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1.
David S. Harburger Mohamed Bouaouina David A. Calderwood 《The Journal of biological chemistry》2009,284(17):11485-11497
Integrin activation, the rapid conversion of integrin adhesion receptors
from low to high affinity, occurs in response to intracellular signals that
act on the short cytoplasmic tails of integrin β subunits. Talin binding
to integrin β tails provides one key activation signal, but additional
factors are likely to cooperate with talin to regulate integrin activation.
The integrin β tail-binding proteins kindlin-2 and kindlin-3 were
recently identified as integrin co-activators. Here we report an analysis of
kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails
and describe the effect of kindlin expression on integrin activation. We
demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin
β tails in pulldown binding assays. Our mutational analysis shows that
the second conserved NXXY motif (Tyr795), a preceding
threonine-containing region (Thr788 and Thr789) of the
integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the
kindlin FERM domain (kindlin-1 Trp612 and kindlin-2
Trp615) are required for direct kindlin-integrin interactions.
Similar interactions were observed for integrin β3 tails. Using
fluorescence-activated cell sorting we further show that transient expression
of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of
endogenous α5β1 or stably expressed αIIbβ3 integrins.
This inhibition is not dependent on direct kindlin-integrin interactions
because mutant kindlins exhibiting impaired integrin binding activity
effectively inhibit integrin activation. Consistent with previous reports, we
find that when co-expressed with the talin head, kindlin-1 or -2 can activate
αIIbβ3. This effect is dependent on an intact integrin-binding site
in kindlin. Notably however, even when co-expressed with activating levels of
talin head, neither kindlin-1 or -2 can cooperate with talin to activate
β1 integrins; instead they strongly inhibit talin-mediated activation. We
suggest that kindlins are adaptor proteins that regulate integrin activation,
that kindlin expression levels determine their effects, and that kindlins may
exert integrin-specific effects.Integrins are a family of αβ heterodimeric transmembrane
receptors that mediate cell adhesion to extracellular matrix, cell surface, or
soluble protein ligands and modulate a variety of intracellular signaling
cascades. A key feature of integrins is their ability to dynamically regulate
their affinity for extracellular ligands. In a tightly regulated process
generally termed integrin activation, intracellular signals that impinge upon
the β subunit cytoplasmic tail induce conformational rearrangements in
the integrin extracellular domains, increasing the binding affinity for
extracellular ligands
(1-3).
Ligand-bound integrins then recruit additional signaling, adaptor, and
cytoskeletal proteins to the integrin cytoplasmic domains, providing
mechanical connections to the actin cytoskeleton and a link to a variety of
signal transduction pathways
(2-8).Recent years have seen significant advances in our understanding of
integrin activation. Notable among these is the identification of the actin-
and integrin-binding protein talin as a key integrin activator
(1,
9). The 50-kDa talin head
contains the principal integrin-binding site, and expression of the talin head
is sufficient to activate β1 and β3 integrins
(10,
11). The talin head contains a
FERM (four point one ezrin radixin
moesin) domain. FERM domains consist of trefoil arrangement of
three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3
subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in
integrin β tails, and this interaction is necessary for integrin
activation in vitro and in vivo
(10,
12-19).
However, although abundant evidence supports the importance of talin binding
to integrin β tails during integrin activation, differences in
sensitivity of integrins to talin activation and submaximal activation by
overexpressed talin suggested that other activating factors may cooperate with
talin (10,
20). In an attempt to identify
and characterize potential co-activators, we investigated the kindlin family
of FERM domain-containing proteins.Kindlin family proteins
(21) were first characterized
in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112,
was identified in an embryonic screen for defective motility and shown to be
essential for the assembly of proper cell-matrix adhesion structures, where it
normally co-localized with β integrin
(22-24).
UNC-112 is conserved across many species, because the nematode, fly, and human
homologs are ∼60% similar (∼41% identical) over their entire length
(24). Humans express three
known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2
(Mig2 and mig-2), and kindlin-3 (Mig2B and URP2)
(25-27).
Kindlin-1 and -2 are most closely related, sharing 60% identity and 74%
similarity, whereas kindlin-3 shares 53% identity and 69% similarity to
kindlin-1 and 49% identity and 67% similarity to kindlin-2
(28). The kindlin proteins all
contain a predicted Pleckstrin homology domain and a FERM domain that is most
closely related to the talin FERM domain, particularly within the
integrin-binding F3 subdomain
(29). Based on this sequence
similarity we proposed that kindlin FERM domains may directly bind integrin
β tails, and we previously showed that kindlin-1 could be pulled down
from cell lysates using recombinant integrin β1 and β3 tails and
that kindlin-1 co-localized with integrins in focal adhesions
(29). A similar localization
was reported for kindlin-2
(26,
30), and recent reports
provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of
integrin activation
(31-33).
Here, we have used integrin pulldown assays to demonstrate direct binding of
full-length kindlin-1 to the cytoplasmic tails of β1A and β3
integrins and to identify key binding residues within the integrin tails and
the kindlin F3 subdomain. We confirm that these interactions are important for
recruiting kindlin-1 to focal adhesions and show that, contrary to
expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3
integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate
with expressed talin head to activate β3 but not β1 integrins. We
therefore provide the first data suggesting that kindlin-1 and -2 effects on
integrin activation may show β subunit specificity. 相似文献
2.
Ho-Sup Lee Chinten James Lim Wilma Puzon-McLaughlin Sanford J. Shattil Mark H. Ginsberg 《The Journal of biological chemistry》2009,284(8):5119-5127
Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule
(RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to
promote talin-dependent integrin activation. Here, we show that MRL proteins
function as scaffolds that connect the membrane targeting sequences in Ras
GTPases to talin, thereby recruiting talin to the plasma membrane and
activating integrins. The MRL proteins bound directly to talin via short,
N-terminal sequences predicted to form amphipathic helices. RIAM-induced
integrin activation required both its capacity to bind to Rap1 and to talin.
Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the
talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A.
This minimized Rap-RIAM module was sufficient to target talin to the plasma
membrane and to mediate integrin activation, even in the absence of Rap1
activity. We identified a short talin binding sequence in Lamellipodin (Lpd),
another MRL protein; talin binding Lpd sequence joined to a Rap1
membrane-targeting sequence is sufficient to recruit talin and activate
integrins. These data establish the mechanism whereby MRL proteins interact
with both talin and Ras GTPases to activate integrins.Increased affinity (“activation”) of cellular integrins is
central to physiological events such as cell migration, assembly of the
extracellular matrix, the immune response, and hemostasis
(1). Each integrin comprises a
type I transmembrane α and β subunit, each of which has a large
extracellular domain, a single transmembrane domain, and a cytoplasmic domain
(tail). Talin binds to most integrin β cytoplasmic domains and the
binding of talin to the integrin β tail initiates integrin activation
(2–4).
A small, PTB-like domain of talin mediates activation via a two-site
interaction with integrin β tails
(5), and this PTB domain is
functionally masked in the intact talin molecule
(6). A central question in
integrin biology is how the talin-integrin interaction is regulated to control
integrin activation; recent work has implicated Ras GTPases as critical
signaling modules in this process
(7).Ras proteins are small monomeric GTPases that cycle between the GTP-bound
active form and the GDP-bound inactive form. Guanine nucleotide exchange
factors (GEFs) promote Ras activity by exchanging bound GDP for GTP, whereas
GTPase-activating proteins
(GAPs)3 enhance the
hydrolysis of Ras-bound GTP to GDP (for review, see Ref.
8). The Ras subfamily members
Rap1A and Rap1B stimulate integrin activation
(9,
10). For example, expression
of constitutively active Rap1 activates integrin αMβ2 in
macrophage, and inhibition of Rap1 abrogated integrin activation induced by
inflammatory agonists
(11–13).
Murine T-cells expressing constitutively active Rap1 manifest enhanced
integrin dependent cell adhesion
(14). In platelets, Rap1 is
rapidly activated by platelet agonists
(15,
16). A knock-out of Rap1B
(17) or of the Rap1GEF,
RasGRP2 (18), resulted in
impairment of αIIbβ3-dependent platelet aggregation, highlighting
the importance of Rap1 in platelet aggregation in vivo. Thus, Rap1
GTPases play important roles in the activation of several integrins in
multiple biological contexts.Several Rap1 effectors have been implicated in integrin activation
(19–21).
Rap1-GTP-interacting adaptor molecule (RIAM) is a Rap1 effector that is a
member of the MRL (Mig-10/RIAM/Lamellipodin) family of adaptor proteins
(20). RIAM contains Ras
association (RA) and pleckstrin homology (PH) domains and proline-rich
regions, which are defining features of the MRL protein family. In Jurkat
cells, RIAM overexpression induces β1 and β2 integrin-mediated cell
adhesion, and RIAM knockdown abolishes Rap1-dependent cell adhesion
(20), indicating RIAM is a
downstream regulator of Rap1-dependent signaling. RIAM regulates actin
dynamics as RIAM expression induces cell spreading; conversely, its depletion
reduces cellular F-actin content
(20). Whereas RIAM is greatly
enriched in hematopoietic cells, Lamellipodin (Lpd) is a paralogue present in
fibroblasts and other somatic cells
(22).Recently we used forward, reverse, and synthetic genetics to engineer and
order an integrin activation pathway in Chinese hamster ovary cells expressing
a prototype activable integrin, platelet αIIbβ3. We found that Rap1
induced formation of an “integrin activation complex” containing
RIAM and talin (23). Here, we
have established the mechanism whereby Ras GTPases cooperate with MRL family
proteins, RIAM and Lpd, to regulate integrin activation. We find that MRL
proteins function as scaffolds that connect the membrane targeting sequences
in Ras GTPases to talin, thereby recruiting talin to integrins at the plasma
membrane. 相似文献
3.
4.
Mikael K. Schnizler Katrin Schnizler Xiang-ming Zha Duane D. Hall John A. Wemmie Johannes W. Hell Michael J. Welsh 《The Journal of biological chemistry》2009,284(5):2697-2705
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and
peripheral neurons where it generates transient cation currents when
extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic
dendritic spines and has critical effects in neurological diseases associated
with a reduced pH. However, knowledge of the proteins that interact with
ASIC1a and influence its function is limited. Here, we show that
α-actinin, which links membrane proteins to the actin cytoskeleton,
associates with ASIC1a in brain and in cultured cells. The interaction
depended on an α-actinin-binding site in the ASIC1a C terminus that was
specific for ASIC1a versus other ASICs and for α-actinin-1 and
-4. Co-expressing α-actinin-4 altered ASIC1a current density, pH
sensitivity, desensitization rate, and recovery from desensitization.
Moreover, reducing α-actinin expression altered acid-activated currents
in hippocampal neurons. These findings suggest that α-actinins may link
ASIC1a to a macromolecular complex in the postsynaptic membrane where it
regulates ASIC1a activity.Acid-sensing ion channels
(ASICs)2 are
H+-gated members of the DEG/ENaC family
(1–3).
Members of this family contain cytosolic N and C termini, two transmembrane
domains, and a large cysteine-rich extracellular domain. ASIC subunits combine
as homo- or heterotrimers to form cation channels that are widely expressed in
the central and peripheral nervous systems
(1–4).
In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have
two splice forms, a and b. Central nervous system neurons express ASIC1a,
ASIC2a, and ASIC2b
(5–7).
Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2,
and half-maximal activation occurs at pH 6.5–6.8
(8–10).
These channels desensitize in the continued presence of a low extracellular
pH, and they can conduct Ca2+
(9,
11–13).
ASIC1a is required for acid-evoked currents in central nervous system neurons;
disrupting the gene encoding ASIC1a eliminates H+-gated currents
unless extracellular pH is reduced below pH 5.0
(5,
7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions
and present in dendritic spines, the site of excitatory synapses
(5,
14,
15). Consistent with this
localization, ASIC1a null mice manifested deficits in hippocampal
long term potentiation, learning, and memory, which suggested that ASIC1a is
required for normal synaptic plasticity
(5,
16). ASICs might be activated
during neurotransmission when synaptic vesicles empty their acidic contents
into the synaptic cleft or when neuronal activity lowers extracellular pH
(17–19).
Ion channels, including those at the synapse often interact with multiple
proteins in a macromolecular complex that incorporates regulators of their
function (20,
21). For ASIC1a, only a few
interacting proteins have been identified. Earlier work indicated that ASIC1a
interacts with another postsynaptic scaffolding protein, PICK1
(15,
22,
23). ASIC1a also has been
reported to interact with annexin II light chain p11 through its cytosolic N
terminus to increase cell surface expression
(24) and with
Ca2+/calmodulin-dependent protein kinase II to phosphorylate the
channel (25). However, whether
ASIC1a interacts with additional proteins and with the cytoskeleton remain
unknown. Moreover, it is not known whether such interactions alter ASIC1a
function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic
residues that might bind α-actinins. α-Actinins cluster membrane
proteins and signaling molecules into macromolecular complexes and link
membrane proteins to the actincytoskeleton (for review, Ref.
26). Four genes encode
α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an
N-terminal head domain that binds F-actin, a C-terminal region containing two
EF-hand motifs, and a central rod domain containing four spectrin-like motifs
(26–28).
The C-terminal portion of the rod segment appears to be crucial for binding to
membrane proteins. The α-actinins assemble into antiparallel homodimers
through interactions in their rod domain. α-Actinins-1, -2, and -4 are
enriched in dendritic spines, concentrating at the postsynaptic membrane
(29–35).
In the postsynaptic membrane of excitatory synapses, α-actinin connects
the NMDA receptor to the actin cytoskeleton, and this interaction is key for
Ca2+-dependent inhibition of NMDA receptors
(36–38).
α-Actinins can also regulate the membrane trafficking and function of
several cation channels, including L-type Ca2+ channels,
K+ channels, and TRP channels
(39–41).To better understand the function of ASIC1a channels in macromolecular
complexes, we asked if ASIC1a associates with α-actinins. We were
interested in the α-actinins because they and ASIC1a, both, are present
in dendritic spines, ASIC1a contains a potential α-actinin binding
sequence, and the related epithelial Na+ channel (ENaC) interacts
with the cytoskeleton (42,
43). Therefore, we
hypothesized that α-actinin interacts structurally and functionally with
ASIC1a. 相似文献
5.
6.
Jacamo R Sinnett-Smith J Rey O Waldron RT Rozengurt E 《The Journal of biological chemistry》2008,283(19):12877-12887
Protein kinase D (PKD) is a serine/threonine protein kinase rapidly
activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C
(PKC)-dependent pathway. Recently, PKD has been implicated in the regulation
of long term cellular activities, but little is known about the mechanism(s)
of sustained PKD activation. Here, we show that cell treatment with the
preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid
(1–5-min) PKD activation induced by bombesin stimulation, but this
inhibition was greatly diminished at later times of bombesin stimulation
(e.g. 45 min). These results imply that GPCR-induced PKD activation
is mediated by early PKC-dependent and late PKC-independent mechanisms.
Western blot analysis with site-specific antibodies that detect the
phosphorylated state of the activation loop residues Ser744 and
Ser748 revealed striking PKC-independent phosphorylation of
Ser748 as well as Ser744 phosphorylation that remained
predominantly but not completely PKC-dependent at later times of bombesin or
vasopressin stimulation (20–90 min). To determine the mechanisms
involved, we examined activation loop phosphorylation in a set of PKD mutants,
including kinase-deficient, constitutively activated, and PKD forms in which
the activation loop residues were substituted for alanine. Our results show
that PKC-dependent phosphorylation of the activation loop Ser744
and Ser748 is the primary mechanism involved in early phase PKD
activation, whereas PKD autophosphorylation on Ser748 is a major
mechanism contributing to the late phase of PKD activation occurring in cells
stimulated by GPCR agonists. The present studies identify a novel mechanism
induced by GPCR activation that leads to late, PKC-independent PKD
activation.A rapid increase in the synthesis of lipid-derived second messengers with
subsequent activation of protein phosphorylation cascades has emerged as a
fundamental signal transduction mechanism triggered by multiple extracellular
stimuli, including hormones, neurotransmitters, chemokines, and growth factors
(1). Many of these agonists
bind to G protein-coupled receptors
(GPCRs),4 activate
heterotrimeric G proteins and stimulate isoforms of the phospholipase C
family, including β, γ, δ, and ε (reviewed in Refs.
1 and
2). Activated phospholipase Cs
catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce
the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG).
Inositol 1,4,5-trisphosphate mobilizes Ca2+ from intracellular
stores (3,
4) whereas DAG directly
activates the classic (α, β, and γ) and novel (δ,
ε, η, and θ) isoforms of PKC
(5–7).
Although it is increasingly recognized that each PKC isozyme has specific
functions in vivo
(5–8),
the mechanisms by which PKC-mediated signals are propagated to critical
downstream targets remain incompletely defined.PKD, also known initially as PKCμ
(9,
10), and two recently
identified serine protein kinases termed PKD2
(11) and PKCν/PKD3
(12,
13), which are similar in
overall structure and primary amino acid sequence to PKD
(14), constitute a new protein
kinase family within the Ca2+/calmodulin-dependent protein kinase
group (15) and separate from
the previously identified PKCs
(14). Salient features of PKD
structure include an N-terminal regulatory region containing a tandem repeat
of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain)
that confers high affinity binding to phorbol esters and DAG
(9,
16,
17), followed by a pleckstrin
homology (PH) domain that negatively regulates catalytic activity
(18,
19). The C-terminal region of
the PKDs contains its catalytic domain, which is distantly related to
Ca2+-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity
maintained by the N-terminal domain, which represses the catalytic activity of
the enzyme by autoinhibition. Consistent with this model, deletions or single
amino acid substitutions in the PH domain result in constitutive kinase
activity
(18–20).
Physiological activation of PKD within cells occurs via a
phosphorylation-dependent mechanism first identified in our laboratory
(21). In response to cellular
stimuli, PKD is converted from a low activity form into a persistently active
form that is retained during isolation from cells, as shown by in
vitro kinase assays performed in the absence of lipid co-activators
(21,
22). PKD activation has been
demonstrated in response to engagement of specific GPCRs either by regulatory
peptides
(23–30)
or lysophosphatidic acid (27,
31,
32); signaling through
Gq, G12, Gi, and Rho
(27,
31–34);
activation of receptor tyrosine kinases, such as the platelet-derived growth
factor receptor (23,
35,
36); cross-linking of B-cell
receptor and T-cell receptor in B and T lymphocytes, respectively
(37–40);
and oxidative stress
(41–44).Throughout these studies, multiple lines of evidence indicated that PKC
activity is necessary for rapid PKD activation within intact cells. For
example, rapid PKD activation was selectively and potently blocked by cell
treatment with preferential PKC inhibitors (e.g. GF 109203X or
Gö 6983) that do not directly inhibit PKD catalytic activity
(21,
22), implying that PKD
activation in intact cells is mediated, directly or indirectly, through PKCs.
In line with this conclusion, cotransfection of PKD with active mutant forms
of “novel” PKCs (PKCs δ, ε, η, and θ)
resulted in robust PKD activation in the absence of cell stimulation
(21,
44–46).
Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade
in response to multiple GPCR agonists in a broad range of cell types,
including normal and cancer cells (reviewed in Ref.
14). Our previous studies
identified Ser744 and Ser748 in the PKD activation loop
(also referred as the activation segment or T-loop) as phosphorylation sites
critical for PKC-mediated PKD activation (reviewed in Ref.
14). Collectively, these
findings demonstrated the existence of rapidly activated PKC-PKD protein
kinase cascade(s) and raised the possibility that some PKC-dependent
biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular
activities and processes, including signal transduction
(30,
47–49),
chromatin modification (50),
Golgi organization and function
(51,
52), c-Jun function
(47,
53,
54), NFκB-mediated gene
expression (43,
55,
56), and cell survival,
migration, and differentiation and DNA synthesis and proliferation (reviewed
in Ref. 14). Thus, mounting
evidence indicates that PKD has a remarkable diversity of both its signal
generation and distribution and its potential for complex regulatory
interactions with multiple downstream pathways, leading to multiple responses,
including long term cellular events. Despite increasing recognition of its
importance, very little is known about the mechanism(s) of sustained PKD
activation as opposed to the well documented rapid, PKC-dependent PKD
activation.The results presented here demonstrate that prolonged GPCR-induced PKD
activation is mediated by sequential PKC-dependent and PKC-independent phases
of regulation. We report here, for the first time, that PKD
autophosphorylation on Ser748 is a major mechanism contributing to
the late phase of PKD activation occurring in cells stimulated by GPCR
agonists. The present studies expand previous models of PKD regulation by
identifying a novel mechanism induced by GPCR activation that leads to late,
PKC-independent PKD activation. 相似文献
7.
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
8.
Benjamin T. Goult Neil Bate Nicholas J. Anthis Kate L. Wegener Alexandre R. Gingras Bipin Patel Igor L. Barsukov Iain D. Campbell Gordon C. K. Roberts David R. Critchley 《The Journal of biological chemistry》2009,284(22):15097-15106
Talin is a large flexible rod-shaped protein that activates the integrin
family of cell adhesion molecules and couples them to cytoskeletal actin. It
exists in both globular and extended conformations, and an intramolecular
interaction between the N-terminal F3 FERM subdomain and the C-terminal part
of the talin rod contributes to an autoinhibited form of the molecule. Here,
we report the solution structure of the primary F3 binding domain within the
C-terminal region of the talin rod and use intermolecular nuclear Overhauser
effects to determine the structure of the complex. The rod domain (residues
1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into
a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3
(residues 316–326) interacts with a cluster of acidic residues in the
middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain
competes with β3-integrin tails for binding to F3, and the structure of
the complex suggests that the rod is also likely to sterically inhibit binding
of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in
regulating the affinity of the integrin family of cell adhesion molecules for
ligand (1) and in coupling
integrins to the actin cytoskeleton
(2). Thus, depletion of talin
results in defects in integrin activation
(3), integrin signaling through
focal adhesion kinase, the maintenance of cell spreading, and the assembly of
focal adhesions in cultured cells
(4). In the whole organism,
studies on the single talin gene in worms
(5) and flies
(6) show that talin is
essential for a variety of integrin-mediated events that are crucial for
normal embryonic development. In vertebrates, there are two talin
genes, and mice carrying a talin1 null allele fail to complete
gastrulation (7).
Tissue-specific inactivation of talin1 results in an inability to activate
integrins in platelets (8,
9), defects in the
membrane-cytoskeletal interface in megakaryocytes
(10), and disruption of the
myotendinous junction in skeletal muscle
(11). In contrast, mice
homozygous for a talin2 gene trap allele have no phenotype, although
the allele may be hypomorphic
(12).Recent structural studies have provided substantial insights into the
molecular basis of talin action. Talin is composed of an N-terminal globular
head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The
talin head contains a
FERM2 domain (made up
of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0
(2). Studies by Wegener et
al. (30) have shown how
the F3 FERM subdomain, which has a phosphotyrosine binding domain fold,
interacts with both the canonical NPXY motif and the
membrane-proximal helical region of the cytoplasmic tails of integrin
β-subunits (13). The
latter interaction apparently activates the integrin by disrupting the salt
bridge between the integrin α- and β-subunit tails that normally
keeps integrins locked in a low affinity state. The observation that the F0
region is also important in integrin activation
(14) may be explained by our
recent finding that F0 binds, albeit with low affinity,
Rap1-GTP,3 a known
activator of integrins (15,
16). The talin rod is made up
of a series of amphipathic α-helical bundles
(17–20)
and contains a second integrin binding site (IBS2)
(21), numerous binding sites
for the cytoskeletal protein vinculin
(22), at least two actin
binding sites (23), and a
C-terminal helix that is required for assembly of talin dimers
(20,
24).Both biochemical (25) and
cellular studies (16) suggest
that the integrin binding sites in full-length talin are masked, and both
phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in
exposing these sites. It is well established that some members of the FERM
domain family of proteins are regulated by a head-tail interaction
(26); gel filtration,
sedimentation velocity, and electron microscopy studies all show that talin is
globular in low salt buffers, although it is more elongated (∼60 nm in
length) in high salt (27). By
contrast, the talin rod liberated from full-length talin by calpain-II
cleavage is elongated in both buffers, indicating that the head is required
for talin to adopt a more compact state. Direct evidence for an interaction
between the talin head and rod has recently emerged from NMR studies by Goksoy
et al. (28), who
demonstrated binding of 15N-labeled talin F3 to a talin rod
fragment spanning residues 1654–2344, an interaction that was confirmed
by surface plasmon resonance (Kd = 0.57 μm)
(28). Chemical shift data also
showed that this segment of the talin rod partially masked the binding site in
F3 for the membraneproximal helix of the β3-integrin tail
(28), directly implicating the
talin head-rod interaction in regulating the integrin binding activity of
talin. Goksoy et al.
(28) subdivided the F3 binding
site in this rod fragment into two sites with higher affinity
(Kd ∼3.6 μm; residues 1654–1848)
and lower affinity (Kd ∼78 μm; residues
1984–2344). Here, we define the rod domain boundaries and determine the
NMR structure of residues 1655–1822, a five-helix bundle. We further
show that this domain binds F3 predominantly via surface-exposed residues on
helix 4, with an affinity similar to the high affinity site reported by Goksoy
et al. (28). We also
report the structure of the complex between F3 and the rod domain and show
that the latter masks the known binding site in F3 for the β3-integrin
tail and is expected to inhibit the association of the talin FERM domain with
the membrane. 相似文献
9.
10.
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
11.
12.
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
13.
14.
Yamini S. Bynagari Bela Nagy Jr. Florin Tuluc Kamala Bhavaraju Soochong Kim K. Vinod Vijayan Satya P. Kunapuli 《The Journal of biological chemistry》2009,284(20):13413-13421
The novel class of protein kinase C (nPKC) isoform η is expressed in
platelets, but not much is known about its activation and function. In this
study, we investigated the mechanism of activation and functional implications
of nPKCη using pharmacological and gene knock-out approaches. nPKCη
was phosphorylated (at Thr-512) in a time- and concentration-dependent manner
by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1
receptor antagonist, or YM-254890, a Gq blocker, abolished
2MeSADP-induced phosphorylation of nPKCη. Similarly, ADP failed to
activate nPKCη in platelets isolated from P2Y1 and
Gq knock-out mice. However, pretreatment of platelets with
P2Y12 receptor antagonist, AR-C69331MX did not interfere with
ADP-induced nPKCη phosphorylation. In addition, when platelets were
activated with 2MeSADP under stirring conditions, although nPKCη was
phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by
activated integrin αIIbβ3 mediated outside-in
signaling. Moreover, in the presence of SC-57101, a
αIIbβ3 receptor antagonist, nPKCη
dephosphorylation was inhibited. Furthermore, in murine platelets lacking
PP1cγ, a catalytic subunit of serine/threonine phosphatase,
αIIbβ3 failed to dephosphorylate nPKCη.
Thus, we conclude that ADP activates nPKCη via P2Y1 receptor
and is subsequently dephosphorylated by PP1γ phosphatase activated by
αIIbβ3 integrin. In addition, pretreatment of
platelets with η-RACK antagonistic peptides, a specific inhibitor of
nPKCη, inhibited ADP-induced thromboxane generation. However, these
peptides had no affect on ADP-induced aggregation when thromboxane generation
was blocked. In summary, nPKCη positively regulates agonist-induced
thromboxane generation with no effects on platelet aggregation.Platelets are the key cellular components in maintaining hemostasis
(1). Vascular injury exposes
subendothelial collagen that activates platelets to change shape, secrete
contents of granules, generate thromboxane, and finally aggregate via
activated αIIbβ3 integrin, to prevent further
bleeding (2,
3). ADP is a physiological
agonist of platelets secreted from dense granules and is involved in feedback
activation of platelets and hemostatic plug stabilization
(4). It activates two distinct
G-protein-coupled receptors (GPCRs) on platelets, P2Y1 and
P2Y12, which couple to Gq and Gi,
respectively
(5–8).
Gq activates phospholipase Cβ (PLCβ), which leads to
diacyl glycerol (DAG)2
generation and calcium mobilization
(9,
10). On the other hand,
Gi is involved in inhibition of cAMP levels and PI 3-kinase
activation (4,
6). Synergistic activation of
Gq and Gi proteins leads to the activation of the
fibrinogen receptor integrin αIIbβ3.
Fibrinogen bound to activated integrin αIIbβ3
further initiates feed back signaling (outside-in signaling) in platelets that
contributes to the formation of a stable platelet plug
(11).Protein kinase Cs (PKCs) are serine/threonine kinases known to regulate
various platelet functional responses such as dense granule secretion and
integrin αIIbβ3 activation
(12,
13). Based on their structure
and cofactor requirements, PKCs are divided in to three classes: classical
(cofactors: DAG, Ca2+), novel (cofactors: DAG) and atypical
(cofactors: PIP3) PKC isoforms
(14). All the members of the
novel class of PKC isoforms (nPKC), viz. nPKC isoforms δ, θ,
η, and ε, are expressed in platelets
(15), and they require DAG for
activation. Among all the nPKCs, PKCδ
(15,
16) and PKCθ
(17–19)
are fairly studied in platelets. Whereas nPKCδ is reported to regulate
protease-activated receptor (PAR)-mediated dense granule secretion
(15,
20), nPKCθ is activated
by outside-in signaling and contributes to platelet spreading on fibrinogen
(18). On the other hand, the
mechanism of activation and functional role of nPKCη is not addressed as
yet.PKCs are cytoplasmic enzymes. The enzyme activity of PKCs is modulated via
three mechanisms (14,
21): 1) cofactor binding: upon
cell stimulus, cytoplasmic PKCs mobilize to membrane, bind cofactors such as
DAG, Ca2+, or PIP3, release autoinhibition, and attain an active
conformation exposing catalytic domain of the enzyme. 2) phosphorylations:
3-phosphoinositide-dependent kinase 1 (PDK1) on the membrane phosphorylates
conserved threonine residues on activation loop of catalytic domain; this is
followed by autophosphorylations of serine/threonine residues on turn motif
and hydrophobic region. These series of phosphorylations maintain an active
conformation of the enzyme. 3) RACK binding: PKCs in active conformation bind
receptors for activated C kinases (RACKs) and are lead to various subcellular
locations to access the substrates
(22,
23). Although various leading
laboratories have elucidated the activation of PKCs, the mechanism of
down-regulation of PKCs is not completely understood.The premise of dynamic cell signaling, which involves protein
phosphorylations by kinases and dephosphorylations by phosphatases has gained
immense attention over recent years. PP1, PP2A, PP2B, PHLPP are a few of the
serine/threonine phosphatases reported to date. Among them PP1 and PP2
phosphatases are known to regulate various platelet functional responses
(24,
25). Furthermore, PP1c, is the
catalytic unit of PP1 known to constitutively associate with
αIIb and is activated upon integrin engagement with
fibrinogen and subsequent outside-in signaling
(26). Among various PP1
isoforms, recently PP1γ is shown to positively regulate platelet
functional responses (27).
Thus, in this study we investigated if the above-mentioned phosphatases are
involved in down-regulation of nPKCη. Furthermore, reports from other cell
systems suggest that nPKCη regulates ERK/JNK pathways
(28). In platelets ERK is
known to regulate agonist induced thromboxane generation
(29,
30). Thus, we also
investigated if nPKCη regulates ERK phosphorylation and thereby
agonist-induced platelet functional responses.In this study, we evaluated the activation of nPKCη downstream of ADP
receptors and its inactivation by an integrin-associated phosphatase
PP1γ. We also studied if nPKCη regulates functional responses in
platelets and found that this isoform regulates ADP-induced thromboxane
generation, but not fibrinogen receptor activation in platelets. 相似文献
15.
Kelly J. Inglis David Chereau Elizabeth F. Brigham San-San Chiou Susanne Sch?bel Normand L. Frigon Mei Yu Russell J. Caccavello Seth Nelson Ruth Motter Sarah Wright David Chian Pamela Santiago Ferdie Soriano Carla Ramos Kyle Powell Jason M. Goldstein Michael Babcock Ted Yednock Frederique Bard Guriqbal S. Basi Hing Sham Tamie J. Chilcote Lisa McConlogue Irene Griswold-Prenner John P. Anderson 《The Journal of biological chemistry》2009,284(5):2598-2602
Several neurological diseases, including Parkinson disease and dementia
with Lewy bodies, are characterized by the accumulation of α-synuclein
phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for
this phosphorylation have been the subject of intense investigation. Here we
submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible
kinase or SNK) is a principle contributor to α-synuclein phosphorylation
at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at
Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases
inhibited α-synuclein phosphorylation both in primary cortical cell
cultures and in mouse brain in vivo. Finally, specific knockdown of
PLK2 expression by transduction with short hairpin RNA constructs or by
knock-out of the plk2 gene reduced p-Ser-129 levels. These results
indicate that PLK2 plays a critical role in α-synuclein phosphorylation
in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson
disease (PD)4 and the
related disorder, dementia with Lewy bodies (DLB), is suggested by its
association with Lewy bodies and Lewy neurites, the inclusions that
characterize these diseases
(1–3),
and demonstrated by the existence of mutations that cause syndromes mimicking
sporadic PD and DLB
(4–6).
Furthermore, three separate mutations cause early onset forms of PD and DLB.
It is particularly telling that duplications or triplications of the gene
(7–9),
which increase levels of α-synuclein with no alteration in sequence,
also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine
residues, at Ser-87 and Ser-129
(10), although to date only
the Ser-129 phosphorylation has been identified in the central nervous system
(11,
12). Phosphorylation at
tyrosine residues has been observed by some investigators
(13,
14) but not by others
(10–12).
Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the
majority of synuclein in Lewy bodies contains this modification
(15). In addition, p-Ser-129
was found to be the most extensive and consistent modification in a survey of
synuclein in Lewy bodies (11).
Results have been mixed from studies investigating the function of
phosphorylation using S129A and S129D mutations to respectively block and
mimic the modification. Although the phosphorylation mimic was associated with
pathology in studies in Drosophila
(16) and in transgenic mouse
models (17,
18), studies using
adeno-associated virus vectors to overexpress α-synuclein in rat
substantia nigra found an exacerbation of pathology with the S129A mutation,
whereas the S129D mutation was benign, if not protective
(19). Interpretation of these
studies is complicated by a recent study showing that the S129D and S129A
mutations themselves have effects on the aggregation properties of
α-synuclein independent of their effects on phosphorylation, with the
S129A mutation stimulating fibril formation
(20). Clearly, determination
of the role of p-Ser-129 phosphorylation would be helped by identification of
the responsible kinase. In addition, identification will provide a
pathologically relevant way to increase phosphorylation in a cell or animal
model.Several kinases have been proposed to phosphorylate α-synuclein,
including casein kinases 1 and 2
(10,
12,
21) and members of the
G-protein-coupled receptor kinase family
(22). In this report, we offer
evidence that a member of the polo-like kinase (PLK) family, PLK2 (or
serum-inducible kinase, SNK), functions as an α-synuclein kinase. The
ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is
established by overexpression in cell culture and by in vitro
reaction with the purified kinase. We show that PLK2 phosphorylates
α-synuclein in cells, including primary neuronal cultures, using a
series of kinase inhibitors as well as inhibition of expression with RNA
interference. In addition, inhibitor and knock-out studies in mouse brain
support a role for PLK2 as an α-synuclein kinase in vivo. 相似文献
16.
Lilly Y. W. Bourguignon Weiliang Xia Gabriel Wong 《The Journal of biological chemistry》2009,284(5):2657-2671
17.
Sharareh Emadi Srinath Kasturirangan Min S. Wang Philip Schulz Michael R. Sierks 《The Journal of biological chemistry》2009,284(17):11048-11058
Neuropathologic and genetics studies as well as transgenic animal models
have provided strong evidence linking misfolding and aggregation of
α-synuclein to the progression of Parkinson disease (PD) and other
related disorders. A growing body of evidence implicates various oligomeric
forms of α-synuclein as the toxic species responsible for
neurodegeneration and neuronal cell death. Although numerous different
oligomeric forms of α-synuclein have been identified in vitro,
it is not known which forms are involved in PD or how, when, and where
different forms contribute to the progression of PD. Reagents that can
interact with specific aggregate forms of α-synuclein would be very
useful not only as tools to study how different aggregate forms affect cell
function, but also as potential diagnostic and therapeutic agents for PD. Here
we show that a single chain antibody fragment (syn-10H scFv) isolated from a
phage display antibody library binds to a larger, later stage oligomeric form
of α-synuclein than a previously reported oligomeric specific scFv
isolated in our laboratory. The scFv described here inhibits aggregation of
α-synuclein in vitro, blocks extracellular
α-synuclein-induced toxicity in both undifferentiated and differentiated
human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes
naturally occurring aggregates in PD but not in healthy human brain
tissue.Parkinson disease
(PD)2 is the second
most common neurodegenerative disorder of the elderly, affecting more than
500,000 people in the United States
(1), with 50,000 new cases
reported each year at an annual cost estimated at 10 billion dollars per year.
Pathologically, PD is characterized by the progressive loss of dopaminergic
neurons in the substantia nigra and formation of fibrillar cytoplasmic
inclusions known as Lewy bodies and Lewy neurites
(2,
3). The protein
α-synuclein has been strongly linked to PD
(4,
5) and other related
neurodegenerative disorders (6,
7) by several lines of
evidence. 1) It is the major component of the hallmark Lewy body aggregates
associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human
A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human
E46K α-synuclein) or multiplication in the α-synuclein gene have
been linked to familial PD
(8–10).
3) Overexpression of α-synuclein in transgenic mice and
Drosophila has been shown to induce the formation of PD-like
pathological phenotypes and behavior, although the animal models do not in
general replicate neuronal loss patterns
(11,
12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain
tissues and is primarily localized at the presynaptic terminals of neurons
(13). The primary structure of
α-synuclein consists of three distinct regions. The N-terminal region of
α-synuclein includes the mutation sites associated with familial PD
(A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV)
that may facilitate protein-protein binding. This repeat section is predicted
to form amphipathic α-helices, typical of the lipid-binding domain of
apolipoproteins (14). The
central region, non-amyloid component, is extremely hydrophobic and includes a
12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation
(15). The C-terminal region is
enriched with acidic glutamate and aspartate residues and is responsible for
the chaperone function of α-synuclein
(16).α-Synuclein normally exists as an unfolded protein, but it can adopt
several different folded conformations depending on the environment, including
small aggregates or oligomers, spherical and linear protofibrils, as well as
the fibrillar structure found in Lewy bodies
(14,
15). A growing body of
evidence implicates the oligomeric forms of α-synuclein as the toxic
species responsible for neurodegeneration and neuronal cell death
(16–18).
Several different oligomeric forms of α-synuclein including spherical,
annular (19), pore-like
(20), and dopamine-stabilized
structures have been identified in vitro
(21).α-Synuclein is considered a cytosolic protein, and consequently its
pathogenic effect was assumed to be limited to the cytoplasm of single cells.
However, recent studies have suggested that α-synuclein also has
extracellular pathogenic effects
(22–25).
α-Synuclein was detected in blood plasma and cerebrospinal fluid in both
monomeric and oligomeric forms
(22–25),
and the presence of significantly elevated levels of oligomeric species of
α-synuclein has been reported extracellularly in plasma and
cerebrospinal fluid samples from patients with PD
(23). Furthermore, various
studies have shown that aggregated α-synuclein added extracellularly to
the culture medium is cytotoxic
(26–32).Despite all these studies, it is still not clear how the various aggregate
forms of α-synuclein are involved in the progression of PD. Therefore,
reagents that can interact with specific aggregate forms of α-synuclein
would be very useful not only for fundamental studies of how α-synuclein
aggregates affect cell function but also as potential diagnostic and
therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular
toxicity of α-synuclein in vitro by a single chain variable
domain antibody fragment (scFv) that specifically recognized an oligomeric
form of α-synuclein
(32). In this study, we
describe a second scFv (syn-10H) that binds a larger later stage oligomeric
form of α-synuclein than the previously reported scFv. The syn-10H scFv
neutralizes α-synuclein-induced toxicity in both undifferentiated and
differentiated SH-SY5Y human neuroblastoma cell line and inhibits
α-synuclein aggregation in vitro. The syn-10H scFv reacts
specifically with homogenized PD brain tissue but does not cross-react with
similarly treated samples taken from Alzheimer disease (AD) or healthy brain
samples. Such scFvs therefore have potential value as diagnostic reagents to
identify the presence of specific oligomeric species in PD tissue and fluid
samples. The scFvs also have value as therapeutic agents as they can be used
either extracellularly or expressed intracellularly (intrabodies) to prevent
formation of toxic aggregates in vivo whether inside or outside of
cells. Intrabodies have been used efficiently to neutralize toxic effects of
different pathogenic agents, including α-synuclein
(33–36).
Moreover, immunization studies in mouse models of PD have shown that
extracellular antibodies can reduce accumulation of intracellular aggregates
of α-synuclein (37),
thereby providing precedent for the use of scFvs in potential passive
vaccination strategies for treating PD. 相似文献
18.
The inhalation anesthetic desflurane induces caspase activation and increases amyloid beta-protein levels under hypoxic conditions 总被引:1,自引:0,他引:1
Zhang B Dong Y Zhang G Moir RD Xia W Yue Y Tian M Culley DJ Crosby G Tanzi RE Xie Z 《The Journal of biological chemistry》2008,283(18):11866-11875
Perioperative factors including hypoxia, hypocapnia, and certain
anesthetics have been suggested to contribute to Alzheimer disease (AD)
neuropathogenesis. Desflurane is one of the most commonly used inhalation
anesthetics. However, the effects of desflurane on AD neuropathogenesis have
not been previously determined. Here, we set out to assess the effects of
desflurane and hypoxia on caspase activation, amyloid precursor protein (APP)
processing, and amyloid β-protein (Aβ) generation in H4 human
neuroglioma cells (H4 naïve cells) as well as those overexpressing APP
(H4-APP cells). Neither 12% desflurane nor hypoxia (18% O2) alone
affected caspase-3 activation, APP processing, and Aβ generation.
However, treatment with a combination of 12% desflurane and hypoxia (18%
O2) (desflurane/hypoxia) for 6 h induced caspase-3 activation,
altered APP processing, and increased Aβ generation in H4-APP cells.
Desflurane/hypoxia also increased levels of β-site APP-cleaving enzyme in
H4-APP cells. In addition, desflurane/hypoxia-induced Aβ generation could
be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the
Aβ aggregation inhibitor clioquinol and γ-secretase inhibitor
L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In
summary, desflurane can induce Aβ production and caspase activation, but
only in the presence of hypoxia. Pending in vivo confirmation, these
data may have profound implications for anesthesia care in elderly patients,
and especially those with AD.An estimated 200 million patients worldwide undergo surgery each year.
Several reports have suggested that anesthesia and surgery may facilitate
development of Alzheimer disease
(AD)4
(1–3).
A recent study also reported that patients having coronary artery bypass graft
surgery under general anesthesia are at increased risk for AD as compared with
those having percutaneous transluminal coronary angioplasty under local
anesthesia (4).Genetic evidence, confirmed by neuropathological and biochemical findings,
indicates that excessive production and/or accumulation of amyloid
β-protein (Aβ) play a fundamental role in the pathology of AD
(reviewed in Refs. 5 and
6). Aβ is produced via
serial proteolysis of amyloid precursor protein (APP) by aspartyl protease
β-site APP-cleaving enzyme (BACE), or β-secretase,
andγ-secretase. BACE cleaves APP to generate a 99-residue
membrane-associated C terminus fragment (APP-C99). APP-C99 is further cleaved
by γ-secretase to release 4-kDa Aβ and β-amyloid precursor
protein intracellular domain
(7–9).
Presenilin and γ-secretase co-fractionate as a detergent-sensitive, high
molecular weight complex (10)
that includes at least three other proteins, nicastrin/APH-2, APH-1, and
PEN-2, all of which are necessary and sufficient for γ-secretase
activity
(11–13).
Increasing evidence indicates that apoptosis is associated with a variety of
neurodegenerative disorders, including AD (Refs.
14–17;
reviewed in Ref. 18). Aβ
has been shown to cause caspase activation and apoptosis, which can in turn
potentiate Aβ generation
(16,
19–28).
Finally, fibrillar aggregates of Aβ and oligomeric species of Aβ are
more neurotoxic
(29–37).Perioperative factors, including hypoxia
(38–42),
hypocapnia (43), and
anesthetics
(44–47),
have been reported to potentially contribute to AD neuropathogenesis. These
perioperative factors may also cause post-operative cognitive dysfunction, a
dementia associated with surgery and anesthesia, by triggering AD
neuropathogenesis.Isoflurane, sevoflurane, and desflurane are the most commonly used
inhalation anesthetics. It has been reported that isoflurane enhances the
oligomerization and cytotoxicity of Aβ
(44) and induces apoptosis
(48–51).
Our recent studies have shown that a clinically relevant concentration of
isoflurane can lead to caspase-3 activation, decrease cell viability, alter
APP processing, and increase Aβ generation in human H4 neuroglioma cells
overexpressing human APP
(45–47).
Loop et al. (49)
reported that isoflurane and sevoflurane, but not desflurane, can induce
caspase activation and apoptosis in human T lymphocytes. However, effects of
desflurane and desflurane plus other perioperative risk factors, e.g.
hypoxia, on APP processing and Aβ generation have not been assessed.In the present study, we set out to determine effects of desflurane,
hypoxia, and the combination of the two (desflurane/hypoxia) on caspase-3
activation, APP processing, and Aβ generation in H4 human neuroglioma
cells (H4 naïve cells) and H4 naïve cells stably transfected to
express full-length (FL) APP (H4-APP cells). We also investigated whether the
caspase inhibitor, Z-VAD, the γ-secretase inhibitor L-685,458, and the
Aβ aggregation inhibitor clioquinol could attenuate
desflurane/hypoxia-induced caspase-3 activation and Aβ generation. 相似文献
19.
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