevant physiological
extracellular ligand; our results indicate APP as receptor for neuroprotective
sAPPα, as sAPPα binding disrupts APP dimers, and this disruption
of APP dimers by sAPPα is necessary for the protection of neuroblastoma
cells against starvation-induced cell death. Only cells expressing reversibly
dimerized wild-type, but not covalently dimerized mutant APP are protected by
sAPPα. These findings suggest a potentially beneficial effect of
increasing sAPPα production or disrupting APP dimers for neuronal
survival.The amyloid precursor protein
(APP)4 is known both
for its important role in the development and plasticity of the nervous system
(1–6)
and for its involvement in Alzheimer disease (AD)
(7,
8). Despite intensive research
efforts, the initial events that lead to the prevalent sporadic, i.e.
non-familial, forms of AD are still unclear. Furthermore, although a higher
gene dose of APP (9) or the
presence of pathological APP mutations is sufficient to induce familial AD
(for review, see Ref. 10), the
exact pathological mechanism that is triggered by APP is still under
debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are
thought to contribute to synaptic dysfunction and neurotoxicity
(11,
12). On the other hand, the
α-secretase-derived extracellular fragment of APP (sAPPα), which
is present at lower levels in AD patients than in controls
(13), has been shown to be
beneficial for memory function, to possess neuroprotective properties, and to
counteract the effects of Aβ
(14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration
in AD
(19–24);
however, the signal transduction pathway for transmembrane APP remains
unknown, although several potential regulatory proteins, glycosaminoglycans,
and metal ions are known to bind with high affinity to APP and sAPPα
(25,
26). The most common form of
signal transduction for single-pass transmembrane proteins is the
ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the
dimerization of transmembrane APP has been implied several times in the past.
Several studies have investigated the effects of presumed dimer-breaking
perturbations on biological read-outs, such as the production of Aβ
(27,
28), but without directly
measuring the APP aggregation state, or have investigated the aggregation
state of APP subdomains, often reconstituted in cell-free systems
(27–32).
Dimerization interfaces in both the extracellular and the transmembrane domain
have been suggested.In the studies investigating the aggregation state of full-length APP, most
of the employed methods, such as chemical cross-linking and
co-immunoprecipitation, do not lend themselves readily to a rigorous
quantitative analysis of the abundance of potentially instable dimers
(31,
33), whereas in other cases
the use of chimeras may have influenced the dimerization potential or
precluded the search for a natural stimulus
(23,
34). The only previously
reported direct observation of APP dimerization by Förster resonance
energy transfer (FRET) microscopy uses an assay in which the FRET efficiency
varies with the level of overexpression
(35). Therefore, a
concentration-dependent FRET component due to nonspecific stochastic
encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the
selective detection of APP dimers on the surface of live cells, where they
would encounter ligands, they could not differentiate between subpopulations
of APP. This may be one reason why no natural ligand of APP has ever been
shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective
sAPPα
(36–39).
The ligand-dependent dimerization of sAPPα in solution
(40) and its origination from
transmembrane APP suggest that APP might serve as receptor for sAPPα,
but this binding has never been experimentally shown. 相似文献
11.
Greg Brown Alexander Singer Vladimir V. Lunin Michael Proudfoot Tatiana Skarina Robert Flick Samvel Kochinyan Ruslan Sanishvili Andrzej Joachimiak Aled M. Edwards Alexei Savchenko Alexander F. Yakunin 《The Journal of biological chemistry》2009,284(6):3784-3792
Gluconeogenesis is an important metabolic pathway, which produces glucose
from noncarbohydrate precursors such as organic acids, fatty acids, amino
acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of
gluconeogenesis, is found in all organisms, and five different classes of
these enzymes have been identified. Here we demonstrate that Escherichia
coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which
show different catalytic properties. We present the first crystal structure of
a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and
in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor
(phosphate). The crystal structure of the ligand-free GlpX revealed a compact,
globular shape with two α/β-sandwich domains. The core fold of GlpX
is structurally similar to that of Li+-sensitive phosphatases
implying that they have a common evolutionary origin and catalytic mechanism.
The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that
the active site is located between two domains and accommodates several
conserved residues coordinating two metal ions and the substrate. The third
metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate
strongly inhibited activity of both GlpX and YggF, and the crystal structure
of the GlpX complex with phosphate demonstrated that the inhibitor molecule
binds to the active site. Alanine replacement mutagenesis of GlpX identified
12 conserved residues important for activity and suggested that
Thr90 is the primary catalytic residue. Our data provide insight
into the molecular mechanisms of the substrate specificity and catalysis of
GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase
(FBPase,2 EC
3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of
fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It
is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis,
and the product, fructose 6-phosphate, is an important precursor in various
biosynthetic pathways (1). In
all organisms, gluconeogenesis is an important metabolic pathway that allows
the cells to synthesize glucose from noncarbohydrate precursors, such as
organic acids, amino acids, and glycerol. FBPases are members of the large
superfamily of lithium-sensitive phosphatases, which includes three families
of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167
sequences, Pfam data base). These enzymes show metal-dependent and
lithium-sensitive phosphomonoesterase activity and include inositol
polyphosphate 1-phosphatases, inositol monophosphatases (IMPases),
3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting
on both inositol 1,4-bisphosphate and PAP (PIPases)
(2). They possess a common
structural core with the active site lying between α+β and
α/β domains (3).
Li+-sensitive phosphatases are putative targets for lithium therapy
in the treatment of manic depressive patients
(4), whereas FBPases are
targets for the development of drugs for the treatment of noninsulin-dependent
diabetes (5,
6). In addition, FBPase is
required for virulence in Mycobacterium tuberculosis and
Leishmania major and plays an important role in the production of
lysine and glutamate by Corynebacterium glutamicum
(7,
8).Presently, five different classes of FBPases have been proposed based on
their amino acid sequences (FBPases I to V)
(9–11).
Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in
various prokaryotes. Types I, II, and III are primarily in bacteria, type IV
in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in
thermophilic prokaryotes from both domains
(11). Many organisms have more
than one FBPase, mostly the combination of types I + II or II + III, but no
bacterial genome has a combination of types I and III FBPases
(9). The type I FBPase is the
most widely distributed among living organisms and is the primary FBPase in
Escherichia coli, most bacteria, a few archaea, and all
eukaryotes (9,
11–15).
The type II FBPases are represented by the E. coli GlpX and FBPase
F-I from Synechocystis PCC6803
(9,
16); type III is represented
by the Bacillus subtilis FBPase
(17); type IV is represented
by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus
furiosus (18), MJ0109
from Methanococcus jannaschii
(19), and AF2372 from
Archaeoglobus fulgidus
(20); and type V is
represented by the FBPases TK2164 from Pyrococcus
(Thermococcus) kodakaraensis and ST0318 from Sulfolobus
tokodai (10,
21).Three-dimensional structures of the type I (from pig kidney, spinach
chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V
(ST0318) FBPases have been solved
(10,
11,
19,
20,
22,
23). FBPases I and IV and
inositol monophosphatases share a common sugar phosphatase fold organized in
five layered interleaved α-helices and β-sheets
(α-β-α-β-α)
(2,
19,
24). ST0318 (an FBPase V
enzyme) is composed of one domain with a completely different four-layer
α-β-β-α fold
(10). The FBPases from these
three classes (I, IV, and V) require divalent cations for activity
(Mg2+, Mn2+, or Zn2+), and their structures
have revealed the presence of three or four metal ions in the active site.E. coli has five Li+-sensitive phosphatases as follows:
CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase
II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a
3′-phosphoadenosine 5′-phosphatase involved in the cysteine
biosynthesis pathway (25,
26), whereas SuhB is an
inositol monophosphatase (IMPase) that is also known as a suppressor of
temperature-sensitive growth phenotypes in E. coli
(27,
28). Fbp is required for
growth on gluconeogenic substrates and probably represents the main
gluconeogenic FBPase (12).
This enzyme has been characterized both biochemically and structurally and
shown to be inhibited by low concentrations of AMP (IC50 15
μm) (11,
29,
30). The E. coli
GlpX, a class II enzyme FBPase, has been shown to possess a
Mn2+-dependent FBPase activity
(9). The increased expression
of glpX from a multicopy plasmid complemented the Fbp-
phenotype; however, the glpX knock-out strain grew normally on
gluconeogenic substrates (succinate or glycerol)
(9).In this study, we present the first structure of a class II FBPase, the
E. coli GlpX, in a free state and in the complex with FBP + metals or
phosphate. We have demonstrated that the fold of GlpX is similar to that of
the lithium-sensitive phosphatases. We have identified the GlpX residues
important for activity and proposed a catalytic mechanism. We have also showed
that YggF is a third FBPase in E. coli, which has distinct catalytic
properties and is more sensitive than GlpX to the inhibition by lithium or
phosphate. 相似文献
12.
Christopher P. Gayer Lakshmi S. Chaturvedi Shouye Wang David H. Craig Thomas Flanigan Marc D. Basson 《The Journal of biological chemistry》2009,284(4):2001-2011
The intestinal epithelium is repetitively deformed by shear, peristalsis,
and villous motility. Such repetitive deformation stimulates the proliferation
of intestinal epithelial cells on collagen or laminin substrates via ERK, but
the upstream mediators of this effect are poorly understood. We hypothesized
that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this
mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β
(GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10%
deformation, and pharmacologic blockade of these molecules or reduction by
small interfering RNA (siRNA) prevented the mitogenic effect of strain in
Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required
PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that
AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain
mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera
including the PH domain and hinge region of AKT2 and the catalytic domain and
C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression
of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the
catalytic domain and C-tail of AKT2 did not. These data delineate a role for
PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is
required for both ERK and AKT2 activation, whereas AKT2 is sequentially
required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic
domain and tail region. Manipulating this pathway may prevent mucosal atrophy
and maintain the mucosal barrier in conditions such as ileus, sepsis, and
prolonged fasting when peristalsis and villous motility are decreased and the
mucosal barrier fails.Mechanical forces are part of the normal intestinal epithelial environment.
Numerous different forces deform these cells including shear stress from
endoluminal chyme, bowel peristalsis, and villous motility
(1,
2). During normal bowel
function the mucosa is subjected to injury that must be repaired to maintain
the mucosal barrier (3,
4). Deformation patterns of the
bowel are altered in conditions such as prolonged fasting, post-surgical
ileus, and sepsis states, resulting in profoundly reduced mucosal deformation.
When such states are prolonged, proliferation slows, the mucosa becomes
atrophic, and bacterial translocation may ensue as the mucosal barrier of the
gut breaks down
(5–7).In vitro, repetitive deformation is trophic for intestinal
epithelial cells (8) cultured
on type I or type IV collagen or laminin. Human Caco-2 intestinal epithelial
cells (9), non-transformed rat
IEC-6 intestinal epithelial cells
(10), and primary human
intestinal epithelial cells isolated from surgical specimens
(11) proliferate more rapidly
in response to cyclic strain
(12) unless substantial
quantities of fibronectin are added to the media or matrix
(11) to mimic the acute phase
reaction of acute or chronic inflammation and injury. Cyclic strain also
stimulates proliferation in HCT 116 colon cancer cells
(13) and differentiation of
Caco-2 cells cultured on a collagen substrate
(9). This phenomenon has also
been observed in vivo
(14). Thus, repetitive
deformation may help to maintain the normal homeostasis of the gut mucosa
under non-inflammatory conditions. Previous work in our laboratory has
implicated Src, focal adhesion kinase, and the mitogen-activated protein
kinase (MAPK)2
extracellular signal-related kinase (ERK) in the mitogenic effect of strain
(10). Although p38 is also
activated in Caco-2 cells subjected to cyclic strain on a collagen matrix, its
activity is not required for the mitogenic effect of strain
(12).Although often the PI3K/AKT pathway is thought of as a parallel pathway to
the MAPK, this is not always the case. Protein kinase C isoenzymes
differentially modulate thrombin effect on MAPK-dependent retinal pigment
epithelial cell (RPE) proliferation, and it has been shown that PI3K or AKT
inhibition prevented thrombin-induced ERK activation and RPE proliferation
(15).PI3K, AKT, and glycogen synthase kinase (GSK), a downstream target of AKT
(16), have been implemented in
intestinal epithelial cell proliferation in numerous cell systems not
involving strain
(17–19)
including uncontrolled proliferation in gastrointestinal cancers
(20–22).
Mechanical forces activate this pathway as well. PI3K and AKT are required for
increased extracellular pressure to stimulate colon cancer cell adhesion
(23), although the pathway by
which pressure stimulates colon cancer cells in suspension differs from the
response of adherent intestinal epithelial cells to repetitive deformation
(24), and GSK is not involved
in this effect.3
Repetitive strain also stimulates vascular endothelial cell proliferation via
PI3K and AKT (25,
26), whereas respiratory
strain stimulates angiogenic responses via PI3K
(27). We, therefore,
hypothesized that the PI3K/AKT/GSK axis would be involved in the mitogenic
effects of repetitive deformation on a collagen matrix.To test this hypothesis, we used the Flexcell apparatus to rhythmically
deform Caco-2 intestinal epithelial cells. IEC-6 cells were used to confirm
key results. A frequency of 10 cycles per min was used, which is similar in
order of magnitude to the frequency that the intestinal mucosa might be
deformed by peristalsis or villous motility in vivo
(28,
29). Mechanical forces such as
repetitive deformation are likely cell-type and frequency-specific, as
different cell types respond to different frequencies. Vascular endothelial
cells respond to frequencies of 60–80 cycles/min
(25), whereas intestinal
epithelial cells may actually decrease proliferation in response to
frequencies of 5 cycles/min
(30). We characterized PI3K,
AKT, and GSK phosphorylation with strain, blocked these molecules
pharmacologically or by siRNA, and delineated the specificity of the AKT
effect using isozyme-specific siRNA and transfection of AKT1/2 chimeras. We
also characterized the interaction of this pathway with the activation of ERK
by strain, which has previously been implicated in the mitogenic response
(12). 相似文献
13.
Andrés Norambuena Claudia Metz Lucas Vicu?a Antonia Silva Evelyn Pardo Claudia Oyanadel Loreto Massardo Alfonso González Andrea Soza 《The Journal of biological chemistry》2009,284(19):12670-12679
Galectins have been implicated in T cell homeostasis playing complementary
pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent
pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase
D/phosphatidic acid signaling pathway that has not been reported for any
galectin before. Gal-8 increases phosphatidic signaling, which enhances the
activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a
subsequent decrease in basal protein kinase A activity. Strikingly, rolipram
inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4
activation releases a negative influence of cAMP/protein kinase A on ERK1/2.
The resulting strong ERK1/2 activation leads to expression of the death factor
Fas ligand and caspase-mediated apoptosis. Several conditions that decrease
ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking
antibodies. In addition, experiments with freshly isolated human peripheral
blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28,
show that Gal-8 is pro-apoptotic on activated T cells, most likely on a
subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic
lupus erythematosus block the apoptotic effect of Gal-8. These results
implicate Gal-8 as a novel T cell suppressive factor, which can be
counterbalanced by function-blocking autoantibodies in autoimmunity.Glycan-binding proteins of the galectin family have been increasingly
studied as regulators of the immune response and potential therapeutic agents
for autoimmune disorders (1).
To date, 15 galectins have been identified and classified according with the
structural organization of their distinctive monomeric or dimeric carbohydrate
recognition domain for β-galactosides
(2,
3). Galectins are secreted by
unconventional mechanisms and once outside the cells bind to and cross-link
multiple glycoconjugates both at the cell surface and at the extracellular
matrix, modulating processes as diverse as cell adhesion, migration,
proliferation, differentiation, and apoptosis
(4–10).
Several galectins have been involved in T cell homeostasis because of their
capability to kill thymocytes, activated T cells, and T cell lines
(11–16).
Pro-apoptotic galectins might contribute to shape the T cell repertoire in the
thymus by negative selection, restrict the immune response by eliminating
activated T cells at the periphery
(1), and help cancer cells to
escape the immune system by eliminating cancer-infiltrating T cells
(17). They have also a
promising therapeutic potential to eliminate abnormally activated T cells and
inflammatory cells (1). Studies
on the mostly explored galectins, Gal-1, -3, and -9
(14,
15,
18–20),
as well as in Gal-2 (13),
suggest immunosuppressive complementary roles inducing different pathways to
apoptosis. Galectin-8
(Gal-8)4 is one of the
most widely expressed galectins in human tissues
(21,
22) and cancerous cells
(23,
24). Depending on the cell
context and mode of presentation, either as soluble stimulus or extracellular
matrix, Gal-8 can promote cell adhesion, spreading, growth, and apoptosis
(6,
7,
9,
10,
22,
25). Its role has been mostly
studied in relation to tumor malignancy
(23,
24). However, there is some
evidence regarding a role for Gal-8 in T cell homeostasis and autoimmune or
inflammatory disorders. For instance, the intrathymic expression and
pro-apoptotic effect of Gal-8 upon CD4highCD8high
thymocytes suggest a role for Gal-8 in shaping the T cell repertoire
(16). Gal-8 could also
modulate the inflammatory function of neutrophils
(26), Moreover Gal-8-blocking
agents have been detected in chronic autoimmune disorders
(10,
27,
28). In rheumatoid arthritis,
Gal-8 has an anti-inflammatory action, promoting apoptosis of synovial fluid
cells, but can be counteracted by a specific rheumatoid version of CD44
(CD44vRA) (27). In systemic
lupus erythematosus (SLE), a prototypic autoimmune disease, we recently
described function-blocking autoantibodies against Gal-8
(10,
28). Thus it is important to
define the role of Gal-8 and the influence of anti-Gal-8 autoantibodies in
immune cells.In Jurkat T cells, we previously reported that Gal-8 interacts with
specific integrins, such as α1β1, α3β1, and
α5β1 but not α4β1, and as a matrix protein promotes cell
adhesion and asymmetric spreading through activation of the extracellular
signal-regulated kinases 1 and 2 (ERK1/2)
(10). These early effects
occur within 5–30 min. However, ERK1/2 signaling supports long term
processes such as T cell survival or death, depending on the moment of the
immune response. During T cell activation, ERK1/2 contributes to enhance the
expression of interleukin-2 (IL-2) required for T cell clonal expansion
(29). It also supports T cell
survival against pro-apoptotic Fas ligand (FasL) produced by themselves and by
other previously activated T cells
(30,
31). Later on, ERK1/2 is
required for activation-induced cell death, which controls the extension of
the immune response by eliminating recently activated and restimulated T cells
(32,
33). In activation-induced
cell death, ERK1/2 signaling contributes to enhance the expression of FasL and
its receptor Fas/CD95 (32,
33), which constitute a
preponderant pro-apoptotic system in T cells
(34). Here, we ask whether
Gal-8 is able to modulate the intensity of ERK1/2 signaling enough to
participate in long term processes involved in T cell homeostasis.The functional integration of ERK1/2 and PKA signaling
(35) deserves special
attention. cAMP/PKA signaling plays an immunosuppressive role in T cells
(36) and is altered in SLE
(37). Phosphodiesterases
(PDEs) that degrade cAMP release the immunosuppressive action of cAMP/PKA
during T cell activation (38,
39). PKA has been described to
control the activity of ERK1/2 either positively or negatively in different
cells and processes (35). A
little explored integration among ERK1/2 and PKA occurs via phosphatidic acid
(PA) and PDE signaling. Several stimuli activate phospholipase D (PLD) that
hydrolyzes phosphatidylcholine into PA and choline. Such PLD-generated PA
plays roles in signaling interacting with a variety of targeting proteins that
bear PA-binding domains (40).
In this way PA recruits Raf-1 to the plasma membrane
(41). It is also converted by
phosphatidic acid phosphohydrolase (PAP) activity into diacylglycerol (DAG),
which among other functions, recruits and activates the GTPase Ras
(42). Both Ras and Raf-1 are
upstream elements of the ERK1/2 activation pathway
(43). In addition, PA binds to
and activates PDEs of the type 4 subfamily (PDE4s) leading to decreased cAMP
levels and PKA down-regulation
(44). The regulation and role
of PA-mediated control of ERK1/2 and PKA remain relatively unknown in T cell
homeostasis, because it is also unknown whether galectins stimulate the PLD/PA
pathway.Here we found that Gal-8 induces apoptosis in Jurkat T cells by triggering
cross-talk between PKA and ERK1/2 pathways mediated by PLD-generated PA. Our
results for the first time show that a galectin increases the PA levels,
down-regulates the cAMP/PKA system by enhancing rolipram-sensitive PDE
activity, and induces an ERK1/2-dependent expression of the pro-apoptotic
factor FasL. The enhanced PDE activity induced by Gal-8 is required for the
activation of ERK1/2 that finally leads to apoptosis. Gal-8 also induces
apoptosis in human peripheral blood mononuclear cells (PBMC), especially after
activating T cells with anti-CD3/CD28. Therefore, Gal-8 shares with other
galectins the property of killing activated T cells contributing to the T cell
homeostasis. The pathway involves a particularly integrated signaling context,
engaging PLD/PA, cAMP/PKA, and ERK1/2, which so far has not been reported for
galectins. The pro-apoptotic function of Gal-8 also seems to be unique in its
susceptibility to inhibition by anti-Gal-8 autoantibodies. 相似文献
14.
15.
16.
17.
Kelvin B. Luther Hermann Schindelin Robert S. Haltiwanger 《The Journal of biological chemistry》2009,284(5):3294-3305
The Notch receptor is critical for proper development where it orchestrates
numerous cell fate decisions. The Fringe family of
β1,3-N-acetylglucosaminyltransferases are regulators of this
pathway. Fringe enzymes add N-acetylglucosamine to O-linked
fucose on the epidermal growth factor repeats of Notch. Here we have analyzed
the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis
strategy for Lfng was guided by a multiple sequence alignment of Fringe
proteins and solutions from docking an epidermal growth factor-like
O-fucose acceptor substrate onto a homology model of Lfng. We
targeted three main areas as follows: residues that could help resolve where
the fucose binds, residues in two conserved loops not observed in the
published structure of Manic Fringe, and residues predicted to be involved in
UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a
kinetic analysis of mutant enzyme activity toward the small molecule acceptor
substrate 4-nitrophenyl-α-l-fucopyranoside to judge their
effect on Lfng activity. Our results support the positioning of
O-fucose in a specific orientation to the catalytic residue. We also
found evidence that one loop closes off the active site coincident with, or
subsequent to, substrate binding. We propose a mechanism whereby the ordering
of this short loop may alter the conformation of the catalytic aspartate.
Finally, we identify several residues near the UDP-GlcNAc-binding site, which
are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases,
including multiple sclerosis
(1), several forms of cancer
(2-4),
cerebral autosomal dominant arteriopathy with sub-cortical infarcts and
leukoencephalopathy (5), and
spondylocostal dysostosis
(SCD)3
(6-8).
The transmembrane Notch signaling receptor is activated by members of the DSL
(Delta, Serrate, Lag2) family of ligands
(9,
10). In the endoplasmic
reticulum, O-linked fucose glycans are added to the epidermal growth
factor-like (EGF) repeats of the Notch extracellular domain by protein
O-fucosyltransferase 1
(11-13).
These O-fucose monosaccharides can be elongated in the Golgi
apparatus by three highly conserved
β1,3-N-acetylglucosaminyltransferases of the Fringe family
(Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals)
(14-16).
The formation of this GlcNAc-β1,3-Fuc-α1,
O-serine/threonine disaccharide is necessary and sufficient for
subsequent elongation to a tetrasaccharide
(15,
19), although elongation past
the disaccharide in Drosophila is not yet clear
(20,
21). Elongation of
O-fucose by Fringe is known to potentiate Notch signaling from Delta
ligands and inhibit signaling from Serrate ligands
(22). Delta ligands are termed
Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate
are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on
Drosophila Notch can be recapitulated in Notch ligand in
vitro binding assays using purified components, suggesting that the
elongation of O-fucose by Fringe alters the binding of Notch to its
ligands (21). Although Fringe
also appears to alter Notch-ligand interactions in mammals, the effects of
elongation of the glycan past the O-fucose monosaccharide is more
complicated and appears to be cell type-, receptor-, and ligand-dependent (for
a recent review see Ref.
23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate
UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc
disaccharide
(14-16).
They belong to the GT-A-fold of inverting glycosyltransferases, which includes
N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase
I (17,
18). The mechanism is presumed
to proceed through the abstraction of a proton from the acceptor substrate by
a catalytic base (Asp or Glu) in the active site. This creates a nucleophile
that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its
configuration from α (on the nucleotide sugar) to β (in the
product) (24,
25). The enzyme then releases
the acceptor substrate modified with a disaccharide and UDP. The Mfng
structure (26) leaves little
doubt as to the identity of the catalytic residue, which in all likelihood is
aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe
throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc
soaked into the crystals (26)
showed density only for the UDP portion of the nucleotide-sugar donor and no
density for two loops flanking either side of the active site. The presence of
flexible loops that become ordered upon substrate binding is a common
observation with glycosyltransferases in the GT-A fold family
(18,
25). Density for the entire
donor was observed in the structure of rabbit
N-acetylglucosaminyltransferase I
(27). In this case, ordering
of a previously disordered loop upon UDP-GlcNAc binding may have contributed
to increased stability of the donor. In the case of bovine
β1,4-galactosyltransferase I, a section of flexible random coil from the
apo-structure was observed to change its conformation to α-helical upon
donor substrate binding (28).
Both loops in Lfng are highly conserved, and we have mutated a number of
residues in each to test the hypothesis that they interact with the
substrates. The mutagenesis strategy was also guided by docking of an
EGF-O-fucose acceptor substrate into the active site of the Lfng
model as well as comparison of the Lfng model with a homology model of the
β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on
thrombospondin type 1 repeats
(29,
30). The β3GlcT is
predicted to be a GT-A fold enzyme related to the Fringe family
(17,
18,
29). 相似文献
18.
19.