|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 22, 20070-20078, May 31, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, August 30, 2001, and in revised form, February 20, 2002
We have isolated a novel protein based
on its association with Drosophila APP-like protein (APPL),
a homolog of the The cytoplasmic domains of APPs are more highly conserved than the
other domains among a wide variety of species and are important for
intracellular metabolism (10, 11) and for physiological function
(12-15). Several proteins have been reported to interact with the
cytoplasmic domain of APP (APPcyt) in mammals (16-21), although the
importance and physiological function(s) of these interactions are not
well understood. In invertebrates the protein interaction of APPs has
been scarcely identified. In this study we identified a novel gene in
Drosophila, named APLIP1, which encodes a
protein that interacts with the cytoplasmic domain of APPL (APPLcyt).
APLIP1 was homologous to mammalian scaffold proteins JIP1b and JIP2,
which were implicated in the JNK signaling cascade and/or intracellular
transport (22-25). They could interact with APPs and in addition
shared abundant expression in neural tissue and the properties to bind
JNK kinase and kinesin. Moreover, we propose a possible function of
mammalian JIP to modulate the phosphorylation of APP. Analysis of
this evolutionarily conserved interaction of APPs with
APLIP1 and JIP contributes to our knowledge of the mechanism of
Alzheimer's disease progression.
Yeast Two-hybrid System--
The yeast two-hybrid screening was
executed with the MATCH MAKER two-hybrid system (Clontech) as described
(18). The pGBT9APPLcyt "bait" plasmid encoding the cytoplasmic
domain (amino acids 834-886) of APPL (4) and a Drosophila
whole adult cDNA library cloned into the vector pACT2 (Clontech)
were cotransfected into the yeast HF7c strain. Cotransfectants were
grown on a selective medium lacking Trp, Leu, and His and were assayed
for their activation of lacZ reporter genes. Positive clones
were recloned into Escherichia coli HB101, and their
nucleotide sequences were determined. Quantification of the
Molecular Cloning of APLIP1, JIP1b, and JIP2--
The cDNA
encoding full-length APLIP1 was isolated from a Drosophila
embryonic cDNA library (a kind gift from Dr. Ueda) using a
radiolabeled probe prepared from a partial fragment of APLIP1 (nucleotides 841-1788). Hybridizations were carried out with a standard procedure (26). Positive clones were subjected to excision of
the pBluescript phagemid from the Plasmid Construction--
The cDNA encoding
APPLcyt was produced by RT-PCR using adult total RNA, and the resulting
cDNA fragment encoding amino acid positions 834-886 of APPL (4)
was cloned into pGBT9 (Clontech) at EcoRI/BamHI
sites for yeast two-hybrid screening. Various deletions of APPLcyt were
produced by PCR and cloned into pGBT9 for two-hybrid analysis: Northern Blot Hybridization and RT-PCR
Analysis--
Poly(A)+ RNA was isolated from various
developmental stages of embryos, larvae, pupae, and adult flies
(Canton-S) using a µMACS RNA isolation kit (PerkinElmer Life
Sciences). For Northern blotting, 1 µg of poly(A)+ RNA
was loaded onto a 1% (w/v) agarose gel under denatured conditions, electrophoresed, and transferred to a membrane. Radiolabeled probes (>108 cpm/µg DNA) of APLIP1 and ribosomal protein 49 (rp49) were prepared with [ In Situ Hybridization--
A digoxigenin-labeled RNA probe was
prepared with T7 RNA polymerase (Roche Diagnostics),
digoxigenin-11-UTP, and APLIP1 cDNA in pBluescript SKII digested
with EcoRI as a template for the antisense probe. Embryos
were dechorionated and fixed as described previously (27).
Hybridization was performed with antisense probes and visualized using
an anti-digoxigenin antibody conjugated to alkaline phosphatase and an
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate
(Roche Diagnostics).
Antibodies--
The rabbit polyclonal anti-APP antibody G369 has
been described previously (28). A rabbit polyclonal anti-APPL antibody raised against the extracellular domain of APPL (Ab952) was a kind gift
from Dr. K. White (29). Anti-FLAG (M2; Sigma), anti-HA (12CA5; Roche
Diagnostics), and anti-c-Myc (Invitrogen) monoclonal antibodies
were purchased. Polyclonal APP phosphorylation
state-specific antibody (pAbThr-668) UT-33 was raised against a
phosphopeptide APP665-673[Cys][PiThr-668] of APP695
and described previously (12, 30, 31).
Expression of Proteins in Cultured Cells--
African green
monkey kidney COS-7 and mouse neuroblastoma Neuro-2a cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
(v/v) heat-inactivated fetal bovine serum. To express proteins, 5 × 105 ~1 × 106 COS-7 or ~1 × 106 Neuro-2a cells were transiently transfected with 0.5-2
µg of each plasmid in LipofectAMINE 2000 (Invitrogen) or
LipofectAMINE (Invitrogen) according to the manufacturer's protocol
and cultured for 24-48 h in Dulbecco's modified Eagle's medium
containing 10% (v/v) fetal bovine serum. The cells were harvested and
lysed in CHAPS lysis buffer (phosphate-buffered saline containing 10 mM CHAPS, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml pepstatin A, 1 mM Na3VO4,
and 1 mM NaF) for 0.5 h at 4 °C. Microcystine-LR (1 µM) was added to the buffer to analyze the
phosphorylation. The lysed cells were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was used for
the following analysis.
In Vitro Binding Assay Using GST Fusion Proteins--
A
pGEX-4T-1 cDNA construct was introduced into E. coli BL21, and a GST fusion protein was prepared. The protein was
purified with glutathione-Sepharose 4B (Amersham Biosciences) as
described (18), and the purity of the protein was examined by staining with Coomassie Brilliant Blue R-250 after SDS-PAGE. The purified GST
fusion proteins (~1 nmol) coupled to glutathione-Sepharose 4B were
incubated for 2 h at 4 °C with cell lysates derived from COS-7
cells transiently expressing the recombinant protein. The beads were
washed with phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, 140 mM NaCl) three times, and the
protein bound to the GST fusion protein was eluted with SDS sample
buffer and analyzed by Western blot with ECL (Amersham Biosciences) or
125I-protein A (Amersham Biosciences).
Coimmunoprecipitation Assay--
The supernatant prepared from
double transfected COS-7 cells was incubated with 5-10 µg of
antibody at 4 °C for 1 h. The resulting immunocomplex bound to
mouse antibodies was recovered using protein G-Sepharose beads
(Amersham Biosciences) and analyzed by Western blot with the
antibody indicated.
Isolation of APLIP1--
APPs are thought to function through
interaction of their cytoplasmic domains with cytoplasmic proteins.
Therefore, we screened a Drosophila adult cDNA library
with the yeast two-hybrid system using APPLcyt as bait and obtained
clones encoding part of a novel protein. The full-length cDNA was
isolated from an embryonic cDNA library by plaque hybridization
using partial cDNA as a probe. The full-length cDNA obtained
encoded a protein consisting of 490 amino acids and containing putative
SH3 and PI domains in its carboxyl-terminal half (Fig.
1, A and B). We called
this novel protein APLIP1, for APP-like protein
interacting protein 1. From an
analysis of the Drosophila genomic data base (32), the
APLIP1 gene was revealed to be composed of five exons
and to be located on region 61F1-61F4 of chromosome 3L. No other gene
resembling APLIP1 was found in the data base. In this
two-hybrid screening, a cDNA encoding the carboxyl-terminal
fragment of the Drosophila homolog of X11L (dX11L) (GenBank
AF208839)2 and a putative Ser/Thr
protein kinase, Bin4 (GenBank AF096866), were also selected as positive
clones. The binding activities of these clones with APPL examined in
yeast are comparable with that of
APLIP1.3
Expression of APLIP1--
APPL is expressed specifically in neural
tissues during development (4, 7). If APLIP1 interacts with the
cytoplasmic domain of APPL in vivo, we would expect APLIP1
to be expressed in neural tissues as well. Therefore, we examined the
expression profile of APLIP1. Northern blot analysis was performed
against poly(A)+ RNA prepared from various developmental
stages (Fig. 2A). A 2.4-kb transcript showed strong expression during late embryonic (12-18 h
after egg deposition (E12-18)) to adult stages. No signal could be
detected in the lane containing RNA from an E0-6 embryo, and only a
weak signal was observed in E6-12 embryos. This expression profile is
similar to that of APPL, although expression of APPL is weak in larval
stages (4). Next we examined whether the expression of APLIP1 is
enriched in the nervous system. We prepared poly(A)+ RNA
from the heads and bodies of adult flies and quantified the amount of
transcript of the APLIP1 and rp49 by
RT-PCR using specific primer sets (Fig. 2B). A larger amount
of the APLIP1 PCR product was obtained from the heads than from the
bodies, whereas comparable amounts of the PCR product derived from the
rp49 RNA were observed in the heads and bodies. This result suggests
that APLIP1 is expressed largely in neural tissues, as is APPL. The
abundant expression of the APLIP1 in the neural tissues was
confirmed by in situ hybridization (Fig. 2C).
APLIP1 expression was detected in the brain and central nervous system of late embryos (Fig. 2C), whereas no signal
was observed in early embryos (data not shown). These observations suggest the possibility that APLIP1 interacts with APPL in neural tissues.
Interaction between APLIP1 and APPL--
The APLIP1 protein
interacted with APPL in yeast cells. To investigate whether they
interact in other systems, we examined protein-protein interactions
between APLIP1 and APPL in vitro using a GST fusion protein.
A GST-APLIP1 fusion protein was prepared in bacteria, purified,
immobilized on glutathione-Sepharose, and incubated with a lysate of
COS-7 cells expressing APPL. APPL expressed in COS-7 cells exhibits
several bands probably differing in glycosylation. APPL attached to the
beads was washed, eluted together with GST-APLIP1, and detected by
Western blot analysis using an anti-APPL antibody (Fig.
3A). APPL bound to the beads
coupled with the GST-APLIP1 fusion protein but not to GST alone.
We examined the interaction of APLIP1 with APPL in cells (Fig.
3B). APLIP1 tagged with FLAG epitope (FLAG-APLIP1) was
expressed in COS-7 cells together with or without APPL (left
panel of Fig. 3B). The proteins were immunoprecipitated
from the cell lysate with an anti-FLAG antibody and analyzed by
immunoblot with anti-APPL and anti-FLAG antibodies. We observed that
APPL was coimmunoprecipitated from the cell lysate with the FLAG-APLIP1
(middle panel of Fig. 3B). Conversely, when we
immunoprecipitated APPL from the cell lysates with anti-APPL antibody,
FLAG-APLIP1 was recovered with APPL (right panel of Fig.
3B). These results indicate that APLIP1 binds to APPL both
in vitro and in the cell.
Identifying the Binding Domains of APLIP1 and APPL--
The region
required for the interaction between APLIP1 and APPL was determined.
The ability of various truncated protein constructs derived from APLIP1
to bind to the APPLcyt was examined using the yeast two-hybrid system.
Growth in a selective medium and
Conversely, we examined the region of the APPLcyt required for the
interaction with APLIP1 (Fig. 4B). Truncated proteins
lacking the amino-terminal side of APPLcyt (
We confirmed these binding properties by an in vitro protein
binding analysis with a GST fusion protein (Fig. 4C). Cell
lysates containing the Myc-tagged full-length, N Identification of Mammalian Counterparts of APLIP1--
The region
around the GYENPTY motif of APPL, which was required for the
interaction with APLIP1, is almost completely conserved in mammalian
APP. Because we observed that human APP interacts with APLIP1 in a
yeast two-hybrid assay (data not shown), we suspected that genes
corresponding to APLIP1 are conserved in mammal, although no homologous
gene could be found in the data base at the time we cloned the
APLIP1 gene. Therefore, we screened a mouse brain cDNA
library by plaque hybridization using a fragment of APLIP1 as a probe.
This screen resulted in the isolation of two cDNAs homologous to
APLIP1. A BLAST search revealed that one is identical to JIP1b, a
splicing variant of the scaffold protein JIP1 (22, 23), which was also
reported as islet brain-1 (34). The other is mouse JIP2, which is
almost identical to the recently cloned human JIP2 (24). JIP1b and JIP2
are expressed abundantly in brain (data not shown) (22, 24). When the
primary structures of APLIP1, JIP1b, and JIP2 were aligned (Fig. 1,
A and B), the mammalian JIP1b and JIP2 were found
to resemble APLIP1, especially in the carboxyl-terminal structure
containing the SH3 and PI domains. This structural resemblance of JIP1b
and JIP2 to APLIP1 is expected to correlate with functional similarity.
Characterization of APLIP1--
The structural similarity between
APLIP1 and the JIPs suggests that they have a conserved function.
Mammalian JIP1 and JIP2 have already been reported to bind several
proteins to act as scaffold proteins in the JNK signaling pathway
and/or as cargos for intracellular protein transport (23-25).
JIP1 and JIP2 selectively bind components of the JNK signaling cascade
such as JNK (MAP kinase), MKK7 (MAP kinase kinase), and mixed lineage
kinase family proteins (MAP kinase kinase kinase) (23, 24). The JNK
signaling pathway is basically conserved in Drosophila. The
Drosophila JNK pathway consists of Drosophila JNK/basket (DJNK) and JNK kinase Hep, which correspond to JNK and MKK7
in mammals, respectively (for review, see Ref. 35). Thus, we examined
whether APLIP1 could bind these protein kinases in the
Drosophila JNK pathway as well as JIP1 and JIP2 can bind to
the homologous proteins in mammals. First we investigated whether APLIP1 could bind DJNK. A GST-DJNK fusion protein was prepared, immobilized on glutathione beads, and incubated with a lysate of COS-7
cells expressing FLAG-tagged APLIP1, Drosophila Hep, or
mouse JIP1b protein (Fig. 5A).
Both Drosophila Hep, which is known to interact with DJNK
(36), and mouse JIP1b bound to the GST-DJNK fusion protein, but binding
of APLIP1 to GST-DJNK could not be detected in the same experiment. We
also could not detect an interaction between APLIP1 and DJNK by
coimmunoprecipitation analysis (data not shown). Mammalian JIPs share
the JNK binding domain consensus sequence in the amino-terminal region
(37), which also exists in other JNK-binding proteins such as c-Jun and
is responsible for the binding with JNK (22). However, consistent with
the results of the in vitro binding assay (Fig.
5A), we did not observe the sequence corresponding to this
consensus in APLIP1 (Fig. 1B). We conclude that APLIP1 does
not bind to DJNK as mammalian JIPs bind to JNK. On the other hand, we
found that Hep bound to the GST-APLIP1 fusion protein but not to GST
alone in vitro (upper panel of Fig.
5B). The interaction of APLIP1 with Hep was also observed in
cells. COS-7 cells expressing HA-tagged APLIP1 and FLAG-tagged Hep were
lysed and subjected to immunoprecipitation with an anti-FLAG antibody.
The immunoprecipitates were analyzed by Western blot with anti-HA and
anti-FLAG antibodies (Fig. 5C). APLIP1 was
coimmunoprecipitated with FLAG-tagged Hep, and without FLAG-tagged Hep
the anti-FLAG antibody failed to precipitate HA-tagged APLIP1. These
results revealed that Hep, a protein kinase upstream of DJNK, could
bind APLIP1.
Next we tried to verify whether molecules of APLIP1 can interact with
each other because JIP family proteins are known to form homo- or
hetero-oligomeric complexes in mammals (24). We found that APLIP1 bound
to the GST-APLIP1 fusion protein but not to GST alone in
vitro (lower panel of Fig. 5B). Formation of
homo-oligomeric complexes of APLIP1 was observed in cultured cells
expressing HA-tagged and FLAG-tagged APLIP1 in cells (Fig.
5D). The anti-FLAG antibody coimmunoprecipitated HA-tagged
APLIP1 from the cell lysate with FLAG-tagged APLIP1. These results
suggest that APLIP1 can form homo-oligomers as JIP1 and JIP2 do.
Finally, the interaction of APLIP1 with the DKLC was examined. JIP1 and
JIP2 were recently reported to bind the tetratricopeptide repeat of the
kinesin light chain, a component of the motor protein kinesin, at its
carboxyl terminus (Fig. 1B) (25). APLIP1 and a fragment of
DKLC containing a tetratricopeptide repeat were expressed in COS-7
cells, and a coimmunoprecipitation assay was performed (Fig.
6). APLIP1 was coprecipitated with DKLC as
JIP1 was, whereas APLIP1C
Thus, APLIP1 is not only similar to JIP1b and JIP2 structurally but
also shares most of the features of their protein interactions, with
the exception of binding to JNK.
Mammalian JIP1b and JIP2 Interact with APP--
APLIP1 shared most
of the features with JIP1b and JIP2. We showed that the amino acid
sequence containing the GYENPTY motif of APPL is responsible for its
interaction with APLIP1 and that this sequence is highly conserved in
mammalian APPs. The carboxyl-half of APLIP1, containing the domain
interacting with APPL, is also highly conserved in the JIP1b and JIP2
proteins. These facts suggest that JIP1b and JIP2 may be able to bind
APP. In fact, in our laboratory, JIP1b was isolated in a yeast
two-hybrid screening of a human fetal cDNA library using the APPcyt
as bait.3 To examine this possibility, we performed
coimmunoprecipitation analysis using COS-7 cells expressing FLAG-tagged
APP and HA-tagged JIP1b and JIP2 (Fig.
7A). JIP1a, a splicing variant of
JIP1 lacking a part of the PI domain, and X11L, which is known as one
of the APP-binding proteins in mammal (18), were also subjected to the
analysis. JIP1b, as well as X11L, was coimmunoprecipitated from the
cell lysate with FLAG-APP by anti-FLAG antibody. JIP2 and JIP1a were
also coprecipitated with APP, although their amounts were much smaller
than that of JIP1b and X11L. These interactions were examined further
by an in vitro protein binding assay (Fig. 7B). A
COS-7 cell lysate including HA-JIP1b, HA-JIP1a, HA-JIP2, or HA-X11L was
incubated with a GST-APPcyt fusion protein coupled to beads. The beads
were washed, and proteins bound to the beads were analyzed by
immunoblot with an anti-HA antibody. Both JIP1b and JIP2 were eluted
from the beads coupled with the GST-APPcyt fusion protein, as X11L was.
Interestingly, less binding between JIP2 and APP was detected than
between JIP1b and APP in both analyses, suggesting that the interaction
of APP with JIP2 is weaker than that with JIP1b, even though their
carboxyl-terminal regions are similar to each other. In this assay
JIP1a could not be detected to bind APPcyt. These results suggest that
at least mammalian JIP1b and JIP2 could bind the cytoplasmic domain of
APP, whereas the preferences of JIP1b and JIP2 to bind APP are a
little different.
Different Effect of JIP1b and JIP2 on APP
Phosphorylation--
JIP1 and JIP2 have been reported as scaffold
proteins that associate with JNK signaling cascade. The cytoplasmic
domain of APP is known to be phosphorylated at Thr-668 by
cyclin-dependent protein kinases such as Cdc2 and Cdk5 (30,
38). Recently it was revealed that JNK family proteins, JNK1, 2, and 3, also have the ability to phosphorylate APP at Thr-668 in
vitro and in cultured cells (39).3 Therefore, we
assumed that JIP1 and JIP2 could affect the phosphorylation of APP
induced by the activation of JNK cascades. In cultured cells,
phosphorylation of APP was induced by the overexpression of DLK, one of
the upstream kinases of JNK (40), after the activation of JNK cascade
(Fig. 8). APP was expressed together with or
without DLK, JIP1b, JIP1a, and JIP2 in Neuro-2a cells, and the
phosphorylation of APP at Thr-668 was detected and quantified by
immunoblot with the APP phosphorylation state-specific antibody. This
antibody selectively recognizes APP phosphorylated at Thr-668. The
phosphorylated APP was detected in the cells expressing APP together
with DLK but not in the cells expressing APP alone or DLK alone. In the cells expressing JIP1b the phosphorylation level of APP increased slightly when compared with it in the cell not expressing JIPs. Conversely, expression of JIP1a, a splicing variant of JIP1 lacking part of the PI domain, resulted in a decreased level of the
phosphorylation. Moreover, expression of JIP2 also induced the decrease
of APP phosphorylation. It seems interesting that the effects of JIP1b, JIP1a, and JIP2 on APP phosphorylation were different, although these
proteins have been reported to play a similar role in JNK signaling
(24). Because JIP1b can bind both APP and JNK, it is probable
that JIP1b recruits JNK to the APPcyt and contributes to the
phosphorylation of APP at Thr-668. Conversely, JIP1a and JIP2 have the
ability to bind JNK as well as JIP1b, but they lack the ability to
associate with APPcyt efficiently. Most likely, JIP1a and JIP2 isolated
JNK from APPcyt.
Phosphorylation of APP at Thr-668 causes a conformational change of
APPcyt and affects the interaction with the binding proteins such as
Fe65 (31). Thus, the phosphorylation of APP at Thr-668 is suggested to
be important for the metabolism and/or putative function(s) of APP. It
is possible that JIPs are involved in APP metabolism and/or function(s)
by regulating APP phosphorylation.
APPs possess a membrane-associated receptor-like structure, and
the amino acid sequence of their short cytoplasmic region is highly
conserved among a wide variety of species (for review, see Refs. 1 and
2). Protein interactions between the cytoplasmic domains of APPs and
cytoplasmic proteins are thought to be important for regulating the
metabolism of APPs and/or for putative physiological function of APP
(6, 10-15). Here, we isolated a novel gene in Drosophila
named APLIP1 and indicated that APLIP1 and its putative mammalian homologs JIP1b and JIP2 could interact with the cytoplasmic domain of the APPs. These proteins, APLIP1, JIP1b, and JIP2, resemble each other in their structure, especially in their carboxyl-terminal regions that contain SH3 and PI domains. We revealed that they also
share properties such as interactions with APPs, MAP kinase kinase, and
kinesin; an abundant expression in the nervous system; and the
formation of homo-oligomers. These similarities suggest that APLIP1,
JIP1b, and JIP2 belong to same protein family functionally conserved in
various species. In mammals, JIP3/JSAP was reported as another member
of the JIP family of proteins (37, 41), which displayed scaffold
function in the JNK signaling pathway as JIP1 and JIP2 did. In
Drosophila, a putative homolog of JIP3, designed Sunday
Driver protein (SYD), has been reported (42). However, they differ from
APLIP1, JIP1b, and JIP2 in their domain structure; they do not possess
the SH3 and PI domains that are important regions for binding several
proteins including APPs (43-45), and they may have some different
roles from APLIP1, JIP1, and JIP2.
In mammal several proteins bind the APPcyt (16-21), whereas the
physiological role(s) of these interaction have not been sufficiently revealed. Here we showed that JIP1b and JIP2, mammalian counterparts of
APLIP1, could interact with APPcyt. Regarding JIP1b, the binding to
APPcyt was also reported recently to be relatively lower than the
binding of other APP-binding proteins, such as mDab1, X11, and Fe65
(46). We also observed that the binding of JIP1b was slightly lower
than that of the other binding protein X11L in vitro but not
in the cell. However, the faint differences in the binding activities
do not necessarily deny the physiological importance of JIP1b for APP.
In fact, we found a novel function of JIP1 and JIP2 to be the
modulation of the phosphorylation of APP at Thr-668 residue induced by
the activation of JNK. Expression of JIP1b slightly enhanced the
phosphorylation of APP, whereas the expression of JIP2 or JIP1a
suppressed the phosphorylation. From the previous reports that JIP1b,
JIP1a, or JIP2 could equally facilitate the activation of JNK signaling
(23, 24), it was expected that these proteins similarly regulate the
phosphorylation of APP when JNK was activated. Nevertheless, only JIP1b
facilitated the phosphorylation, and others decreased the level of the
phosphorylation of APP. We indicated that the interaction of JIP2 and
JIP1a with APP was remarkably weaker than that of JIP1b in the cell.
Therefore, it is conceivable that the effect of JIP1a or JIP2 to
decrease the level of the phosphorylation of APP reflects their weaker
binding properties to APP rather than their properties of regulating
the JNK signaling cascade. It is assumed that formation of the complex between JIP1a or JIP2 and JNK may suppress JNK to approach to the
phosphorylation site of APP, whereas the complex of JIP1b with JNK can
easily approach APPcyt. Indeed a recent report showed that formation of
the tripartite complex composed of JIP1b, JNK, and APP could be
observed in cultured cells (46). Phosphorylation of APP at Thr-668 has
been implicated in the metabolism and/or putative function(s) of APP
(12, 31), and modulation of the phosphorylation level of APP by JIPs in
mammal possibly has physiological importance.
In invertebrates only this APLIP1 and dX11L have been reported to
interact with APPs (for dX11L, GenBank AF208839),2 except
kinesin interacts genetically with APPL (47, 48). They may be
implicated in evolutionarily conserved roles relative to metabolism
and/or function of APPs, besides the role of mammalian JIP in the
phosphorylation of APP. In Drosophila, APPL does not have a
phosphorylation site corresponding to the Thr-668 residue of mammalian
APP695. In addition, there are some differences in the function of
APLIP1 on the JNK signaling pathway from that of mammalian JIP1 and
JIP2 because APLIP1 could not interact with DJNK, whereas it could
interact with Drosophila JNK kinase Hep (Fig. 5). Thus
APLIP1 cannot form a complex with DJNK and facilitate JNK activation in
Drosophila in the same manner as JIPs do in mammals, whereas
a possibility of regulating JNK signaling through an interaction with
Hep still remains. Therefore, the effect modulating the phosphorylation
of APP by JIP1 and JIP2 may be acquired in the evolutionary process.
Questions of what the evolutionarily conserved role(s) of the
interaction of APLIP1, JIP1b, and JIP2 with APPs are remain to be
elucidated. Several physiological roles for the mammalian JIP family
proteins have been proposed other than acting as scaffold molecules of
JNK cascades (23, 24): as a transactivator of the
GLUT2 gene (34) and as cargo for kinesin to mediate
the transportation of several transmembrane proteins (25). In
Drosophila APLIP1 interacts with the kinesin light chain as
well as mammalian JIP1 and JIP2 do, but interaction with the molecules
of the JNK cascades is only partly conserved. The metabolic scheme of
APPs is basically conserved between Drosophila and mammal.
Kinesin is involved in intracellular transport and metabolism of APP in
mammal and is associated with APPL in Drosophila (47, 48).
Accordingly, we assume that Drosophila APLIP1 and mammalian
JIP share a role in the intracellular metabolism of APPs.
In conclusion, we identified a novel protein in Drosophila
named APLIP1 and mammalian JIP1b and JIP2 as binding proteins of APPs.
APLIP1, JIP1b, and JIP2 comprise an evolutionary conserved protein
family and share properties in their domain structure, expression
pattern, and interaction profiles with proteins such as APPs, kinesin,
and JNK kinase, although a few exceptions are observed (Fig.
9). We propose that a novel function of
mammalian JIP1 and JIP2 is to modulate the phosphorylation of
APP. Further analysis of conserved or different roles of
APLIP1, JIP1b, and JIP2 may contribute to our understanding of the
mechanisms of APPs metabolism and the pathogenesis of Alzheimer's
disease.
We thank Dr. S. Takeda (the University of
Tokyo, Japan) for helpful discussions. We also thank Yo-ko Morimoto,
Kazumi Arikawa, and Akio Sumioka for technical assistance. We thank Dr.
H. Ueda (National Institute of Genetics, Mishima, Japan) for the
cDNA library, Dr. K. White (Princeton University) for anti-APPL
antibody, and Drs. P. Greengard (Rockefeller University) and Samuel
Gandy (New York University) for anti-APP antibody G369.
*
This work was supported by Grant-in-aid for Scientific
Research on Priority Areas, Advanced Brain Science Project, TS 13210031 and Grants in-aid for Scientific Research TS 12470494 and TS 13877369 from the Ministry of Education, Science, Culture, Sports, and Science,
and Technology, Japan.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF220194 (Drosophila APLIP1), AF054611
(mouse JIP1b), and AF220195 (mouse JIP2).
¶
To whom correspondence should be addressed: Laboratory of
Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku Kita-12 Nishi-6, Sapporo 060-0812, Japan. Tel.: 81-11-706-3250; Fax: 81-11-706-4991; E-mail:
tsuzuki@pharm.hokudai.ac.jp.
Published, JBC Papers in Press, March 22, 2002, DOI 10.1074/jbc.M108372200
2
M. Hase, Y. Yagi, H. Taru, S. Tomita, A. Sumioka, K. Hori, Y. Miyamoto, T. Sasamura, M. Nakamura, K. Matsuno,
and T. Suzuki, J. Neurochem. (2002) in press.
3
H. Taru, K. Iijima, M. Hase, Y. Kirino,
Y. Yagi, and T. Suzuki, unpublished observation.
The abbreviations used are:
APP, Alzheimer's
Interaction of Alzheimer's
-Amyloid Precursor
Family Proteins with Scaffold Proteins of the JNK Signaling
Cascade*
,§,,,, and§¶
Laboratory of Neurobiophysics,
School of Pharmaceutical Sciences, the University of Tokyo, Hongo
7-3-1, Bunkyo-ku, Tokyo 113-0033 and the § Laboratory of
Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido
University, Kita-ku Kita-12 Nishi-6, Sapporo 060-0812, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid precursor protein (APP) that is implicated
in Alzheimer's disease. This novel
APPL-interacting
protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed
abundantly in neural tissues. The phosphotyrosine interaction domain of
APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic
domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal
halves of mammalian c-Jun NH2-terminal kinase
(JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain
Src homology 3 and phosphotyrosine interaction domains. The similarity
of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of
the JNK signaling pathway and with the motor protein kinesin and the
formation of homo-oligomers. JIP1b interacts strongly with the
cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the
interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b
slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the
regulation of APP phosphorylation by JNK. Analysis of APP family
proteins and their associated proteins is expected to contribute to
understanding the molecular process of neural degeneration in
Alzheimer's disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amyloid precursor protein
(APP),1 implicated in the cause
and progression of Alzheimer's disease, is a receptor-like
transmembrane protein consisting of an extracellular domain, a
transmembrane domain, and a short cytoplasmic domain (for review, see
Ref. 1). APP belongs to the conserved APP family (APPs), which includes amyloid precursor-like protein 1 and 2 (APLP1 and APLP2) in mammals, APP747 in Xenopus, elAPP in electric ray,
APL-1 in Caenorhabditis elegans, and APP-like (APPL)
in Drosophila (for review, see Ref. 2). Besides the
involvement of APP in the pathogenic process of Alzheimer's disease,
APPs are expected to be functionally important because mice lacking all
of the APP family genes die in the early postnatal period (3), but the
physiological role of APPs has not been analyzed sufficiently in
mammal. Except for mammalian APPs, Drosophila APPL is the
most characterized (4). APPL is expressed abundantly in neural tissue
(5) and has been associated with synaptic differentiation at the
neuromuscular junction (6). APPL is metabolized in the same manner as
mammalian APPs (7), and human APP expressed in fly is transported
normally in neurons (8). Furthermore, the behavioral defect of
APPL-null flies is partially rescued by the expression of human APP
(9). These facts suggest that the molecular mechanism of metabolism and
physiological roles of APPs may be basically conserved among species.
Revealing the characteristics of APPs which have been conserved among
species may be helpful in understanding the character of APP and the
molecular mechanism of the pathogenic process of Alzheimer's disease.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity of cotransfectants was executed with a
liquid assay using o-nitrophenyl galactopyranoside
according to the manufacturer's protocol (Clontech) and was described
in Miller units.
ZAPII vector using the
ExAssist/SOLR system (Stratagene). Mouse clones were isolated by
cross-hybridization with a mouse brain cDNA library cloned into the
UNIZAP XR phage vector (Stratagene) using an APLIP1 fragment as a
probe. The 5'-upstream sequence of mouse JIP2 cDNA was obtained by
5'-rapid amplification of cDNA ends (version 2; Invitrogen) from
mouse whole brain total RNA using a reverse primer
(5'-CCTTTTCACAGTGGTCCGAG-3') and a nested PCR reverse primer
(5'-TAAGGCCCAGGCCACAGT-3').
N12
(846-886), N21 (855-886), C25 (834-861), and 873-882
(internal 873-882 deletion in APPLcyt 834-886). cDNAs encoding
APPLcyt(834-886) and APPLcyt873-882 were inserted into a
pGEX-4T-1 vector (Amersham Biosciences) at
EcoRI/SalI sites to produce GST fusion proteins.
The cDNA encoding the full-length APLIP1 protein (APLIP1) and the
PCR-produced carboxyl-terminal deletion construct C137 (amino acids
1-353) were cloned into pGAD424 (Clontech) at
SmaI/PstI sites. Other various amino-terminal truncated proteins of APLIP1 (N84 (amino acids 85-490), N207 (208-490), N280 (281-490), N330 (331-490), and N387
(388-490)) were also produced by PCR and cloned into pGAD424
(Clontech) at EcoRI/PstI. cDNAs of APLIP1,
N207, and C137 were cloned into the mammalian expression vector
pcDNA3.1Myc/HisA (Invitrogen) to produce pcDNA3.1APLIP1Myc/HisA,
pcDNA3.1APLIP1-N207Myc/HisA, and
pcDNA3.1APLIP1-C137Myc/HisA, respectively; these constructs express proteins tagged with a c-Myc epitope at the carboxyl
terminus in mammalian cells. Full-length cDNAs of
Drosophila APPL (GenBank J04516),
Drosophila JNK (DJNK; GenBank U49249), and
Drosophila hemipterous (Hep; GenBank
U93032), and a partial cDNA encoding amino acids 162-508 of the
Drosophila kinesin light chain (DKLC; GenBank L11013) were
obtained by RT-PCR from poly(A)+ RNA of 12-18-h embryos.
APPL cDNA was inserted into pcDNA3 at EcoRI/XbaI. Hep and DKLC cDNA were inserted
into pcDNA3 at EcoRI/XhoI with a FLAG tag at
the amino terminus. DJNK and Hep were cloned into pGEX-4T-1 (Amersham
Biosciences) at SmaI/XhoI and
EcoRI/XhoI sites, respectively, to produce GST
fusion proteins. The constructs pcDNA3APP695 (18) and
pcDNA3-FLAG-APP695 (12) have been described. Full-length
cDNAs of JIP1 (GenBank AF054611) and JIP2 (GenBank AF220195) were
obtained from mouse brain total RNA by RT-PCR and cloned into
pcDNA3 at EcoRI/XbaI and
EcoRI/XhoI sites, respectively, with FLAG or
hemagglutinin (HA) tags at the their amino termini. The cDNA
encoding JIP1a (GenBank AF003115), which is also known as a splicing
variant of JIP1 with an internal deletion of amino acids 557-603 in
JIP1b (22, 23), was produced by PCR and cloned into the
EcoRI and XbaI sites of pcDNA3 (Invitrogen),
with the HA sequence at the amino terminus. pcDNA3-HA-X11L were
produced by inserted HA sequence into amino terminus of full-length
cDNA of X11L cloned into pcDNA3 (18). The cDNA encoding
dual leucine zipper-bearing kinase (DLK; GenBank NM006301) was obtained
from human brain total RNA (Clontech) by RT-PCR and cloned into
pcDNA3.1Myc/HisA to express proteins tagged with a c-Myc
epitope at the carboxyl terminus.
-32P]dCTP (3,000 Ci/mmol,
PerkinElmer Life Sciences) and a random primer labeling kit (Roche
Diagnostics). Hybridization was carried out using ExpresHyb
hybridization solution (Clontech) according to the user's manual, and
signals were detected by autoradiography. A cDNA of rp49 (GenBank
U92431) was obtained by RT-PCR with adult head poly(A)+
RNA. For RT-PCR analysis, poly(A)+ RNA, prepared from adult
heads and bodies separated using glass beads and a sieve, were reverse
transcribed with SuperScript II (Invitrogen) using an oligo(dT) primer
for 2 h at 37 °C and treated with RNase H (Takara). A
PCR was performed with Ex-Taq (Takara) using
[-32P]dCTP and primer sets specific for APLIP1
and rp49 as follows: APLIP1, 5'-AATGCGGCTACTTGATG-3' and
5'-GTGGTGCCGCGGCACAAA-3'; rp49, 5'-AGTCGGATCGATATGCTAAG-3' and
5'-AGTAAACGCGGTTCTGCATG-3'. The resulting products were
electrophoresed on 5% (w/v) acrylamide gels and quantified with a Fuji
BAS image analyzer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (47K):
[in a new window]
Fig. 1.
Primary structure of
Drosophila APLIP1 and mouse JIP1b and JIP2 and
comparison of the amino acid sequences and homology among them.
The Drosophila APLIP1 primary structure and amino
acid sequence are compared with those of mouse JIP1b and JIP2.
A, schematic diagram of Drosophila APLIP1, mouse
JIP1b, and mouse JIP2 proteins. The ratio of amino acid residues of
APLIP1 identical to JIP2 or JIP1b is indicated in each region as
percent homology. The two-hybrid indicates a region of
cDNA isolated originally using yeast two-hybrid screening.
B, alignment was performed with the ClustalW algorithm (49).
The protein sequences are presented using the single-letter code for
amino acids. Gaps ( 
) were introduced to optimize the alignment. The
amino acid residues of APLIP1 identical to either JIP1b or JIP2 are
indicated with a plus (+); those identical to both JIP1 and
JIP2 are indicated with an asterisk (*). The amino acid
residues of JIP1b which are identical to JIP2 but not to APLIP1 are
indicated with a period (.). The JNK binding motifs
(JBD) of JIP1b and JIP2 are boxed in
orange, and the conserved core sequence is indicated in
red. The amino acid sequence of JIP1b required for
interaction with kinesin is boxed in pink. The
residues corresponding to the SH3 and PI domains are boxed
in green and blue, respectively.
View larger version (27K):
[in a new window]
Fig. 2.
Expression of Drosophila
APLIP1. A, Northern blot analysis of APLIP1.
Membranes containing 1 µg of poly(A)+ RNA were hybridized
with APLIP1 (upper panel) or rp49 (lower panel)
probes. E0-6, E6-12, E12-18, and
E18-24 indicate embryos 0-6, 6-12, 12-18, and 18-24 h
after egg deposition, respectively. L3, third instar larva;
PP, pre-pupa; P, pupa; A, adult flies.
A 2.4-kb transcript is the most prominent on the autoradiogram
(upper panel). Numbers indicate molecular sizes
of the RNA standard (kb). B, semiquantitative RT-PCR
analysis of APLIP1. Poly(A)+ RNA from the heads and bodies
of adult flies was analyzed by RT-PCR with specific primer sets for
APLIP1 (upper panel) and rp49 (lower panel). The
resulting PCR products were detected and quantified with a Fuji BAS
2000 imaging analyzer. C, expression of APLIP1 in an embryo.
Whole mount in situ hybridization of embryos was carried out
with antisense probes against the APLIP1 gene. The arrow
designates the brain region, and arrowheads point to the
central nervous system.
View larger version (25K):
[in a new window]
Fig. 3.
Interaction of APPL with APLIP1.
A, interaction of APPL with APLIP1 in vitro. A
whole cell lysate of COS-7 cells expressing APPL was incubated with
glutathione beads bearing the GST-APLIP1 fusion protein
(APLIP1) or GST protein alone (GST). APPL in
crude lysate (crude) and APPL attached to beads with
(APLIP1) or without (GST) APLIP1 were detected by
Western blot analysis with an anti-APPL antibody. B,
coimmunoprecipitation of APPL with APLIP1. The lysate of COS-7 cells
transiently expressing APPL and APLIP1 tagged with FLAG
(FLAG-APLIP1), APPL alone, or FLAG-APLIP1 alone (expression
is indicated as +) were immunoprecipitated (IP) with an
anti-FLAG (middle, IP: FLAG) or anti-APPL
(right, IP: APPL) antibody. The crude lysate
(left) and the immunoprecipitates were analyzed by
immunoblot analysis with anti-APPL (upper panels) and
anti-FLAG (lower panels) antibodies. Numbers
indicate the molecular masses (kDa) of the protein standards.
-galactosidase activity were
analyzed (Fig. 4A). Proteins that were truncated but still contained the putative SH3 and PI domains in
the carboxyl-terminal half (N84, N207, and N280) were able to
bind APPLcyt. A transformant harboring a protein lacking the SH3 region
(N330) showed no significant difference in -galactosidase activity compared with the control (mock) but still could grow in the
selective medium. However, N387, which lacked the amino-terminal side of the PI domain, and C137, which lacks the carboxyl-terminal region including the PI domain, exhibited neither -galactosidase activity nor growth in the selective medium. This result suggests that
the carboxyl-terminal region containing the PI domain of APLIP1 is
essential for binding to APPLcyt and that the SH3 domain may be
involved in the preservation of the structure of the PI domain
necessary for binding to APPL.
View larger version (41K):
[in a new window]
Fig. 4.
Identification of regions that are
responsible for the interaction between APLIP1 and the cytoplasmic
domain of APPL. A, protein constructs derived from
APLIP1 (indicated in the schematic structure) were expressed in yeast,
and their ability to bind to APPLcyt was examined. APLIP1,
full-length APLIP1; N 84, amino acids 85-490;
N207, 208-490; N280, 281-490;
N330, 331-490; N387, 388-490;
C137, 1-353. The ability to grow in the selective medium
was observed (indicated as + or - under Growth).
-Galactosidase activity (-gal) was quantified by
liquid assay and is indicated in Miller units ± S.D.
(n = 4). * indicates p < 0.01; **,
p < 0.005; ***, p < 0.001 compared
with the value of the mock treated study with plasmid alone.
B, protein constructs derived from APPLcyt, indicated in the
schematic structure (left), were expressed in yeast, and
their ability to bind to APLIP1 was examined. APPLcyt, amino acids
834-886. N12, 846-886; N21, 855-886;
C25, 834-861; 873-882, the internal
deletion of amino acids 873-882 of APPLcyt. The ability to grow in the
selective medium was observed as above. -Galactosidase activity was
quantified by liquid assay and is indicated in Miller units ± S.D. (n = 3). * indicates p < 0.01;
**, p < 0.005; ***, p < 0.001 compared with the value of the mock treated study with plasmid alone.
C, binding of APLIP1 protein constructs to the APPLcyt
in vitro. A whole cell lysate of COS-7 cells expressing the
protein constructs derived from APLIP1 tagged with c-Myc
(crude) was incubated with glutathione beads bearing the
GST-APPLcyt fusion protein (APPLcyt), GST-APPLcyt deleted in
873-882 (873-882), or GST protein alone
(GST). The protein constructs attached to beads were
detected by Western blot analysis with an anti-c-Myc monoclonal
antibody. APLIP1-myc, full-length APLIP1 tagged with Myc;
N207-myc, amino acids 208-490 of APLIP1 tagged with Myc;
and C137-myc, amino acids 1-353 of APLIP1 tagged with
Myc. Numbers indicate the molecular masses (kDa) of the
protein standards.
N12 and N21)
interacted with APLIP1 as well as APPLcyt did. However, deletion of the
carboxyl-terminal half of APPLcyt (C25) resulted in the complete
loss of both the -galactosidase activity and the growth in the
selective medium. We further showed that an APPLcyt construct with an
internal deletion of 10 amino acids (873-882) around the GYENPTY
motif lost completely both the -galactosidase activity and the
growth ability in the selective medium. These observations indicate
that an amino acid sequence containing the GYENPTY motif, which in
mammalian APP is known as a motif that interacts with several
cytoplasmic proteins (33), interacts with the PI domain of APLIP1.
207, and C137
constructs of APLIP1 were incubated with beads coupled to GST fusion
proteins harboring APPLcyt, the 873-882 construct of APPLcyt, or
GST alone. Proteins bound to the beads were analyzed by immunoblot with
an anti-Myc antibody. The Myc-tagged APLIP1 and N207 bound the
GST-APPLcyt fusion protein. In contrast, we could not detect the
binding of the Myc-tagged C137 protein to the GST-APPLcyt fusion
protein. Furthermore, we found that APLIP1 did not interact with the
GST-APPL873-882 fusion protein. The in vitro protein
binding assays agreed with the results obtained from the yeast
two-hybrid assay, which confirmed that the carboxyl-terminal region
containing the PI domain of APLIP1 interacts with a sequence containing
the GYENPTY motif in APPLcyt.
View larger version (43K):
[in a new window]
Fig. 5.
Interaction of APLIP1 with components of the
JNK signaling pathway and oligomer formation of APLIP1.
A, whole cell lysates of COS-7 cells expressing the
FLAG-tagged APLIP1, Drosophila JNK kinase Hep, or mouse
JIP1b were incubated with glutathione beads bearing the GST-DJNK fusion
protein (DJNK) or GST protein alone (GST). The
proteins in the crude lysate (crude) and those attached to
beads were detected by Western blot analysis with anti-FLAG monoclonal
antibody. B, whole cell lysates of COS-7 cells expressing
the FLAG-tagged Hep (upper panel) and FLAG-tagged APLIP1
(lower panel) were incubated with glutathione beads bearing
the GST-APLIP1 fusion protein (APLIP1) or GST protein alone.
The proteins in the crude lysates and those attached to beads were
detected by Western blot analysis with anti-FLAG monoclonal antibody.
C, COS-7 cells transiently expressing HA-tagged APLIP1
(HA-APLIP1) together with FLAG-tagged Hep
(FLAG-Hep) were immunoprecipitated with anti-FLAG monoclonal
antibody. The crude lysate and the immunoprecipitates were analyzed by
Western blot with anti-HA (HA, top and
middle panels) and anti-FLAG (FLAG, bottom
panel) monoclonal antibodies. Expression of proteins is indicated
as +. The asterisk (*) designates a nonspecific band.
D, COS-7 cells transiently expressing FLAG-tagged APLIP1
(FLAG-APLIP1) together with HA-tagged APLIP1 were
immunoprecipitated with an anti-FLAG monoclonal antibody and non-immune
IgG (IgG). The crude lysate and the immunoprecipitates
(IP) were analyzed by Western blot with anti-HA (upper
panel) and anti-FLAG (lower panel) monoclonal
antibodies. H indicates the heavy chain of IgG.
Arrows indicate proteins, and numbers indicate
the molecular masses (kDa) of the protein standards.
137, the truncated protein lacking the
carboxyl-terminal region, could not be precipitated. Thus, the
interaction of APLIP1 with kinesin is also conserved in
Drosophila.
View larger version (51K):
[in a new window]
Fig. 6.
Interaction of APLIP1 with the
Drosophila kinesin light chain. COS-7 cells
transiently expressing HA-tagged APLIP1 (HA-APLIP1), C 137
(HA-C137), and JIP1b (HA-JIP1b) together with
FLAG-tagged Drosophila kinesin light chain
(FLAG-DKLC) were immunoprecipitated (IP) with
anti-FLAG monoclonal antibody. The crude lysate (crude,
middle panel) and the immunoprecipitates were analyzed by
Western blot with anti-HA (HA, top and
middle panels) and anti-FLAG (FLAG, bottom
panel) monoclonal antibodies. The expression of proteins is
indicated as +. H and the asterisks (*) indicate
the heavy chain of IgG and nonspecific bands, respectively.
Arrows indicate proteins, and numbers indicate
the molecular masses (kDa) of the protein standards.
View larger version (46K):
[in a new window]
Fig. 7.
Interaction of APP with JIPs and X11L.
A, coimmunoprecipitation of APP with JIP1b, JIP1a, JIP2, and
X11L. The lysate of COS-7 cells transiently expressing FLAG-tagged APP
(APP) together with HA-tagged JIP1b (JIP1b),
JIP1a (JIP1a), JIP2 (JIP2), or X11L
(X11L), or plasmid alone (mock) were
immunoprecipitated (IP) with anti-FLAG monoclonal antibody.
The crude lysate (crude, middle panel) and the
immunoprecipitates (top and bottom panels) were
analyzed by immunoblot with an anti-HA (top and middle
panels) or anti-FLAG (bottom panel) antibody.
B, whole cell lysates of COS-7 cells expressing the
HA-tagged JIP1b, JIP1a, JIP2, or X11L were incubated with glutathione
beads bearing the APPcyt fused with GST (APPcyt) or GST
protein alone (GST). The proteins in the crude lysate and
those attached to beads were detected by immunoblot analysis with
anti-HA monoclonal antibody.
View larger version (28K):
[in a new window]
Fig. 8.
Effect of JIP1 and JIP2 on APP
phosphorylation. FLAG-tagged JIP1b, JIP2, or JIP1a was transiently
expressed together with APP and DLK in Neuro-2a cells (expression is
indicated as +), and the lysates of these cells were analyzed by
immunoblot with anti-phospho-APP antibody UT-33 (top panel,
P-APP), and anti-APP antibody G369 (middle panel,
APP). The amount of phosphorylated APP was quantified,
standardized to the amount of total APP, and is represented as relative
ratio to the value (1.0) of the cells not expressing JIPs (bottom
panel, graph). Bars indicate the means ± S.D. (n = 4). * p < 0.05, ***
p < 0.005.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 9.
Schematic diagram of the protein interaction
of APLIP1, JIP1b, and JIP2. The interactions of proteins with
APLIP1, JIP1b, and JIP2 are indicated with double-headed
arrows. The phosphorylation and activation cascade of the JNK
signaling components are designated with arrowheads. The
interaction of JIP2 with APP (small double headed gray
arrow) is weaker than that of JIP1. Phosphorylation of APP at
Thr-668 by JNK is indicated as P. JIP2 suppresses the
phosphorylation of APP. The interaction of APLIP1 with hypothetical
homologs of mixed lineage kinases in Drosophila
(DMLK) is assumed (gray arrow). Kinesin binds to
APLIP1, JIP1b, and JIP2 and may associate further with microtubules.
MKK7, MAP kinase kinase; MLK, mixed lineage
kinase (MAP kinase kinase kinase).
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-amyloid precursor protein;
APPcyt, the cytoplasmic domain of APP;
APPs, APP family proteins;
APLP1, amyloid precursor-like protein 1;
APLP2, amyloid precursor-like protein 2;
APPL, Drosophila
APP-like protein;
APPLcyt, the cytoplasmic domain of APPL;
APLIP1, APPL-interacting protein 1;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
DJNK, Drosophila JNK;
DKLC, Drosophila kinesin light
chain;
DLK, dual leucine zipper-bearing kinase;
GST, glutathione
S-transferase;
HA, hemagglutinin;
Hep, Hemipterous;
JNK, c-Jun NH2-terminal kinase;
JIP, JNK-interacting protein;
MAP, mitogen-activated protein;
PI domain, phosphotyrosine interaction
domain;
rp49, ribosomal protein 49;
RT-PCR, reverse transcription PCR;
SH3 domain, Src homology domain 3.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Selkoe, D. J.
(1999)
Nature
399,
A23-A31[CrossRef][Medline]
[Order article via Infotrieve]
2.
Coulson, E. J.,
Paliga, K.,
Beyreuther, K.,
and Masters, C. L.
(2000)
Neurochem. Int.
36,
175-184[CrossRef][Medline]
[Order article via Infotrieve]
3.
Heber, S.,
Herms, J.,
Gajic, V.,
Hainfellner, J.,
Aguzzi, A.,
Rulicke, T.,
Kretzschmar, H.,
von Koch, C.,
Sisodia, S.,
Tremml, P.,
Lipp, H. P.,
Wolfer, D. P.,
and Müller, U.
(2000)
J. Neurosci.
20,
7951-7963 4.
Rosen, D. R.,
Martin-Morris, L.,
Luo, L. Q.,
and White, K.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
2478-2482 5.
Martin-Morris, L. E.,
and White, K.
(1990)
Development
110,
185-195[Abstract]
6.
Torroja, L.,
Packard, M.,
Gorczyca, M.,
White, K.,
and Budnik, V.
(1999)
J. Neurosci.
19,
7793-7803 7.
Luo, L. Q.,
Martin-Morris, L. E.,
and White, K.
(1990)
J. Neurosci.
10,
3849-3861[Abstract]
8.
Yagi, Y.,
Tomita, S.,
Nakamura, M.,
and Suzuki, T.
(2000)
Mol. Cell. Biol. Res. Commun.
4,
43-49[CrossRef][Medline]
[Order article via Infotrieve]
9.
Luo, L.,
Tully, T.,
and White, K.
(1992)
Neuron
9,
595-605[CrossRef][Medline]
[Order article via Infotrieve]
10.
Perez, R. G.,
Soriano, S.,
Hayes, J. D.,
Ostaszewski, B.,
Xia, W.,
Selkoe, D. J.,
Chen, X.,
Stokin, G. B.,
and Koo, E. H.
(1999)
J. Biol. Chem.
274,
18851-18856 11.
Tomita, S.,
Kirino, Y.,
and Suzuki, T.
(1998)
J. Biol. Chem.
273,
19304-19310 12.
Ando, K.,
Oishi, M.,
Takeda, S.,
Iijima, K.,
Isohara, T.,
Nairn, A. C.,
Kirino, Y.,
Greengard, P.,
and Suzuki, T.
(1999)
J. Neurosci.
19,
4421-4427 13.
Allinquant, B.,
Hantraye, P.,
Mailleux, P.,
Moya, K.,
Bouillot, C.,
and Prochiantz, A.
(1995)
J. Cell Biol.
128,
919-927 14.
Perez, R. G.,
Zheng, H.,
Van der Ploeg, L. H.,
and Koo, E. H.
(1997)
J. Neurosci.
17,
9407-9414 15.
Xu, X.,
Yang, D.,
Wyss-Coray, T.,
Yan, J.,
Gan, L.,
Sun, Y.,
and Mucke, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
7547-7552 16.
Borg, J. P.,
Ooi, J.,
Levy, E.,
and Margolis, B.
(1996)
Mol. Cell. Biol.
16,
6229-6241[Abstract]
17.
Guenette, S. Y.,
Chen, J.,
Jondro, P. D.,
and Tanzi, R. E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10832-10837 18.
Tomita, S.,
Ozaki, T.,
Taru, H.,
Oguchi, S.,
Takeda, S.,
Yagi, Y.,
Sakiyama, S.,
Kirino, Y.,
and Suzuki, T.
(1999)
J. Biol. Chem.
274,
2243-2254 19.
Zheng, P.,
Eastman, J.,
Vande Pol, S.,
and Pimplikar, S. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14745-14750 20.
Trommsdorff, M.,
Borg, J. P.,
Margolis, B.,
and Herz, J.
(1998)
J. Biol. Chem.
273,
33556-33560 21.
Watanabe, T.,
Sukegawa, J.,
Sukegawa, I.,
Tomita, S.,
Iijima, K.,
Oguchi, S.,
Suzuki, T.,
Nairn, A. C.,
and Greengard, P.
(1999)
J. Neurochem.
72,
549-556[CrossRef][Medline]
[Order article via Infotrieve]
22.
Dickens, M.,
Rogers, J. S.,
Cavanagh, J.,
Raitano, A.,
Xia, Z.,
Halpern, J. R.,
Greenberg, M. E.,
Sawyers, C. L.,
and Davis, R. J.
(1997)
Science
277,
693-696 23.
Whitmarsh, A. J.,
Cavanagh, J.,
Tournier, C.,
Yasuda, J.,
and Davis, R. J.
(1998)
Science
281,
1671-1674 24.
Yasuda, J.,
Whitmarsh, A. J.,
Cavanagh, J.,
Sharma, M.,
and Davis, R. J.
(1999)
Mol. Cell. Biol.
19,
7245-7254 25.
Verhey, K. J.,
Meyer, D.,
Deehan, R.,
Blenis, J.,
Schnapp, B. J.,
Rapoport, T. A.,
and Margolis, B.
(2001)
J. Cell Biol.
152,
959-970 26.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Chap. 12, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
27.
Yagi, Y.,
and Hayashi, S.
(1997)
Genes Cells
2,
41-53[Abstract]
28.
Oishi, M.,
Nairn, A. C.,
Czernik, A. J.,
Lim, G. S.,
Isohara, T.,
Gandy, S. E.,
Greengard, P.,
and Suzuki, T.
(1997)
Mol. Med.
3,
111-123[Medline]
[Order article via Infotrieve]
29.
Torroja, L.,
Luo, L.,
and White, K.
(1996)
J. Neurosci.
16,
4638-4650 30.
Iijima, K.,
Ando, K.,
Takeda, S.,
Satoh, Y.,
Seki, T.,
Itohara, S.,
Greengard, P.,
Kirino, Y.,
Nairn, A. C.,
and Suzuki, T.
(2000)
J. Neurochem.
75,
1085-1091[CrossRef][Medline]
[Order article via Infotrieve]
31.
Ando, K.,
Iijima, K. I.,
Elliott, J. I.,
Kirino, Y.,
and Suzuki, T.
(2001)
J. Biol. Chem.
276,
40353-40361 32.
Celera Genomics, Inc.,
and Berkeley Drosophila Genome Project team.
(2000)
Science
287,
2185-2195 33.
Margolis, B.,
Borg, J. P.,
Straight, S.,
and Meyer, D.
(1999)
Kidney Int.
56,
1230-1237[CrossRef][Medline]
[Order article via Infotrieve]
34.
Bonny, C.,
Nicod, P.,
and Waeber, G.
(1998)
J. Biol. Chem.
273,
1843-1846 35.
Noselli, S.,
and Agnes, F.
(1999)
Curr. Opin. Genet. Dev.
9,
466-472[CrossRef][Medline]
[Order article via Infotrieve]
36.
Sluss, H. K.,
Han, Z.,
Barrett, T.,
Davis, R. J.,
and Ip, Y. T.
(1996)
Genes Dev.
10,
2745-2758 37.
Kelkar, N.,
Gupta, S.,
Dickens, M.,
and Davis, R. J.
(2000)
Mol. Cell. Biol.
20,
1030-1043 38.
Suzuki, T.,
Oishi, M.,
Marshak, D. R.,
Czernik, A. J.,
Nairn, A. C.,
and Greengard, P.
(1994)
EMBO J.
13,
1114-1122[Medline]
[Order article via Infotrieve]
39.
Standen, C. L.,
Brownlees, J.,
Grierson, A. J.,
Kesavapany, S.,
Lau, K. F.,
McLoughlin, D. M.,
and Miller, C. C.
(2001)
J. Neurochem.
76,
316-320[CrossRef][Medline]
[Order article via Infotrieve]
40.
Fan, G.,
Merritt, S. E.,
Kortenjann, M.,
Shaw, P. E.,
and Holzman, L. B.
(1996)
J. Biol. Chem.
271,
24788-24793 41.
Ito, M.,
Yoshioka, K.,
Akechi, M.,
Yamashita, S.,
Takamatsu, N.,
Sugiyama, K.,
Hibi, M.,
Nakabeppu, Y.,
Shiba, T.,
and Yamamoto, K. I.
(1999)
Mol. Cell. Biol.
19,
7539-7548 42.
Bowman, A. B.,
Kamal, A.,
Ritchings, B. W.,
Philip, A. V.,
McGrail, M.,
Gindhart, J. G.,
and Goldstein, L. S. B.
(2000)
Cell
103,
583-594[CrossRef][Medline]
[Order article via Infotrieve]
43.
Meyer, D.,
Liu, A.,
and Margolis, B.
(1999)
J. Biol. Chem.
274,
35113-35118 44.
Gotthardt, M.,
Trommsdorff, M.,
Nevitt, M. F.,
Shelton, J.,
Richardson, J. A.,
Stockinger, W.,
Nimpf, J.,
and Herz, J.
(2000)
J. Biol. Chem.
275,
25616-25624 45.
Stockinger, W.,
Brandes, C.,
Fasching, D.,
Hermann, M.,
Gotthardt, M.,
Herz, J.,
Schneider, W. J.,
and Nimpf, J.
(2000)
J. Biol. Chem.
275,
25625-25632 46.
Matsuda, S.,
Yasukawa, T.,
Homma, Y.,
Ito, Y.,
Niikura, T.,
Hiraki, T.,
Hirai, S.,
Ohno, S.,
Kita, Y.,
Kawasumi, M.,
Kouyama, K.,
Yamamoto, T.,
Kyriakis, J. M.,
and Nishimoto, I.
(2001)
J. Neurosci.
21,
6597-6607 47.
Torroja, L.,
Chu, H.,
Kotovsky, I.,
and White, K.
(1999)
Curr. Biol.
9,
489-492[CrossRef][Medline]
[Order article via Infotrieve]
48.
Gunawardena, S.,
and Goldstein, L. S. B.
(2001)
Neuron
32,
389-401[CrossRef][Medline]
[Order article via Infotrieve]
49.
Thompson, J. D.,
Higgins, D. G.,
and Gibson, T. J.
(1994)
Nucleic Acids Res.
22,
4673-4680
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. J. Park and Y. P. Loh Minireview: How Peptide Hormone Vesicles Are Transported to the Secretion Site for Exocytosis Mol. Endocrinol., December 1, 2008; 22(12): 2583 - 2595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suzuki and T. Nakaya Regulation of Amyloid {beta}-Protein Precursor by Phosphorylation and Protein Interactions J. Biol. Chem., October 31, 2008; 283(44): 29633 - 29637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Krause, S. Karger, S.-Y. Sheu, T. Aigner, R. Kursawe, O. Gimm, K.-W. Schmid, H. Dralle, and D. Fuhrer Evidence for a role of the amyloid precursor protein in thyroid carcinogenesis J. Endocrinol., August 1, 2008; 198(2): 291 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Park, N. X. Cawley, and Y. P. Loh Carboxypeptidase E Cytoplasmic Tail-Driven Vesicle Transport Is Key for Activity-Dependent Secretion of Peptide Hormones Mol. Endocrinol., April 1, 2008; 22(4): 989 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sano, A. Syuzo-Takabatake, T. Nakaya, Y. Saito, S. Tomita, S. Itohara, and T. Suzuki Enhanced Amyloidogenic Metabolism of the Amyloid beta-Protein Precursor in the X11L-deficient Mouse Brain J. Biol. Chem., December 8, 2006; 281(49): 37853 - 37860. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Satpute-Krishnan, J. A. DeGiorgis, M. P. Conley, M. Jang, and E. L. Bearer A peptide zipcode sufficient for anterograde transport within amyloid precursor protein PNAS, October 31, 2006; 103(44): 16532 - 16537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-A Chang, H.-S. Kim, T.-Y. Ha, J.-W. Ha, K. Y. Shin, Y. H. Jeong, J.-P. Lee, C.-H. Park, S. Kim, T.-K. Baik, et al. Phosphorylation of Amyloid Precursor Protein (APP) at Thr668 Regulates the Nuclear Translocation of the APP Intracellular Domain and Induces Neurodegeneration. Mol. Cell. Biol., June 1, 2006; 26(11): 4327 - 4338. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Suzuki, Y. Araki, T. Yamamoto, and T. Nakaya Trafficking of Alzheimer's Disease-Related Membrane Proteins and Its Participation in Disease Pathogenesis J. Biochem., June 1, 2006; 139(6): 949 - 955. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nakaya and T. Suzuki Role of APP phosphorylation in FE65-dependent gene transactivation mediated by AICD Genes Cells, June 1, 2006; 11(6): 633 - 645. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sumioka, S. Nagaishi, T. Yoshida, A. Lin, M. Miura, and T. Suzuki Role of 14-3-3{gamma} in FE65-dependent Gene Transactivation Mediated by the Amyloid {beta}-Protein Precursor Cytoplasmic Fragment J. Biol. Chem., December 23, 2005; 280(51): 42364 - 42374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Muresan and V. Muresan Coordinated transport of phosphorylated amyloid-{beta} precursor protein and c-Jun NH2-terminal kinase-interacting protein-1 J. Cell Biol., November 21, 2005; 171(4): 615 - 625. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Muresan and V. Muresan c-Jun NH2-Terminal Kinase-Interacting Protein-3 Facilitates Phosphorylation and Controls Localization of Amyloid-{beta} Precursor Protein J. Neurosci., April 13, 2005; 25(15): 3741 - 3751. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Asaumi, K. Iijima, A. Sumioka, K. Iijima-Ando, Y. Kirino, T. Nakaya, and T. Suzuki Interaction of N-Terminal Acetyltransferase with the Cytoplasmic Domain of {beta}-Amyloid Precursor Protein and Its Effect on A{beta} Secretion J. Biochem., February 1, 2005; 137(2): 147 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. von Rotz, B. M. Kohli, J. Bosset, M. Meier, T. Suzuki, R. M. Nitsch, and U. Konietzko The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor J. Cell Sci., September 1, 2004; 117(19): 4435 - 4448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Zhou, C. Noviello, C. D'Ambrosio, A. Scaloni, and L. D'Adamio Growth Factor Receptor-bound Protein 2 Interaction with the Tyrosine-phosphorylated Tail of Amyloid {beta} Precursor Protein Is Mediated by Its Src Homology 2 Domain J. Biol. Chem., June 11, 2004; 279(24): 25374 - 25380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Perkinton, C. L. Standen, K.-F. Lau, S. Kesavapany, H. L. Byers, M. Ward, D. M. McLoughlin, and C. C. J. Miller The c-Abl Tyrosine Kinase Phosphorylates the Fe65 Adaptor Protein to Stimulate Fe65/Amyloid Precursor Protein Nuclear Signaling J. Biol. Chem., May 21, 2004; 279(21): 22084 - 22091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Taru and T. Suzuki Facilitation of Stress-induced Phosphorylation of {beta}-Amyloid Precursor Protein Family Members by X11-like/Mint2 Protein J. Biol. Chem., May 14, 2004; 279(20): 21628 - 21636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Araki, S. Tomita, H. Yamaguchi, N. Miyagi, A. Sumioka, Y. Kirino, and T. Suzuki Novel Cadherin-related Membrane Proteins, Alcadeins, Enhance the X11-like Protein-mediated Stabilization of Amyloid {beta}-Protein Precursor Metabolism J. Biol. Chem., December 5, 2003; 278(49): 49448 - 49458. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Lee, K.-F. Lau, M. S. Perkinton, C. L. Standen, S. J. A. Shemilt, L. Mercken, J. D. Cooper, D. M. McLoughlin, and C. C. J. Miller The Neuronal Adaptor Protein X11{alpha} Reduces A{beta} Levels in the Brains of Alzheimer's APPswe Tg2576 Transgenic Mice J. Biol. Chem., November 21, 2003; 278(47): 47025 - 47029. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Hansel, A. Rahman, S. Wehner, V. Herzog, C. J. Yeo, and A. Maitra Increased Expression and Processing of the Alzheimer Amyloid Precursor Protein in Pancreatic Cancer May Influence Cellular Proliferation Cancer Res., November 1, 2003; 63(21): 7032 - 7037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Marques, U. Keil, A. Bonert, B. Steiner, C. Haass, W. E. Muller, and A. Eckert Neurotoxic Mechanisms Caused by the Alzheimer's Disease-linked Swedish Amyloid Precursor Protein Mutation: OXIDATIVE STRESS, CASPASES, AND THE JNK PATHWAY J. Biol. Chem., July 18, 2003; 278(30): 28294 - 28302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Inomata, Y. Nakamura, A. Hayakawa, H. Takata, T. Suzuki, K. Miyazawa, and N. Kitamura A Scaffold Protein JIP-1b Enhances Amyloid Precursor Protein Phosphorylation by JNK and Its Association with Kinesin Light Chain 1 J. Biol. Chem., June 13, 2003; 278(25): 22946 - 22955. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Michaut, S. Flister, M. Neeb, K. P. White, U. Certa, and W. J. Gehring Analysis of the eye developmental pathway in Drosophila using DNA microarrays PNAS, April 1, 2003; 100(7): 4024 - 4029. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schoorlemmer and M. Goldfarb Fibroblast Growth Factor Homologous Factors and the Islet Brain-2 Scaffold Protein Regulate Activation of a Stress-activated Protein Kinase J. Biol. Chem., December 13, 2002; 277(51): 49111 - 49119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Taru, Y. Kirino, and T. Suzuki Differential Roles of JIP Scaffold Proteins in the Modulation of Amyloid Precursor Protein Metabolism J. Biol. Chem., July 19, 2002; 277(30): 27567 - 27574. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |