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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
4.
Yan Yang Xin Wu Peichun Gui Jianbo Wu Jian-Zhong Sheng Shizhang Ling Andrew P. Braun George E. Davis Michael J. Davis 《The Journal of biological chemistry》2010,285(1):131-141
Large conductance, calcium-activated K+ (BK) channels are important regulators of cell excitability and recognized targets of intracellular kinases. BK channel modulation by tyrosine kinases, including focal adhesion kinase and c-src, suggests their potential involvement in integrin signaling. Recently, we found that fibronectin, an endogenous α5β1 integrin ligand, enhances BK channel current through both Ca2+- and phosphorylation-dependent mechanisms in vascular smooth muscle. Here, we show that macroscopic currents from HEK 293 cells expressing murine BK channel α-subunits (mSlo) are acutely potentiated following α5β1 integrin activation. The effect occurs in a Ca2+-dependent manner, 1–3 min after integrin engagement. After integrin activation, normalized conductance-voltage relations for mSlo are left-shifted at free Ca2+ concentrations ≥1 μm. Overexpression of human c-src with mSlo, in the absence of integrin activation, leads to similar shifts in mSlo Ca2+ sensitivity, whereas overexpression of catalytically inactive c-src blocks integrin-induced potentiation. However, neither integrin activation nor c-src overexpression potentiates current in BK channels containing a point mutation at Tyr-766. Biochemical tests confirmed the critical importance of residue Tyr-766 in integrin-induced channel phosphorylation. Thus, BK channel activity is enhanced by α5β1 integrin activation, likely through an intracellular signaling pathway involving c-src phosphorylation of the channel α-subunit at Tyr-766. The net result is increased current amplitude, enhanced Ca2+ sensitivity, and rate of activation of the BK channel, which would collectively promote smooth muscle hyperpolarization in response to integrin-extracellular matrix interactions. 相似文献
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Girish V. Shah Anbalagan Muralidharan Mitan Gokulgandhi Kamal Soan Shibu Thomas 《The Journal of biological chemistry》2009,284(2):1018-1030
Calcitonin, a neuroendocrine peptide, and its receptor are localized in the
basal epithelium of benign prostate but in the secretory epithelium of
malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA
displays positive correlation with the Gleason grade of primary prostate
cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of
multiple prostate cancer cell lines by cyclic AMP-dependent protein
kinase-mediated actions. These actions include increased secretion of matrix
metalloproteinases and urokinase-type plasminogen activator and an increase in
prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor
autocrine loop in prostate cancer cell lines led to the loss of cell-cell
adhesion, destabilization of tight and adherens junctions, and internalization
of key integral membrane proteins. In addition, the activation of
calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition
of prostate cancer cells as characterized by cadherin switch and the
expression of the mesenchymal marker, vimentin. The activated calcitonin
receptor phosphorylated glycogen synthase kinase-3, a key regulator of
cytosolic β-catenin degradation within the WNT signaling pathway. This
resulted in the accumulation of intracellular β-catenin, its
translocation in the nucleus, and transactivation of β-catenin-responsive
genes. These results for the first time identify actions of
calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the
destabilization of cell-cell junctions, epithelial-to-mesenchymal transition,
and activation of WNT/β-catenin signaling. The results also suggest that
cyclic AMP-dependent protein kinase plays a key role in calcitonin
receptor-induced destabilization of cell-cell junctions and activation of
WNT-β-catenin signaling.Prostate cancer
(PC)2 is the most
commonly diagnosed cancer and the second leading cause of cancer deaths in men
in the United States (1,
2). Although androgen ablation
therapy is effective in men with advanced disease for some time, the disease
subsequently progresses to the androgen-independent stage. The population of
prostate cells expressing neuroendocrine factors such as calcitonin (CT) also
increases during this progression
(3–5).
At this stage, the disease is metastatic and chemoresistant. Present evidence
suggests that cancer metastasis is usually preceded by the disruption of
normal cell-cell adhesion and the loss of integrity of the primary tumor site
(6,
7). This process may include
several genetic, molecular, and morphological changes characterized by
epithelial-to-mesenchymal transition (EMT)
(8–10).
The EMT is characterized by the loss of cell polarity, altered cell-cell and
cell-matrix adhesion, and acquisition of migratory, mesenchymal phenotype.
Other reported changes include down-regulation of E-cadherin, induction of
N-cadherin, release of β-catenin from junctional complexes, and its
translocation to the nucleus
(11–13).
However, the precise molecular mechanisms associated with this process are
obscure.Several growth factors, including hepatocyte growth factor, transforming
growth factor-β, vascular endothelial growth factor, and epidermal growth
factor, have been reported to induce EMT in tumor cell lines
(14–16).
We have shown that the expression of CT and its G protein-coupled receptor
(CTR) is remarkably higher in advanced PCs, and the CT-CTR autocrine axis is a
potent stimulator of PC cell tumorigenicity, invasion, and metastasis
(4,
17–19).
Although CT-stimulated increase in the motility and invasion of PC cells may
be mediated by CT-stimulated secretion of matrix metalloproteinases and
urokinase-type plasminogen activator, the precise molecular mechanisms
preceding these CTR actions remain to be elucidated
(18,
20). We tested the hypothesis
that CT induces biochemical and morphological changes associated with EMT to
increase the invasiveness of PC cells.Our results indicate that activation of the CT-CTR autocrine axis in
prostate cancer cells induced several changes associated with EMT such as
remodeling of tight and adherens junctions, cadherin switching, and activation
of WNT/β-catenin signaling. In contrast, the silencing of the CT-CTR axis
reversed this process. Moreover, cyclic AMP-dependent protein kinase (PKA)
plays a key role in this CT-CTR-mediated process. This is the first study
demonstrating the action of prostate CTR on junctional complexes and
WNT/β-catenin signaling of PC cell lines. 相似文献
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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
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Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.The CD2 family of cell surface receptors is differentially expressed on immune cells (1, 2) and is involved in regulating both innate and adaptive immunity (3). These receptors have related extracellular immunoglobulin superfamily domains and interact either homophilically or heterophilically within the CD2 family (1, 2). The CD2 family contains a subgroup of receptors termed the SLAM family that have a conserved tyrosine signaling motif in their cytoplasmic region TXYXX(I/V) referred to as an immunoreceptor tyrosine-based switch motif (ITSM).2 The SLAM family of receptors include CD244 (2B4), NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9). Defects in signaling and aberrant expression of these receptors have been implicated in several immunodeficiency and autoimmune disorders in humans and mice (4–8). Within the SLAM family, CD244 is unusual in that it shares its ligand CD48 with the receptor CD2 in rodents, whereas in humans CD2 has evolved to interact with CD58 (9). The affinity of CD244 for CD48 in rodents is 6–9-fold higher than the still functionally relevant CD2/CD48 interaction (10). CD244 and CD2 have different cytoplasmic regions comprised of tyrosine motifs or proline-rich motifs, respectively.CD244 is predominantly found on NK cells and cytotoxic T cells and primarily characterized as an activating receptor (11–15). CD2 is found on the same cells as CD244 but is also expressed on all T cells, both activated and resting, and has an activating or costimulatory function upon engagement of ligand (9). The tyrosine motifs found in the cytoplasmic tail of CD244 have been shown to bind the SH2 domains of cytoplasmic adaptor proteins SAP and EAT-2 and FYN kinase (16–18) and are important to its function (5, 19–21). In contrast to SH2 interactions of CD244, several SH3 domain-mediated interactions have been reported for the cytoplasmic region of CD2 including CD2AP/CMS, CIN85, FYN, and LCK (22–26).The activating function of CD244 was called into question when a study using cells from a CD244 knock-out mouse showed that CD244 had an inhibitory effect as loss of CD244 resulted in enhanced NK killing of target cells (27). This suggested that previous results in mice where positive effects were seen may have been due to blocking CD244 ligand engagement as opposed to cross-linking with antibodies against CD244 (27). This has led to proposals that there are differences in function between mouse and human CD244 as there is more evidence to suggest that human CD244 is a positive regulator enhancing cytotoxicity and cytokine production (13, 15, 28). However, other more recent studies have shown the mouse CD244/CD48 interaction to be important for cytokine production and effector functions such as cytotoxicity against tumor targets in CD244-deficient mice (29). Long and short forms of CD244 have been cloned from mice with the short form being described as activating and the long form inhibitory (27, 30). Only the long form of CD244 is present in humans and is regarded as activating (14).Positive signaling by CD244 has been attributed to the recruitment of SAP (18), which is a signaling adaptor molecule comprised of a single SH2 domain encoded by the SH2D1A gene and has the ability to recruit the kinase FYN by binding its SH3 domain (31, 32). Loss of the SAP/FYN interaction can lead to X-linked lymphoproliferative disease in humans (17). The molecular basis of in vitro inhibitory effects observed with CD244 in mice on ligation with mAb or ligand remains elusive (33). Protein tyrosine and inositol phosphatases have been reported to associate with CD244 (18, 19, 34) but our studies using surface plasmon resonance found them to be very weak and unlikely to bind competitively compared with the SAP family of adaptors or FYN (16). The SAP-related adaptor EAT-2 has been reported to have an active inhibitory effect that is dependent on tyrosine motifs in the tail of EAT-2 (35) but its mechanism is not understood. The only interaction reported for the tail of EAT-2 is with FYN kinase and studies overexpressing EAT-2 in a T cell hybridoma resulted in increased IL-2 production upon antigen stimulation (16).The conservation between mouse and human CD244 cytoplasmic regions and associated adaptors suggests that both function in a similar way. We have explored the main difference between mouse and human CD244, which is the extracellular interaction through CD48 ligation in the mouse. This has revealed that inhibitory effects of CD244 ligation in mice can be due to competition between CD244 and CD2 for CD48. We have also found that the adaptor protein EAT-2 binds PLCγ1 providing a molecular basis for the important role CD244 plays in regulating cellular cytotoxicity (13, 36). We demonstrate that there is a potentially shared signaling mechanism through the FYN kinase that links CD2 and CD244 intracellularly even though in humans CD2 and CD244 no longer share a cell surface ligand. 相似文献
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Ian G. Ganley Du H. Lam Junru Wang Xiaojun Ding She Chen Xuejun Jiang 《The Journal of biological chemistry》2009,284(18):12297-12305
Autophagy is a degradative process that recycles long-lived and faulty
cellular components. It is linked to many diseases and is required for normal
development. ULK1, a mammalian serine/threonine protein kinase, plays a key
role in the initial stages of autophagy, though the exact molecular mechanism
is unknown. Here we report identification of a novel protein complex
containing ULK1 and two additional protein factors, FIP200 and ATG13, all of
which are essential for starvation-induced autophagy. Both FIP200 and ATG13
are critical for correct localization of ULK1 to the pre-autophagosome and
stability of ULK1 protein. Additionally, we demonstrate by using both cellular
experiments and a de novo in vitro reconstituted reaction that FIP200
and ATG13 can enhance ULK1 kinase activity individually but both are required
for maximal stimulation. Further, we show that ATG13 and ULK1 are
phosphorylated by the mTOR pathway in a nutrient starvation-regulated manner,
indicating that the ULK1·ATG13·FIP200 complex acts as a node for
integrating incoming autophagy signals into autophagosome biogenesis.Macroautophagy (herein referred to as autophagy) is a catabolic process
whereby long-lived proteins and damaged organelles are shuttled to lysosomes
for degradation. This process is conserved in all eukaryotes. Under normal
growth conditions a housekeeping level of autophagy exists. Under stress, such
as nutrient starvation, autophagy is strongly induced resulting in the
engulfment of cytosolic components and organelles in specialized
double-membrane structures termed autophagosomes. Following fusion of the
outer autophagosomal membrane with lysosomes, the inner membrane and its
cytoplasmic cargo are degraded and recycled
(1–3).
Recent work has implicated autophagy in many disease pathologies, including
cancer, neurodegeneration, as well as in eliminating intracellular pathogens
(4–8).The morphology of autophagy was first described in mammalian cells over 50
years ago (9). However, it is
only recently through yeast genetic screens, that multiple autophagy-related
(ATG) genes have been identified
(10–12).
The yeast ATG proteins have been classified into four major groups: the Atg1
protein kinase complex, the Vps34 phosphatidylinositol 3-phosphate kinase
complex, the Atg8/Atg12 conjugation systems, and the Atg9 recycling complex
(13). Even though many ATG
genes are now known, most of which have functional homologs in mammalian cells
(14,
15), the molecular mechanism
by which they sense the initial triggers and subsequently dictate
autophagy-specific intracellular membrane events is far from understood.In yeast, one of the earliest autophagy-specific events is believed to
involve the Atg1 protein kinase complex. Atg1 is a serine/threonine protein
kinase and a key autophagy-regulator
(16). Atg1 is complexed to at
least two other proteins during autophagy, Atg13 and Atg17, both of which are
required for normal Atg1 function and autophagosome generation
(17–19).
Classical signaling pathways such as the cAMP-dependent kinase (PKA) pathway
or the Tor kinase pathway appear to converge upon this complex, placing Atg1
at an early stage during autophagosome biogenesis
(20–22).
Atg1 phosphorylation by PKA blocks its association with the forming
autophagosome (21), while the
Tor pathway hyperphosphorylates Atg13 causing a reduced affinity of Atg13 for
Atg1, resulting in repression of autophagy
(17,
19). In contrast, nutrient
starvation or inhibition of Tor leads to dephosphorylation of Atg13 thus
increased Atg1 complex formation and kinase activity, resulting in stimulation
of autophagy (19).
Surprisingly, the physiological substrates of Atg1 kinase have not been
identified; thus how Atg1 transduces upstream autophagic signaling is
undefined. Recently, mammalian homologs of Atg1 have been identified as ULK1
and ULK2 (Unc-51-like
kinase)2
(23–25).
ULK1 and ULK2 are ubiquitously expressed and localize to the isolation
membrane, or forming autophagosome, upon nutrient starvation
(25); RNAi-mediated depletion
of ULK1 in HEK293 cells compromises autophagy
(23,
24). The exact role of ULK1
versus ULK2 in autophagy is unclear, and it is possible some
redundancy exists between the two isoforms
(26).Given the conservation of autophagy from yeast to man, it is interesting to
note that no mammalian counterpart to yeast Atg13 or Atg17 had been identified
until very recently. The protein FIP200 (focal adhesion kinase
family-interacting protein of 200 kDa) was
identified as an autophagy-essential binding partner of both ULK1 and ULK2
(25), and it has been
speculated that FIP200 might be the equivalent of yeast Atg17, despite low
sequence similarity (25,
27).In this study, we delve deeper into the molecular regulation of ULK1 to
gain a better insight into how mammalian signaling pathways affect autophagy
initiation. We describe here the identification of a triple complex consisting
of ULK1, FIP200, and the mammalian equivalent of Atg13. This complex is
required not only for localization of ULK1 to the isolation membrane but also
for maximal kinase activity. In addition, both ATG13 and ULK1 are kinase
substrates in the mTOR pathway and thus might function to sense nutrient
starvation. Therefore, this study defines the role of mammalian
ULK1-ATG13-FIP200 complex in mediating the initial autophagic triggers and to
transduce the signal to the core autophagic machinery. 相似文献
15.
Omar Ramadan Yongxia Qu Raj Wadgaonkar Ghayath Baroudi Eddy Karnabi Mohamed Chahine Mohamed Boutjdir 《The Journal of biological chemistry》2009,284(8):5042-5049
The novel α1D L-type Ca2+ channel is expressed
in supraventricular tissue and has been implicated in the pacemaker activity
of the heart and in atrial fibrillation. We recently demonstrated that PKA
activation led to increased α1D Ca2+ channel
activity in tsA201 cells by phosphorylation of the channel protein. Here we
sought to identify the phosphorylated PKA consensus sites on the
α1 subunit of the α1D Ca2+
channel by generating GST fusion proteins of the intracellular loops, N
terminus, proximal and distal C termini of the α1 subunit of
α1D Ca2+ channel. An in vitro PKA kinase
assay was performed for the GST fusion proteins, and their phosphorylation was
assessed by Western blotting using either anti-PKA substrate or
anti-phosphoserine antibodies. Western blotting showed that the N terminus and
C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus
sites, were phosphorylated by PKA and identified by mass spectrometry. Site
directed mutagenesis and patch clamp studies revealed that serines 1743 and
1816 were major functional PKA consensus sites. Altogether, biochemical and
functional data revealed that serines 1743 and 1816 are major functional PKA
consensus sites on the α1 subunit of α1D
Ca2+ channel. These novel findings provide new insights into the
autonomic regulation of the α1D Ca2+ channel in
the heart.L-type Ca2+ channels are essential for the generation of normal
cardiac rhythm, for induction of rhythm propagation through the
atrioventricular node and for the contraction of the atrial and ventricular
muscles
(1–5).
L-type Ca2+ channel is a multisubunit complex including
α1, β and α2/δ subunits
(5–7).
The α1 subunit contains the voltage sensor, the selectivity
filter, the ion conduction pore, and the binding sites for all known
Ca2+ channel blockers
(6–9).
While α1C Ca2+ channel is expressed in the atria
and ventricles of the heart
(10–13),
expression of α1D Ca2+ channel is restricted to
the sinoatrial (SA)2
and atrioventricular (AV) nodes, as well as in the atria, but not in the adult
ventricles (2,
3,
10).Only recently it has been realized that α1D along with
α1C Ca2+ channels contribute to L-type
Ca2+ current (ICa-L) and they both play important but
unique roles in the physiology/pathophysiology of the heart
(6–9).
Compared with α1C, α1D L-type
Ca2+ channel activates at a more negative voltage range and shows
slower current inactivation during depolarization
(14,
15). These properties may
allow α1D Ca2+ channel to play critical roles in
SA and AV nodes function. Indeed, α1D Ca2+ channel
knock-out mice exhibit significant SA dysfunction and various degrees of AV
block (12,
16–19).The modulation of α1C Ca2+ channel by
cAMP-dependent PKA phosphorylation has been extensively studied, and the C
terminus of α1 was identified as the site of the modulation
(20–22).
Our group was the first to report that 8-bromo-cAMP (8-Br-cAMP), a
membrane-permeable cAMP analog, increased α1D Ca2+
channel activity using patch clamp studies
(2). However, very little is
known about potential PKA phosphorylation consensus motifs on the
α1D Ca2+ channel. We therefore hypothesized that
the C terminus of the α1 subunit of the α1D
Ca2+ channel mediates its modulation by cAMP-dependent PKA
pathway. 相似文献
16.
17.
Control of TANK-binding Kinase 1-mediated Signaling by the
��134.5 Protein of Herpes Simplex Virus
1
Dustin Verpooten Yijie Ma Songwang Hou Zhipeng Yan Bin He 《The Journal of biological chemistry》2009,284(2):1097-1105
TANK-binding kinase 1 (TBK1) is a key component of Toll-like
receptor-dependent and -independent signaling pathways. In response to
microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and
cytokine expression. Here we show that TBK1 is a novel target of the
γ134.5 protein, a virulence factor whose expression is
regulated in a temporal fashion. Remarkably, the γ134.5
protein is required to inhibit IRF3 phosphorylation, nuclear translocation,
and the induction of antiviral genes in infected cells. When expressed in
mammalian cells, the γ134.5 protein forms complexes with TBK1
and disrupts the interaction of TBK1 and IRF3, which prevents the induction of
interferon and interferon-stimulated gene promoters. Down-regulation of TBK1
requires the amino-terminal domain. In addition, unlike wild type virus, a
herpes simplex virus mutant lacking γ134.5 replicates
efficiently in TBK1-/- cells but not in TBK1+/+ cells.
Addition of exogenous interferon restores the antiviral activity in both
TBK1-/- and TBK+/+ cells. Hence, control of
TBK1-mediated cell signaling by the γ134.5 protein
contributes to herpes simplex virus infection. These results reveal that TBK1
plays a pivotal role in limiting replication of a DNA virus.Herpes simplex virus 1
(HSV-1)3 is a large
DNA virus that establishes latent or lytic infection, in which the virus
triggers innate immune responses. In HSV-infected cells, a number of antiviral
mechanisms operate in a cell type- and time-dependent manner
(1). In response to
double-stranded RNA (dsRNA), Toll-like receptor 3 (TLR3) recruits an adaptor
TIR domain-containing adaptor inducing IFN-β and stimulates cytokine
expression (2,
3). In the cytoplasm, RNA
helicases, RIG-I (retinoid acid-inducible gene-I), and MDA5 (melanoma
differentiation associated gene 5) recognize intracellular viral
5′-triphosphate RNA or dsRNA
(2,
4). Furthermore, a
DNA-dependent activator of IFN-regulatory factor (DAI) senses double-stranded
DNA in the cytoplasm and induces cytokine expression
(5). There is also evidence
that viral entry induces antiviral programs independent of TLR and RIG-I
pathways (6). While recognizing
distinct viral components, these innate immune pathways relay signals to the
two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB
kinase (IKKi) (2).The IKK-related kinases function as essential components that phosphorylate
IRF3 (interferon regulatory factor 3), as well as the closely related IRF7,
which translocates to the nucleus and induces antiviral genes, such as
interferon-α/β and ISG56 (interferon-stimulated gene 56)
(7,
8). TBK1 is constitutively
expressed, whereas IKKi is engaged as an inducible gene product of innate
immune signaling (9,
10). IRF3 activation is
attenuated in TBK1-deficient but not in IKKi-deficient cells
(11,
12). Its activation is
completely abolished in double-deficient cells
(12), suggesting a partially
redundant function of TBK1 and IKKi. Indeed, IKKi also negatively regulates
the STAT-signaling pathway
(13). TBK1/IKKi interacts with
several proteins, such as TRAF family member-associated NF-κB activator
(TANK), NAP1 (NAK-associated protein 1), similar to NAP1TBK1 adaptor
(SINTBAD), DNA-dependent activator of IFN-regulatory factors (DAI), and
secretory protein 5 (Sec5) in host cells
(5,
14–18).
These interactions are thought to regulate TBK1/IKKi, which delineates innate
as well as adaptive immune responses.Upon viral infection, expression of HSV proteins interferes with the
induction of antiviral immunity. When treated with UV or cycloheximide, HSV
induces an array of antiviral genes in human lung fibroblasts
(19,
20). Furthermore, an HSV
mutant, with deletion in immediate early protein ICP0, induces ISG56
expression (21). Accordingly,
expression of ICP0 inhibits the induction of antiviral programs mediated by
IRF3 or IRF7
(21–23).
However, although ICP0 negatively regulates IFN-β expression, it is not
essential for this effect
(24). In HSV-infected human
macrophages or dendritic cells, an immediate early protein ICP27 is required
to suppress cytokine induction involving IRF3
(25). In this context, it is
notable that an HSV mutant, lacking a leaky late gene γ134.5,
replicates efficiently in cells devoid of IFN-α/β genes
(26). Additionally, the
γ134.5 null mutant induces differential cytokine expression
as compared with wild type virus
(27). Thus, HSV modulation of
cytokine expression is a complex process that involves multiple viral
components. Currently, the molecular mechanism governing this event is
unclear. In this study, we show that HSV γ134.5 targets TBK1
and inhibits antiviral signaling. The data herein reveal a previously
unrecognized mechanism by which γ134.5 facilitates HSV
replication. 相似文献
18.
Many human diseases are caused by missense substitutions that result in
misfolded proteins that lack biological function. Here we express a mutant
form of the human cystathionine β-synthase protein, I278T, in
Saccharomyces cerevisiae and show that it is possible to dramatically
restore protein stability and enzymatic function by manipulation of the
cellular chaperone environment. We demonstrate that Hsp70 and Hsp26 bind
specifically to I278T but that these chaperones have opposite biological
effects. Ethanol treatment induces Hsp70 and causes increased activity and
steady-state levels of I278T. Deletion of the SSA2 gene, which
encodes a cytoplasmic isoform of Hsp70, eliminates the ability of ethanol to
restore function, indicating that Hsp70 plays a positive role in proper I278T
folding. In contrast, deletion of HSP26 results in increased I278T
protein and activity, whereas overexpression of Hsp26 results in reduced I278T
protein. The Hsp26-I278T complex is degraded via a
ubiquitin/proteosome-dependent mechanism. Based on these results we propose a
novel model in which the ratio of Hsp70 and Hsp26 determines whether misfolded
proteins will either be refolded or degraded.Cells have evolved quality control systems for misfolded proteins,
consisting of molecular chaperones (heat shock proteins) and proteases. These
molecules help prevent misfolding and aggregation by either promoting
refolding or by degrading misfolded protein molecules
(1). In eukaryotic cells, the
Hsp70 system plays a critical role in mediating protein folding. Hsp70 protein
interacts with misfolded polypeptides along with co-chaperones and promotes
refolding by repeated cycles of binding and release requiring the hydrolysis
of ATP (2). Small heat shock
proteins (sHsp)2 are
small molecular weight chaperones that bind non-native proteins in an
oligomeric complex and whose function is poorly understood
(3). In mammalian cells, the
sHsp family includes the α-crystallins, whose orthologue in
Saccharomyces cerevisiae is Hsp26. Studies suggest that Hsp26 binding
to misfolded protein aggregates is a prerequisite for effective disaggregation
and refolding by Hsp70 and Hsp104
(4,
5).Misfolded proteins can result from missense substitutions such as those
found in a variety of recessive genetic diseases, including cystathionine
β-synthase (CBS) deficiency. CBS is a key enzyme in the
trans-sulfuration pathway that converts homocysteine to cysteine
(6). Individuals with CBS
deficiency have extremely elevated levels of plasma total homocysteine,
resulting in a variety of symptoms, including dislocated lenses, osteoporosis,
mental retardation, and a greatly increased risk of thrombosis
(7). Approximately 80% of the
mutations found in CBS-deficient patients are point mutations that are
predicted to cause missense substitutions in the CBS protein
(8). The most common mutation
found in CBS-deficient patients, an isoleucine to threonine substitution at
amino acid position 278 (I278T), has been observed in nearly one-quarter of
all CBS-deficient patients. Based on the crystal structure of the catalytic
core of CBS, this mutation is located in a β-sheet more than 10 Å
distant from the catalytic pyridoxal phosphate and does not directly affect
the catalytic binding pocket or the dimer interface
(9).Previously, our lab has developed a yeast bioassay for human CBS
in which yeast expressing functional human CBS can grow in media lacking
cysteine, whereas yeast expressing mutant CBS cannot
(10). We have used this assay
to characterize the functional effects of many different CBS missense alleles,
including I278T (7,
11). However, an unexpected
finding was that it was possible to restore function to I278T and a number of
other CBS missense mutations by either truncation or the addition of a second
missense mutation in the C-terminal regulatory domain
(12,
13). The ability to restore
function by a cis-acting second mutation suggested to us that it
might be possible to restore function in trans via either interaction
of mutant CBS with a small molecule (i.e. drug) or a mutation in
another yeast gene. In a previous study, we found that small osmolyte chemical
chaperones could restore function to mutant CBS presumably by directly
stabilizing the mutant CBS protein
(14).In this study we report on the surprising finding that exposure of yeast to
ethanol can restore function of I278T CBS by altering the ratio of the
molecular chaperones Hsp26 and Hsp70. We demonstrate Hsp70 binding promotes
I278T folding and activity, whereas Hsp26 binding promotes I278T degradation
via the proteosome. By manipulating the levels of Hsp26 and Hsp70, we are able
to show that I278T CBS protein can have enzymatic activity restored to near
wild-type levels of activity. Our findings suggest a novel function for
sHsps. 相似文献