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1.
2.
Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic β-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) β-glucosidase isozymes. Southern-blot data suggested that β-glu-cosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root—only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-β-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression. 相似文献
3.
Xiaojing Wang Snezana Levic Michael Anne Gratton Karen Jo Doyle Ebenezer N. Yamoah Anthony E. Pegg 《The Journal of biological chemistry》2009,284(2):930-937
Male gyro (Gy) mice, which have an X chromosomal deletion inactivating the
SpmS and Phex genes, were found to be profoundly hearing
impaired. This defect was due to alteration in polyamine content due to the
absence of spermine synthase, the product of the SpmS gene. It was
reversed by breeding the Gy strain with CAG/SpmS mice, a transgenic line that
ubiquitously expresses spermine synthase under the control of a composite
cytomegalovirus-IE enhancer/chicken β-actin promoter. There was an almost
complete loss of the endocochlear potential in the Gy mice, which parallels
the hearing deficiency, and this was also reversed by the production of
spermine from the spermine synthase transgene. Gy mice showed a striking toxic
response to treatment with the ornithine decarboxylase inhibitor
α-difluoromethylornithine (DFMO). Within 2–3 days of exposure to
DFMO in the drinking water, the Gy mice suffered a catastrophic loss of motor
function resulting in death within 5 days. This effect was due to an inability
to maintain normal balance and was also prevented by the transgenic expression
of spermine synthase. DFMO treatment of control mice or Gy-CAG/SpmS had no
effect on balance. The loss of balance in Gy mice treated with DFMO was due to
inhibition of polyamine synthesis because it was prevented by administration
of putrescine. Our results are consistent with a critical role for polyamines
in regulation of Kir channels that maintain the endocochlear potential and
emphasize the importance of normal spermidine:spermine ratio in the hearing
and balance functions of the inner ear.Polyamines are essential for viability in mammals. Knockouts of the genes
for ornithine decarboxylase and S-adenosylmethionine decarboxylase,
which are enzymes needed for the synthesis of putrescine, spermidine, and
spermine, are lethal at early stages of embryonic development
(1,
2). There is convincing
evidence that the formation of hypusine in eIF5A, which requires spermidine as
a precursor, is essential for eukaryotes
(3). However, the function(s)
of spermine is not so well established. Yeast mutants with inactivated
spermine synthase grow at a normal rate
(4). Mammalian cells in culture
also grow normally in the presence of inhibitors of spermine synthase
(5) or after inactivation of
the spermine synthase gene (SpmS)
(6–8).
Inactivation of both of the genes that were originally described as encoding
spermine synthases in plants leads to profound developmental defects
(9–11),
but recently it was discovered that one of these genes actually encodes a
thermospermine synthase, and it appears that the lack of thermospermine may be
responsible for these defects
(12).In contrast, spermine is clearly required for normal development in
mammals. The rare human Snyder-Robinson syndrome is caused by mutations in
SpmS located in the X chromosome that drastically reduces the amount
of spermine synthase (13,
14). This leads to mental
retardation, hypotonia, cerebellar circuitry dysfunction, facial asymmetry,
thin habitus, osteoporosis, and kyphoscoliosis. Male mice, which have an X
chromosomal deletion that includes SpmS and have no detectable
spermine synthase activity, do survive but are only viable on the B6C3H
background
(15–17).
This mouse strain having an X-linked dominant mutation was isolated from a
female offspring of an irradiated mouse and was termed gyro
(Gy)2 based on a
circling behavior pattern in affected males
(18). Subsequent studies have
shown that the Gy mice have a deletion of part of the X chromosome that
inactivates both Phex, a gene that regulates phosphate metabolism,
and SpmS (16,
19). The lack of SpmS
causes a total absence of spermine
(6,
7,
15,
16). Such Gy mice suffer from
hypophosphatemia, have a greatly reduced size, sterility, and neurological
abnormalities, and have a short life span
(6,
16,
18). All of these changes
except the hypophosphatemia are reversed when spermine synthase activity is
restored (20).The original characterization of Gy mice also reported preliminary
indications that these mice had hearing defects lacking the Preyer reflex
(21,
22). This is of particular
interest in the context of polyamine metabolism because a drug,
α-difluoromethylornithine (DFMO, Eflornithine), that targets ornithine
decarboxylase has been shown to cause occasional hearing loss in some patients
(23–26).
Although DFMO was ineffective for cancer treatment, it is an extremely
promising agent for cancer chemoprevention
(27,
28). When combined with
sulindac, DFMO treatment produced a substantial reduction in the recurrence of
colorectal adenomas in a large clinical trial
(27). DFMO is a major drug for
the treatment of African sleeping sickness caused by Trypanosoma
brucei (29,
30). It is also used as a
topically applied cream for treatment of unwanted facial hair in women
(31,
32). DFMO is generally well
tolerated even at high doses, but reversible hearing loss has been reported in
multiple clinical trials (25,
33), and a rarer irreversible
defect has also been reported
(34). These side effects are
not observed at lower doses of DFMO
(26,
27).Ototoxicity has been demonstrated to occur in experimental animals treated
with DFMO including rats (35),
guinea pigs (36), gerbils
(37), and mice
(38). Using
immunohistochemistry, a high level of ornithine decarboxylase was observed in
the inner ear of the rat, with the highest in the organ of Corti and lateral
wall followed by the cochlear nerve
(39). Measurements of
polyamines in the relevant structures are very difficult due to the small
amount of tissue available, but as expected, DFMO treatment reduced polyamine
levels and ornithine decarboxylase activity in the inner ear of the guinea pig
(36). A plausible explanation
for the importance of polyamines in auditory physiology is based on their well
documented role as regulators of potassium channels
(38). The inward rectification
of Kir channels is caused by blockage of the outward current by polyamines
(40–42).
Studies of the cloned mouse cochlear lateral wall-specific Kir4.1 channel
showed that inward rectification was reduced and that there was a marked
reduction in endocochlear potential (EP). It was proposed that DFMO treatment
increases the outward Kir4.1 current, resulting in a drop in EP
(38).In the experiments reported here, we have studied in more detail the role
of polyamines in auditory physiology using Gy mice and crosses of these mice
with transgenic CAG/SpmS mice
(43). These mice express
spermine synthase under the control of a composite cytomegalovirus-IE
enhancer/chicken β-actin promoter, which was designed to provide
ubiquitous expression
(44–46).
Assays of the spermine synthase activity in CAG/SpmS line 8 confirmed that
there was a high level of expression of the transgene in many different organs
and that this level was maintained for at least 1 year
(43). Our studies confirm that
Gy mice are totally deaf and that this condition is reversed by the expression
of the SpmS gene. These changes are due to a virtually complete loss
of the EP in the Gy mice. We have also examined the effect of DFMO on the Gy
mice. Unexpectedly, it was found that these mice show a rapid and profound
toxicity to this drug, leading to death within a few days. Within 5 days of
exposure to DFMO in the drinking water, the DFMO-treated mice suffered a
catastrophic loss of balance due to inner ear effects. This toxicity was also
prevented by the transgenic expression of spermine synthase in the Gy
background. 相似文献
4.
5.
Annamari Paino Tuuli Ahlstrand Jari Nuutila Indre Navickaite Maria Lahti Heidi Tuominen Hannamari V?limaa Urpo Lamminm?ki Marja T. P?ll?nen Riikka Ihalin 《PloS one》2013,8(7)
Aggregatibacter
actinomycetemcomitans
is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system. 相似文献
6.
Norihisa Nishimichi Fumiko Higashikawa Hiromi H. Kinoh Yoshiko Tateishi Haruo Matsuda Yasuyuki Yokosaki 《The Journal of biological chemistry》2009,284(22):14769-14776
Osteopontin (OPN) is a cytokine and ligand for multiple members of the
integrin family. OPN undergoes the in vivo polymerization catalyzed
by cross-linking enzyme transglutaminase 2, which consequently increases the
bioactivity through enhanced interaction with integrins. The integrin
α9β1, highly expressed on neutrophils, binds to the sequence
SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence
appears to be cryptic in intact OPN because α9β1 does not recognize
intact OPN. Because transglutaminase 2-catalyzed polymers change their
physical and chemical properties, we hypothesized that the SVVYGLR site might
also be exposed on polymeric OPN. As expected, α9β1 turned into a
receptor for polymeric OPN, a result obtained by cell adhesion and migration
assays with α9-transfected cells and by detection of direct binding of
recombinant soluble α9β1 with colorimetry and surface plasmon
resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a
ligand for α9β1, has been reported to attract neutrophils, we next
examined migration of neutrophils to polymeric OPN using time-lapse
microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which
was clearly inhibited by anti-α9β1 antibody. Unexpectedly,
mutagenesis studies showed that α9β1 bound to polymeric OPN
independently of the SVVYGLR sequence, and further, SVVYGLR sequence of
polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize
polymeric OPN. These results demonstrate that polymerization of OPN generates
a novel α9β1-binding site and that the interaction of this site
with the α9β1 integrin is critical to the neutrophil chemotaxis
induced by polymeric OPN.Acidic phosphorylated secreted glycoprotein osteopontin
(OPN),4 known as a
cytokine, has multiple functions, including roles in tissue remodeling,
fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis
(1–3).
OPN is also an integrin ligand. At least nine integrins can function as OPN
receptors. α5β1, α8β1, αvβ1, αvβ3,
αvβ5 (1), and
αvβ6 (4) recognize
the linear tripeptide RGD, and α9β1, α4β1, and
α4β7 recognize the sequence, SVVYGLR
(5), adjacent to RGD but only
after OPN has been cleaved by the protease, thrombin
(Fig. 1).Open in a separate windowFIGURE 1.Schematic diagram of OPN. Two integrin-binding sites
(boxed), a thrombin cleavage site (arrow), and a putative
transglutamination site (circled) are shown. The term
thrombin-cleaved nOPN is defined as in the figure.The overlap of receptors for OPN does not necessarily mean that these
integrins play redundant roles in cellular responses to OPN because the
patterns of integrin expression and utilization vary widely among cell types.
In addition, interactions of different integrins with a single ligand can
exert distinct effects on cell behavior in a single cell type. For example, we
have previously reported that signals by ligation of αvβ3,
αvβ6, or α9β1 to a single ligand, tenascin-C,
differently affected cell adhesion, spreading, and proliferation of the colon
cancer cell line, SW480 (6).
Furthermore, intact OPN or thrombin- or matrix metalloproteinase-cleaved OPN
interact with distinct subsets of integrins and exhibit distinct effects on
cell behavior (4,
7,
8). Collectively, some of the
functional diversity of OPN could be attributed to this multiplicity of
receptors and responses. We have recently shown that polymerization of OPN
results in enhanced biological activity
(9). We thus set out to
determine whether polymerized OPN exerts its effects through unique
interactions with integrins.OPN is polymerized by transglutaminase 2 (TG2, EC 2.3.2.13)
(10) that catalyzes formation
of isopeptide cross-links between glutamine and lysine residues in substrate
proteins (11) including OPN.
Polymeric OPN has been identified in vivo in bone
(12) and calcified aorta
(13). We have previously
reported that upon polymerization, OPN displays increased integrin binding
accompanied by enhanced cell adhesion, spreading, migration, and focal contact
formation (9). However, very
little is known about how polymeric OPN induces its biological effects.Integrin α9β1, highly expressed on neutrophils
(14), does not act as a
receptor for intact OPN but does bind to an N-terminal fragment of OPN (nOPN)
that is generated by thrombin cleavage
(15) through the new
C-terminal sequence, SVVYGLR. Protein polymerization can expose otherwise
cryptic domains (16), so we
hypothesized that the SVVYGLR site might be exposed upon polymerization and
serve as a binding site for α9β1. In the present study, we
demonstrate that α9β1 is indeed a receptor for polymeric OPN and
that neutrophil migration induced by polymeric OPN is largely mediated by this
interaction. However, mutational analysis and antibody studies demonstrate
that this interaction does not involve the SVVYGLR site, suggesting the
presence of de novo binding site in polymeric OPN. 相似文献
7.
Does Leaf Position within a Canopy Affect Acclimation of
Photosynthesis to Elevated CO2?
: Analysis of a Wheat Crop under Free-Air CO2
Enrichment 总被引:1,自引:0,他引:1
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Colin P. Osborne Julie La Roche Richard L. Garcia Bruce A. Kimball Gerard W. Wall Paul J. Pinter Jr. Robert L. La Morte George R. Hendrey Steve P. Long 《Plant physiology》1998,117(3):1037-1045
Previous studies of photosynthetic acclimation to elevated CO2 have focused on the most recently expanded, sunlit leaves in the canopy. We examined acclimation in a vertical profile of leaves through a canopy of wheat (Triticum aestivum L.). The crop was grown at an elevated CO2 partial pressure of 55 Pa within a replicated field experiment using free-air CO2 enrichment. Gas exchange was used to estimate in vivo carboxylation capacity and the maximum rate of ribulose-1,5-bisphosphate-limited photosynthesis. Net photosynthetic CO2 uptake was measured for leaves in situ within the canopy. Leaf contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), light-harvesting-complex (LHC) proteins, and total N were determined. Elevated CO2 did not affect carboxylation capacity in the most recently expanded leaves but led to a decrease in lower, shaded leaves during grain development. Despite this acclimation, in situ photosynthetic CO2 uptake remained higher under elevated CO2. Acclimation at elevated CO2 was accompanied by decreases in both Rubisco and total leaf N contents and an increase in LHC content. Elevated CO2 led to a larger increase in LHC/Rubisco in lower canopy leaves than in the uppermost leaf. Acclimation of leaf photosynthesis to elevated CO2 therefore depended on both vertical position within the canopy and the developmental stage. 相似文献
8.
9.
10.
11.
Ana Paula S. Dornellas 《ZooKeys》2012,(224):89-106
Calliostoma tupinamba isa new species from Southeastern Brazil, ranging from southern Rio de Janeiro to northern São Paulo, and found only on coastal islands, on rocks and sessile invertebrates at 3 to 5 meters of depth. Shell and soft part morphology is described here in detail. Calliostoma tupinamba is mainly characterized by a depressed trochoid shell; eight slightly convex whorls; a sharply suprasutural carina starting on the third whorl and forming a peripheral rounded keel; and a whitish, funnel-shaped and deep umbilicus, measuring about 5%–10% of maximum shell width. Calliostoma tupinamba resembles Calliostoma bullisi Clench & Turner, 1960 in shape, but differs from it in being taller and wider, having a smaller umbilicus and lacking a strong and large innermost spiral cord at its base. Finally, an identification key of Brazilian Calliostoma species is presented. 相似文献
12.
13.
Cell death can be divided into the anti-inflammatory process of apoptosis and the
pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell
death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP)
1/3 are major mediators. We previously showed that absence or inhibition of PARP-1
protects mice from nephritis, however only the male mice. We therefore hypothesized that
there is an inherent difference in the cell death program between the sexes. We show here
that in an immune-mediated nephritis model, female mice show increased apoptosis compared
to male mice. Treatment of the male mice with estrogens induced apoptosis to levels
similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in
both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We
also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male
and female mice are prone to different types of cell death. Our data also suggest that
estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore
propose that targeting cell death based on sex will lead to tailored and better treatments
for each gender. 相似文献
14.
15.
16.
Hardeep Kaur Chitranshu Kumar Christophe Junot Michel B. Toledano Anand K. Bachhawat 《The Journal of biological chemistry》2009,284(21):14493-14502
GSH metabolism in yeast is carried out by the γ-glutamyl cycle as
well as by the DUG complex. One of the last steps in the
γ-glutamyl cycle is the cleavage of Cys-Gly by a peptidase to the
constitutent amino acids. Saccharomyces cerevisiae extracts carry
Cys-Gly dipeptidase activity, but the corresponding gene has not yet been
identified. We describe the isolation and characterization of a novel Cys-Gly
dipeptidase, encoded by the DUG1 gene. Dug1p had previously been
identified as part of the Dug1p-Dug2p-Dug3p complex that operates as an
alternate GSH degradation pathway and has also been suggested to function as a
possible di- or tripeptidase based on genetic studies. We show here that Dug1p
is a homodimer that can also function in a Dug2-Dug3-independent manner as a
dipeptidase with high specificity for Cys-Gly and no activity toward tri- or
tetrapeptides in vitro. This activity requires zinc or manganese
ions. Yeast cells lacking Dug1p (dug1Δ) accumulate Cys-Gly.
Unlike all other Cys-Gly peptidases, which are members of the metallopeptidase
M17, M19, or M1 families, Dug1p is the first to belong to the M20A family. We
also show that the Dug1p Schizosaccharomyces pombe orthologue
functions as the exclusive Cys-Gly peptidase in this organism. The human
orthologue CNDP2 also displays Cys-Gly peptidase activity, as seen by
complementation of the dug1Δ mutant and by biochemical
characterization, which revealed a high substrate specificity and affinity for
Cys-Gly. The results indicate that the Dug1p family represents a novel class
of Cys-Gly dipeptidases.GSH is a thiol-containing tripeptide
(l-γ-glutamyl-l-cysteinyl-glycine) present in
almost all eukaryotes (barring a few protozoa) and in a few prokaryotes
(1). In the cell, glutathione
exists in reduced (GSH) and oxidized (GSSG) forms. Its abundance (in the
millimolar range), a relatively low redox potential (-240 mV), and a high
stability conferred by the unusual peptidase-resistant γ-glutamyl bond
are three of the properties endowing GSH with the attribute of an important
cellular redox buffer. GSH also contributes to the scavenging of free radicals
and peroxides, the chelation of heavy metals, such as cadmium, the
detoxification of xenobiotics, the transport of amino acids, and the
regulation of enzyme activities through glutathionylation and serves as a
source of sulfur and nitrogen under starvation conditions
(2,
3). GSH metabolism is carried
out by the γ-glutamyl cycle, which coordinates its biosynthesis,
transport, and degradation. The six-step cycle is schematically depicted in
Fig. 1
(2).Open in a separate windowFIGURE 1.γ-Glutamyl cycle of glutathione metabolism.
γ-Glutamylcysteine synthetase and GSH synthetase carry out the first two
steps in glutathione biosynthesis. γ-glutamyltranspeptidase,
γ-glutamylcyclotransferase, 5-oxoprolinase, and Cys-Gly dipeptidase are
involved in glutathione catabolism. Activities responsible for
γ-glutamylcyclotransferase and 5-oxoprolinase have not been detected in
S. cerevisiae.In Saccharomyces cerevisiae, γ-glutamyl cyclotransferase and
5-oxoprolinase activities have not been detected, which has led to the
suggestion of the presence of an incomplete, truncated form of the
γ-glutamyl cycle (4) made
of γ-glutamyl transpeptidase
(γGT)4 and
Cys-Gly dipeptidase and only serving a GSH catabolic function. Although
γGT and Cys-Gly dipeptidase activities were detected in S.
cerevisiae cell extracts, only the γGT gene (ECM38) has
been identified so far. Cys-Gly dipeptidase activity has been identified in
humans (5,
6), rats
(7–10),
pigs (11,
12), Escherichia coli
(13,
14), and other organisms
(15,
16), and most of them belong
to the M17 or the M1 and M19 metallopeptidases gene families
(17).S. cerevisiae has an alternative γGT-independent GSH
degradation pathway (18) made
of the Dug1p, Dug2p, and Dug3p proteins that function together as a complex.
Dug1p also seem to carry nonspecific di- and tripeptidase activity, based on
genetic studies (19).We show here that Dug1p is a highly specific Cys-Gly dipeptidase, as is its
Schizosaccharomyces pombe homologue. We also show that the mammalian
orthologue of DUG1, CNDP2, can complement the defective utilization
of Cys-Gly as sulfur source of an S. cerevisiae strain lacking
DUG1 (dug1Δ). Moreover, CNDP2 has Cys-Gly dipeptidase
activity in vitro, with a strong preference for Cys-Gly over all
other dipeptides tested. CNDP2 and its homologue CNDP1 are members of the
metallopeptidases M20A family and have been known to carry carnosine
(β-alanyl-histidine) and carnosine-like (homocarnosine and anserine)
peptidase activity (20,
21). This study thus reveals
that the metallopeptidase M20A family represents a novel Cys-Gly peptidase
family, since only members of the M19, M1, and M17 family were known to carry
this function. 相似文献
17.
Xiufeng Song Sergio Coffa Haian Fu Vsevolod V. Gurevich 《The Journal of biological chemistry》2009,284(1):685-695
Arrestins bind active phosphorylated G protein-coupled receptors,
precluding G protein activation and channeling signaling to alternative
pathways. Arrestins also function as mitogen-activated protein kinase (MAPK)
scaffolds, bringing together three components of MAPK signaling modules. Here
we have demonstrated that all four vertebrate arrestins interact with JNK3,
MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the
functional specificity of arrestins is not determined by differential binding
of the kinases. Using receptor binding-impaired mutant, we have shown that
free arrestin3 readily promotes JNK3 phosphorylation. We identified key
arrestin-binding elements in JNK3 and ASK1 and investigated the molecular
interactions of arrestin2 and arrestin3 and their individual domains with the
components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We
found that both arrestin domains interact with all six kinases. These findings
shed new light on the mechanism of arrestin-mediated MAPK activation and the
spatial arrangement of the three kinases on arrestin molecule.Arrestins are multifunctional regulators of cell signaling
(1,
2). Arrestins, which bind
active phosphorylated G protein-coupled receptors
(GPCRs),2 which play a
major role in receptor desensitization and internalization
(3,
4). With the identification of
numerous non-receptor binding partners, the classical paradigm of arrestin
function has been expanded, implicating arrestins in mitogen-activated protein
kinase (MAPK) activation, protein ubiquitination, chemotaxis, apoptosis, and
other cellular functions (2,
5-11).The first indication that arrestins function as signaling adapters came
from the studies of arrestin-dependent c-Src recruitment to the receptors,
which results in the activation of extracellular signal-regulated kinases
(ERK1/2) (10,
12,
13). Subsequently, arrestin2
and arrestin3 in complex with different receptors were reported to scaffold
JNK3 (9), ERK1/2
(8,
14), and p38
(15,
16) activation cascades.
Although arrestins play an important role in regulating different MAPK
pathways, the mechanism of arrestin-dependent assembly of MAP kinases into a
signaling complex remains largely unexplored. Existing models have limited
predictive value. For example, the idea that JNK3 is activated solely by
arrestin3 because this arrestin subtype has unique ability to bind JNK3
(9,
17) was not supported by
further experimentation
(18-20).
Similarly, the hypothesis that only receptor-bound arrestins interact with MAP
kinases (8,
9) was not confirmed
(17-20).Here we addressed several key mechanistic issues in arrestin-dependent MAPK
signaling. First, we show that the scaffolding function is not limited to
receptor-bound arrestin; free arrestin3 facilitates ASK1-mediated JNK3
activation, indicating that arrestins are not exclusively receptor-regulated
adapters as thought previously. Second, we show that all four mammalian
arrestins bind each component of the JNK3 cascade with comparable affinity,
demonstrating that binding does not necessarily translate into activation.
This finding establishes the mechanistic basis of the
“dominant-negative” effect of certain arrestin subtypes. Third,
using truncated forms of ASK1 and JNK3, we identified the major
arrestin-binding elements of these two kinases. Finally, we show that every
kinase in JNK3 and ERK2 activation cascades binds both arrestin domains. Based
on these findings, we propose a functional model of arrestin-dependent
regulation of MAPK activity and a new structural model of the arrestin-MAPK
multiprotein signaling complex. 相似文献
18.
19.
20.
Usha Padmanabhan D. Eric Dollins Peter C. Fridy John D. York C. Peter Downes 《The Journal of biological chemistry》2009,284(16):10571-10582