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J. Biol. Chem., Vol. 275, Issue 25, 19275-19281, June 23, 2000
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From the Department of Anatomy and Division of Neurosurgery, P. O. Box 980709, Virginia Commonwealth University, Richmond, Virginia 23298
Received for publication, November 4, 1999, and in revised form, March 21, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Expression of the src homology 3 (SH3) domain-containing expressed in tumorigenic astrocytes (SETA) gene
is associated with the tumorigenic state in astrocytes. SETA encodes a
variety of adapter proteins containing either one or two SH3 domains,
as suggested by the sequence heterogeneity of isolated cDNAs. Using both SH3 domains in a yeast two-hybrid screen of a glial
progenitor cell cDNA library, we isolated the rat homolog of
the ALG-2-interacting protein 1 or ALG-2-interacting protein X
(AIP1/Alix). In vitro confrontation experiments showed that
the SH3-N domain of SETA interacted with the proline-rich C terminus of
AIP1. In co-immunoprecipitation experiments, SETA and AIP1 interacted
and could form a complex with apoptosis-linked gene 2 protein.
Endogenous SETA and AIP1 proteins showed similar patterns of
staining in primary rat astrocytes. Misexpression of a variety of SETA
protein isoforms in these astrocytes revealed that they localized to
the actin cytoskeleton. Furthermore, SETA proteins containing the SH3-N
domain were able to sensitize astrocytes to apoptosis induced by UV
irradiation. Expression of the isolated SH3-N domain had the greatest
effect in these experiments, indicating that interference in the
interaction between endogenous SETA and AIP1 sensitizes astrocytes to
apoptosis in response to DNA damage.
The src homology 3 (SH3)1 domain-containing
expressed in tumorigenic astrocytes (SETA) gene was isolated from
differentiating glial cells and implicated in primary brain tumors on
the basis of its expression in malignant astrocytes in culture and
gliomas in the adult brain in vivo (1). While SETA mRNA
is expressed in the developing rodent brain at high levels, it is
barely detectable in the adult rat or mouse cortex or in normal human
brain. However, approximately half of all experimentally induced rat
gliomas as well as human astrocytomas of grade II, III, and IV express
the gene, and it is also found in oligodendrogliomas and
oligoastrocytomas (1). Furthermore, expression of SETA in a
culture model of astrocytoma progression based on astrocytes from p53
knockout mice (2, 3) was closely associated with the ability of these cells to form tumors when reintroduced into animals (1). In this model
system, SETA shared expression patterns with established glioma-associated genes including epidermal growth factor receptor, platelet-derived growth factor receptors The SETA gene encodes proteins that contain SH3 domains, which are
involved in high affinity, specific protein-protein interactions. While
these domains are found in proteins with enzymatic function, such as
Src kinase, they are also common in adapter molecules whose function is
to promote the interaction of members of signal transduction pathways.
SETA appears to belong in this group and to be a member of a new
subfamily of adapter molecules that includes the CD2AP and CMS proteins
(4, 5). In addition to SH3 domains, these proteins appear to have
coiled-coil motifs at their C termini and two PXXP motifs,
which are themselves the cognates of SH3 domains. Therefore, they
appear to have at least three modalities of binding to other proteins,
suggesting involvement in a complex series of protein-protein
interactions. While CD2AP has been shown to interact with the CD2
molecule in T-cells (4), CMS has been shown to interact with actin,
p130cas, the p85 subunit of phosphatidylinositol 3-kinase,
src family kinases, and Grb2 (5), suggesting that this
family of proteins may play a role in cell architecture and mitogenic signaling.
As a first step toward understanding the function of SETA, we have
taken the direct approach of isolating a binding partner with a yeast
two-hybrid library screen. The protein we isolated is the rat homolog
of the mouse gene, apoptosis-linked gene 2 (ALG-2)-interacting protein
1 or ALG-2-interacting protein X (AIP1/Alix), which was recently
isolated by two groups performing yeast two-hybrid screens with the
ALG-2 (6, 7). In vitro confrontation and co-immunoprecipitation from transiently transfected cells demonstrated that the SH3-N domain of SETA interacts with the proline-rich C
terminus of AIP1 and that SETA, AIP1, and ALG-2 can form a complex.
ALG-2 was originally isolated in a "death trap" screen in T-cells,
and an antisense ALG-2 cDNA promoted survival after apoptosis had
been induced by a variety of stimuli (8). Conversely, when ALG-2 was
overexpressed in fibroblasts it sensitized them to cell death.
Therefore, it has been suggested that the 22-kDa calcium-binding protein ALG-2 is a necessary component of the apoptotic machinery (8).
AIP1 is a 105-kDa protein with a proline-rich C terminus that has 10 PXXP sequence motifs of the kind that bind to SH3 domains
(7). While the mechanism of action of AIP1 alone or in conjunction with
ALG-2 is not understood, it is clear that perturbing the levels of
these proteins in a variety of cells alters their response to apoptotic
stimuli without affecting the background level of apoptosis (7, 8). In
this paper, we show that these genes are expressed in astrocytes and
glioma cells, which also express endogenous SETA proteins. Furthermore,
introducing SETA proteins capable of binding to AIP1 sensitized
astrocytes to UV light-induced cell death, while control cells or those
expressing a part of the SETA protein that does not bind AIP1 had no
effect. These data suggest that SETA, AIP1, and ALG-2 represent a new set of proteins with a role in modulating apoptosis in astrocytes and with a potential role in the formation of gliomas.
Yeast Two-hybrid Screen--
The SETA SH3-NC bait construct
insert was generated by polymerase chain reaction from a cDNA
template using proofreading DeepVent DNA polymerase (New England
Biolabs), and the absence of mutations was confirmed by sequencing. It
included amino acids 24-258 (ERQRR ... LPSDF; see Fig.
2A) according to the numbering of the longer SETA isoform
described in Ref. 1 and GenBankTM AF131867. This insert was
introduced into the pBD-GAL4 phagemid vector (Stratagene) and was used
to screen a CG4 rat glial progenitor cell library (1) previously
constructed in HybriZAP and rescued into the pAD-GAL4 plasmid form
(Stratagene). Two rounds of yeast colony screening for the ability to
grow in the absence of histidine and for a positive In Vitro Confrontation--
SETA SH3 cDNAs (see legend to
Fig. 2A for details) were cloned in frame into pGEX-KG, and
glutathione S-transferase (GST) fusion proteins were
generated and purified on glutathione-Sepharose 4B according to the
manufacturer's instructions (Amersham Pharmacia Biotech). The plasmid
encoding the ALG-2 GST fusion protein (pGEX-ALG-2) as well as mouse
AIP1 (pCDNA3-AIP1) were obtained from Luciano D'Adamio and have
been described previously (7). [3H]Leucine-labeled AIP1
and Co-immunoprecipitation following Transient Expression in 293 Cells--
SETA cDNAs (see legend to Fig. 2A for
details) were cloned in frame into pcDNA4-Xpress/His (SH3-N, SH3-C,
and coiled coil (SETA NCcc)) or pcDNA6-V5/His (SETA SH3-NC, -N, and
-NC), which provide the X-press or V5 epitope tag, respectively
(Invitrogen). These plasmids were then co-transfected into 293 cells
with murine AIP1-FLAG, obtained from Luciano D'Adamio, by the calcium
phosphate method. Two days after transfection, cell lysates were
prepared in radioimmune precipitation assay buffer, and
immunoprecipitates were generated using monoclonal antibodies directed
against the V5, Xpress (Invitrogen), or FLAG (Sigma) epitopes and
collected on protein A-agarose (Roche Molecular Biochemicals).
Precipitates were also generated using bacterially made ALG-2 GST
fusion protein, as described above. Precipitates were Western blotted
to polyvinylidene difluoride membrane; exposed to V5, Xpress, FLAG, or
anti-SETA antibodies; and detected by chemiluminescence (Bio-Rad).
Northern Blot Analysis--
Poly(A)+ mRNA was
isolated using poly(dT) beads (Oligotex, Qiagen), and known amounts
were subjected to Northern blotting. Membranes were hybridized with
random primed 32P-labeled (Prime-It; Stratagene) probes
derived from murine AIP1 and ALG-2 cDNAs provided by Luciano
D'Adamio.
Generation and Analysis of SETA-misexpressing Astrocyte
Populations--
Initial attempts at obtaining astrocytes expressing
SETA proteins encoded by pCDNA vectors resulted in the isolation of
clones that showed low levels and percentages of expression. Therefore, pcDNA4-Xpress/His (SETA NCcc)- and pcDNA6-V5/His (SETA SH3-NC, -N, and -C)-derived inserts including the vector-derived epitope tags
were recloned into a retroviral vector, 1726/zeo. This retrovirus is a
modification of 1726, itself a modification of LNL6 encoding the
neo gene, an encephalomyocarditis virus-derived internal
ribosome entry site (9), and the lacZ gene. The
SpeI (870)-XbaI (7093) fragment of 1726 was
isolated using a partial XbaI cut and inserted into
SpeI XbaI double-digested LNCX (another LNL6
derivative modified by, and a kind gift from, Dr. Bob Navaiaux), which
lacked the EcoRI site at position 1 of LNL6. After removal
of the neo gene by EcoRI digestion and
religation, this left a single EcoRI site immediately
downstream of the packaging signal for the insertion of genes of
interest. The lacZ gene was removed and replaced by a
polymerase chain reaction-generated zeo gene, derived from
pSV40/Zeo (Invitrogen), inserted in frame with the internal ribosome
entry site using NcoI and XhoI restriction
enzymes to generate 1726/zeo. SETA cDNAs derived from pCDNA
plasmids were inserted blunt into EcoRI-digested and blunted
1726/zeo, and their orientation was determined by sequencing.
Pseudotyped retrovirus was generated by transient co-transfection of
293GP cells with an expression plasmid encoding vesicular stomatitis
virus G protein (10) and 1726/zeo constructs.
Primary rat astrocytes were plated at a density of 50,000 cells in a
25-cm2 tissue culture flask and infected the following day
with SETA-encoding retrovirus for 4 h in the presence of 8 µg/ml
polybrene. Two days later, cells were passaged and replated in a
75-cm2 tissue culture flask and exposed to 300 µg/ml
zeocin (Invitrogen). Cultures were maintained until uninfected control
cultures contained no viable cells, typically 7-10 days, at which
point they were harvested and replated at nominal passage 1. Cells were
maintained in 300 µg/ml zeocin at all times and analyzed for
expression by Western blot by chemiluminescence detection (Bio-Rad) and
immunohistochemistry using standard protocols. Polyclonal anti-SETA
antibodies are as described (1). Polyclonal anti-AIP1 antibodies were
obtained from Pasquale Vito, and are as described previously (7).
Immunohistochemistry was performed on fixed cells grown on coverslips
and with ALEXA (Molecular Probes, Inc., Eugene, OR) or fluorescein
isothiocyanate-conjugated (Southern Biotechnology Associates) secondary
antibodies. Cells were counterstained with phalloidin-rhodamine or
anti-paxillin antibodies (Sigma).
For analysis of apoptosis, cultures of astrocytes growing on coverslips
in 6-cm tissue culture dishes with 5 ml of medium were exposed to 5 mJ/cm2 of UV irradiation in a GS Genelinker UV chamber
(Bio-Rad). Coverslips were removed 24 h later and prepared for
analysis with annexin V (Pharmingen Becton-Dickinson) or TUNEL (Roche
Biochemicals) according to the manufacturer's instructions.
SETA Interacts with the Apoptosis-linked Genes AIP1 and
ALG-2--
The SETA gene encodes an adapter molecule with SH3 domains,
suggesting that it functions by binding with high affinity and specificity to other proteins. Therefore, a yeast two-hybrid cDNA library screen was performed to isolate potential binding partners of
SETA. A bait construct encoding the two SH3 domains of SETA and the
intervening sequence (for details, see "Experimental Procedures"), fused in frame to the DNA binding domain of the GAL4, was used to
screen a CG4 glial progenitor cell line cDNA library. SETA was
originally isolated from these cells (1), making it likely that
physiologically relevant binding partners would be represented. Analysis of only 250,000 co-transfectants led to the isolation of eight
positive clones after two rounds of screening. Sequence analysis
revealed that they represented two overlapping cDNAs for the rat
homolog of the mouse AIP1/Alix gene (Fig.
1), which had been previously isolated as
a binding partner of ALG-2 (6, 7). Interestingly, the partial rat
cDNA clone isolated by us (Fig. 1) was only one amino acid shorter
than the mouse cDNA isolated by yeast two-hybrid screening with
ALG-2 (7), suggesting that the same region of AIP1 interacts with both
ALG-2 and SETA.
Examination of the predicted protein sequence of the partial rat AIP1
cDNA revealed extensive sequence homology to the mouse AIP1
molecule (Fig. 1). Both predicted proteins encoded a proline-rich region, from amino acid 719 to the C terminus, in which close to 30%
of the residues are proline, as compared with an overall frequency of
8% proline in the long form of AIP1 (6, 7). Ten PXXP
motifs, such as are found at the core of SH3 binding peptides (11), can
be found in this sequence, suggesting that it represents the region
that interacts with SETA. Interestingly, although the rodent AIP1
proteins are highly homologous to the Xenopus gene
Xp95 (12), the similarity is dramatically reduced in this
proline-rich region (Fig. 1).
To establish whether SETA and AIP1 can interact outside of the yeast
two-hybrid system, in vitro confrontation experiments were
performed. Bacterially generated GST fusion proteins, encoding either
one or both of the SETA SH3 domains (Fig.
2A) or the ALG-2 molecule,
were confronted with radiolabeled AIP1 made by coupled in
vitro transcription and translation (Fig. 2B) and then
collected on glutathione-Sepharose. The SETA SH3-NC encoding GST fusion protein (Fig. 2A), which contains the same region of SETA as
was used in the bait construct employed in the yeast two-hybrid screen, interacted with AIP1 in vitro (Fig. 2B).
Furthermore, the isolated N-terminal SETA SH3 domain, fused to GST, was
able to bind to mouse AIP1 in these experiments (Fig. 2B).
However, the C-terminal SH3 domain did not bind to AIP1 (Fig.
2A), which was not due to a general lack of function of this
fusion protein, since it has been shown to bind to another novel SETA
binding protein recently isolated in our laboratory (data not shown).
As expected from previous studies, the ALG-2 protein was able to bind
full-length AIP1 in this assay (6, 7). In contrast to the interaction between ALG-2 and AIP1, the SETA SH3-N protein was able to bind AIP1 in
the absence of calcium, as would be expected from an SH3 domain-mediated interaction. Last, none of the GST fusion proteins interacted with radiolabeled
The use of isolated SETA SH3 domains in these experiments provides the
most precise definition of which regions of this protein are capable of
interacting with AIP1, as functionality depends on correct folding of
the SH3 domain, requiring the presence of their entire sequence.
However, we were able to further define the region of AIP1 that bound
to SETA by determining that neither the SH3-NC nor SH3-N proteins were
able to interact with a truncated AIP1 protein, terminating at the
internal EcoRI site at position 636 (Fig. 1), which lacks
the proline-rich region of AIP1 (data not shown). Together, these data
suggest that the interaction between SETA and AIP1 can occur outside of
yeast and is mediated by SETA's N-terminal SH3 domain and AIP1's
proline-rich C terminus.
To test whether SETA and AIP1 also interact in cells, epitope-tagged
proteins were transiently introduced into 293 cells, which can be
transfected at high efficiency, and cell lysates were analyzed by
immunoprecipitation and Western blotting (Fig. 2C). A
FLAG-tagged AIP1 cDNA was co-transfected with a C-terminally V5-tagged SETA SH3-NC expression construct, an N-terminally
Xpress-tagged SETA NCcc construct or control lacZ constructs
tagged with either epitope tag. Although in our hands the V5 epitope
tag performed better in all experiments than the Xpress tag, the latter
was chosen to tag SETA NCcc N-terminally in order to minimize the possibility of interfering with the structural integrity of the predicted C-terminal coiled-coil. Precipitates from the resultant lysates were collected with anti-V5, anti-Xpress, or anti-FLAG antibodies or with bacterially made ALG-2-GST. The resultant
precipitates were then analyzed in Western blots probed with anti-V5,
anti-Xpress, anti-SETA, or anti-FLAG antibodies.
Antibodies against epitope tags were used in these experiments to
specifically study transfected isoforms of SETA. In order to establish
that immunoprecipitation with these antibodies recovered the
transfected SETA proteins, lysates of 293 cells transfected with SETA
SH3-NC and SETA NCcc were precipitated by anti-V5 or anti-Xpress
antibodies, respectively, and immunoblotted with polyclonal anti-SETA
antibodies. As shown in Fig. 2C, lanes
1 and 2, bands of the expected size were
obtained, demonstrating that these reagents were specifically
recovering SETA proteins.
Analysis of anti-Xpress-generated immunoprecipitates of cells
transfected with AIP1-FLAG and SETA NCcc revealed a band of the size
expected for the AIP1 protein (Fig. 2C, lane
3), which was not present in lysates of cells transfected
with the lacZ control construct (Fig. 2C,
lane 4). Bands common to both lanes 3 and 4 are anti-FLAG antibody cross-reacting
proteins. To further support the suggestion that SETA and AIP1 interact
in cells, lysates were immunoprecipitated with anti-FLAG antibodies
recognizing AIP1. SETA SH3-NC or SETA NCcc was detected in these
lysates with anti-V5 or anti-Xpress antibody, respectively (Fig.
2C, lanes 5 and 6).
Therefore, complexes of AIP1 and SETA proteins could be recovered by
immunoprecipitation with antibodies directed at either protein.
To test whether SETA and AIP1 could bind to ALG-2 in one complex, we
precipitated lysates with bacterially made GST-ALG-2 protein. In line
with previous studies, GST-ALG-2 could precipitate AIP1-FLAG from cell
lysates (Fig. 2C, lane 7) (7).
Furthermore, these lysates also contained SETA SH3-NC or SETA NCcc
protein (Fig. 2C, lanes 8 and
9). Taken together, these data suggest that SETA and AIP1
can interact in cells. Furthermore, the detection of SETA proteins in
precipitates made with GST-ALG-2, which does not interact directly with
SETA, suggests that these two proteins can interact with AIP1 simultaneously.
Wild-type Rat and p53 SETA Modulates Apoptosis in Astrocytes in Response to UV
Irradiation--
To directly examine the question of whether the
association of SETA with the known regulators of apoptosis ALG-2 and
AIP1 allows it to modulate this process, primary rat astrocyte cell lines that expressed various SETA protein forms (shown in Fig. 2A) were established. Initial experiments in astrocytes
demonstrated that the the plasmid expression vectors used in 293 cells
(Fig. 2C) did not achieve high enough efficiencies of
transient transfection or maintain stable expression of SETA or control
proteins in this cell type. Therefore, the epitope-tagged SETA
cDNAs were engineered into a retroviral expression construct
immediately downstream of the packaging signal and upstream of an
internal ribosome entry site and the gene for zeocin resistance (for
details, see "Experimental Procedures"). Infection of primary rat
astrocytes with retroviruses generated from these constructs and
selection with zeocin for 10 days resulted in populations of cells that
showed greater than 98% expression of SETA proteins, which was stable
over time, as determined by immunohistochemistry (data not shown). All
further experiments were performed with these selected cell
populations, thereby eliminating issues relating to clonal variation.
Populations of astrocytes engineered with SETA SH3-NC in the antisense
orientation or with vector alone were also isolated as controls. All of
these populations expressed endogenous AIP1 and ALG-2 mRNA (Fig.
3).
Western blotting of zeocin-selected rat astrocyte populations with a
polyclonal anti-SETA antibody revealed expression of endogenous SETA
that increased with time in culture (Fig.
4). Astrocytes cultured for fewer than
five passages after the completion of selection showed variable, low
levels of endogenous SETA, with bands near 60, 95, and 180 kDa (Fig. 4,
lanes 1-6). At higher than 10 passages, the
level of endogenous SETA was increased, as demonstrated by the
inclusion of cell extract of high passage SETA NCcc-expressing
astrocytes on the same Western blot as the low passage cells (Fig. 4,
lane 7). This higher level of SETA expression was
found in all cell lines at higher passages (Fig. 4, lanes
8-14). In high passage astrocytes, SETA protein bands in
addition to those seen in low passage cells were detected, including
two more bands near 50 kDa, a band at 35 kDa, and some lower molecular
mass bands.
In addition to endogenous SETA, these Western blots revealed the
presence of the introduced SETA SH3-NC and NCcc proteins and showed
that their expression was stable over time. SETA SH3-NC appears as a
strong band near 42 kDa (Fig. 4, lanes 4 and
11), while NCcc is detected just underneath the endogenous
band at 95 kDa (lanes 5 and 12).
Neither SETA SH3-N nor -C proteins were recognized by polyclonal
anti-SETA antibodies, but they could be detected by monoclonal anti-V5
antibody via their V5 epitope tag both at low passage (Fig. 4,
lanes 2 and 3, inset) and
at high passage (data not shown). Epitope tag antibodies V5 for SETA SH3-NC and Xpress for SETA NCcc were also used to confirm the identity
of these proteins on separate blots (data not shown).
The pattern of bands observed in normal rat astrocytes was different
from that seen in p53
To determine the effect of SETA protein misexpression on apoptosis,
populations of astrocytes expressing various SETA proteins were exposed
to 5 mJ/cm2 of ultraviolet irradiation and analyzed by
TUNEL and annexin V labeling 24 h later (Fig.
5). In all experiments, the percentage of
TUNEL-positive or annexin V-positive cells was higher in astrocyte cultures expressing SETA SH3-N, -NC, or -NCcc than in those expressing vector alone, SETA SH3-C, or SETA antisense NC, which showed similar and lower levels of apoptotic cells. Control cultures that received no
irradiation showed barely detectable levels of apoptosis, regardless of
which retroviral vector they contained (not shown). Together, these
experiments demonstrated that the overexpression of SETA isoforms
encoding the SH3-N domain increased the rate of apoptosis in astrocyte
cell cultures in response to UV irradiation.
SETA Proteins Associate with the Actin Cytoskeleton--
Normal
astrocytes harboring the control retroviral vector or expressing
various introduced SETA isoforms were prepared for immunohistochemistry
using either polyclonal anti-SETA or anti-AIP1 antibodies or monoclonal
antibodies directed at the epitope tags encoded by these exogenous
proteins (Fig. 6). Cells transduced with
the vector alone showed no reactivity with the antibodies directed at
the epitope tags (e.g. the V5 antibody; Fig. 6A). However, when stained with anti-SETA antibodies, they did show endogenous SETA expression (Fig. 6B) as expected from the
Western blot results (Fig. 4). Similarly, vector-transduced cells also reacted with anti-AIP1 polyclonal antibodies (Fig. 6C). The
pattern of cell staining obtained with anti-SETA and anti-AIP
antibodies was similar, being cytoplasmic and filamentous in
appearance.
In order to examine the localization of introduced SETA proteins, cells
expressing SETA NCcc were stained with Xpress antibody (Fig.
6D), while those expressing SETA SH3-NC (Fig. 6,
E and H-K), SH3-N (Fig. 6F), or SH3-C
(Fig. 6G) were stained with V5 antibody (Fig. 6,
E-G, I, and K). In all cases, SETA
proteins were found in the cytoplasm of cells, and the smaller SH3-NC,
-N, and -C proteins could also be found in the nucleus in some cells
(Fig. 6, D-G). However, SETA proteins also appeared to
associate with cytoskeletal elements, since a proportion of the signal
had a filamentous appearance. In order to investigate which component of the cytoskeleton SETA proteins associated with, cells were counterstained with phalloidin or anti-paxillin. This analysis revealed
that SETA NCcc, SH3-NC, -N, and -C all localized to the actin
cytoskeleton, including regions that were paxillin-positive as shown in
Fig. 6 for cells expressing SETA SH3-NC. The arrows in Fig.
6, H-K, reveal areas that are both phalloidin-positive (Fig. 6H) and SETA-positive (Fig. 6I) or both
paxillin-positive (Fig. 6J) and SETA-positive (Fig.
6K). Since cells expressing SETA SH3-C (Fig. 6G)
also exhibited this pattern, this suggests that it is unlikely to be
mediated exclusively by interaction with proteins recognized by the
SH3-N domain, such as AIP1.
Expression of SETA in cultured mouse p53 In view of the established role that AIP1 and ALG-2 play in apoptosis
(7, 8, 13), a direct analysis of the role of SETA in apoptosis in
astrocytes was performed. Clonally complex populations of normal rat
astrocytes expressing SETA NCcc as well as partial SETA protein
constructs were isolated using a bicistronic, zeocin
resistance-encoding retrovirus. Analysis of the localization of
introduced SETA proteins in astrocytes revealed that they appeared to
localize to the actin cytoskeleton. The base-line levels of apoptosis
in astrocytes expressing SETA proteins were not changed when compared
with the vector controls and were less than 1% in all cultures tested.
However, when cells were exposed to UV irradiation, it was found that
exogenous SETA proteins that included the SH3-N domain increased the
number of apoptotic cells. Astrocyte cultures expressing SETA NCcc,
SH3-NC, or SH3-N all had elevated levels of apoptosis, as measured by
annexin V staining and TUNEL, when compared with cells expressing
vector alone, SETA SH3-C, or antisense SETA SH3-NC. The isolated SETA
SH3-N protein had the greatest effect in these experiments.
It appears from these data that SETA proteins capable of interacting
with the AIP1·ALG-2 complex modulate apoptosis in response to UV
irradiation in astrocytes, while not affecting their background rate of
apoptosis. Similar findings of a contingent effect on apoptosis have
been made for ALG-2 and AIP1. Reducing levels of ALG-2 protein by
antisense in T-cells protected them from apoptosis induced by a variety
of stimuli including T-cell receptor stimulation, dexamethasone, and
staurosporine (8). Conversely, overexpressing ALG-2 in fibroblasts
sensitized them to cell death in response to phorbol ester and calcium
ionophore without increasing rates of apoptosis in unstimulated cells
(8). In the case of AIP1, overexpression of a truncated form, TH28,
protected HeLa and COS cells from serum starvation, etoposide, and
staurosporine (7). Therefore, it seems likely that SETA, AIP1, and
ALG-2 are involved in a complex that modulates apoptotic signals rather
than generating them de novo.
The information available at present on the mechanism by which AIP1 and
ALG-2 affect apoptosis allows for the simple hypothesis that the
ALG-2-AIP1 complex is a component of a proapoptotic signaling pathway.
The possibility that ALG-2 is the limiting component is suggested by
the observation that reducing levels protects cells, while
overexpression of ALG-2 sensitizes them (8). As proposed by Vito
et al. (7), this model allows the further hypothesis that
TH28, the truncated form of AIP1, is a dominant negative form of AIP1
that protects cells by interfering with the function of the
ALG-2·AIP1 complex. The observation that the protective effect of
overexpressing TH28, which binds ALG-2, can be overcome by
co-expressing additional ALG-2 (7) could then be interpreted as
indicating that under these circumstances enough ALG-2·AIP1 complexes
are formed to allow apoptosis to occur. The data presented here could
fit into this model by suggesting that binding of AIP1 by the
introduced SETA isoforms containing the SH3-N domain promotes the
activity of the AIP1·ALG-2 complex.
The proapoptotic effect of introduced SETA NCcc, SH3-NC, and SH3-N
raises the question as to whether they interfere with the function of
the endogenous SETA protein(s), synergize with it, or have novel and
different functions. While it is difficult to make a strong case for
any of these possibilities in the absence of a detailed understanding
of how SETA functions, the observation that the isolated SH3 domain,
SETA SH3-N, had the greatest effect in these experiments favors the
first possibility. It is difficult to see how this small protein with
only one binding group could act in the same way as larger SETA
proteins with several binding modalities. According to this model, the
SETA isoforms used here interfere with the antiapoptotic influence of
endogenous SETA proteins. Although it is easy to see how partial SETA
proteins such as the SH3-NC and -N isoforms could act as a dominant
negative protein, this is less obvious for SETA NCcc. However,
comparison of SETA cDNA sequences with those of its closest
relatives, CD2AP (4) and CMS (5), both of which have three SH3 domains,
suggests that an additional SH3 domain more N-terminal to SH3-N remains to be isolated. Furthermore, the multiple isoforms of endogenous SETA
evident in the complex pattern revealed by Western blot remain to be
fully characterized. The original cloning of SETA suggested that there
are at least two alternative N termini, encoding both the SH3-N and -C
domains or only the SH3-C domain (1). In addition, the possibility of
two alternate C termini are suggested by cDNAs, with one
encoding a coiled-coil (1). Analysis of additional cDNA clones and
genomic clones for SETA is currently under way. A further consideration
is that, just as overexpression of TH28 only affected apoptosis in
response to some stimuli but not others (7), SETA's effect may be
different in response to different apoptotic stimuli. Experiments using
different stimuli are being performed.
The observation that SETA proteins are associated with the cytoskeleton
suggests that this may be a common characteristic of the subfamily of
SH3 domain-containing adapter molecules that also includes CD2AP and
CMS. CD2AP associates with CD2 in T-cells and is involved in its
clustering and cytoskeletal polarization by linking it to the
cytoskeleton (4). Similarly, the CMS protein is associated with the
cytoskeleton, and its misexpression causes an alteration in actin fiber
arrangement (5). Interestingly, CMS encodes putative actin binding
sequences in its C terminus, which are not conserved in SETA. However,
since SETA proteins, including the isolated SETA SH3-N and -C domains,
were able to localize to actin in rat astrocytes, this suggests that
SETA and CMS may both bind to similar elements of the cytoskeleton but in different ways. Furthermore, the observation that CMS, CD2AP, and
SETA all share two PXXP motifs, which in CMS have been shown to bind to src, p85 subunit of phosphatidylinositol
3-kinase, and Grb2 suggests that members of this group may be
downstream of a variety of signaling pathways. These observations in
combination with the data presented here suggest that the new family of
adapter molecules that includes SETA, CD2AP, and CMS may act at a point of integration of mitogenic signals, cytoskeletal architecture, and
apoptosis (14).
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and 
, vascular
endothelial cell growth factor, and protein kinase C-
(3). These
expression data suggest that the re-expression of the SETA protein in
astrocytes in the mature central nervous system contributes to their
malignant transformation and progression, prompting an investigation of its mode of action at the molecular level.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
reaction were performed. Isolated plasmids were sequenced, and the
sequences were compared with gene data bases.
-galactosidase were generated by coupled in vitro
transcription and translation (TNT; Promega) and confronted with an
excess of SETA or ALG-2 GST fusion proteins in 20 mM Tris, pH 7, 100 mM NaCl, 1 mM EGTA, 0.96 mM CaCl2, and 0.1% Nonidet P-40, which
contains 5 µM free calcium. Complexes were collected using glutathione-Sepharose 4B, washed in several changes of buffer, and separated on an SDS-polyacrylamide gel, which was prepared for
fluorography and exposed to film.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Predicted protein sequence of the partial rat
AIP1 cDNA isolated as binding to SETA. Alignment of the rat
(rAIP1/rAlix; this paper, GenBankTM
accession no. AF192757), mouse (mAIP1, GenBankTM
accession no. AF119955; mAlix, GenBankTM
accession no. AJ005073; and more recently E2F-inducible gene 2, GenBankTM accession no. AF176514), and Xenopus
(Xp95, GenBankTM accession no. AF115497)
proteins is shown. Identical residues are shaded, and the
proline-rich C termini in rat and mouse AIP1 are boxed. In
addition, the truncation in murine AIP1 at residue 636, engineered by
subcloning with EcoRI, and the portion of the proline-rich
region present in the reported short form of Alix (6), starting at
residue 826, are indicated. Numbering is taken from the mouse AIP1
GenBankTM file.
-galactosidase protein (Fig.
2B).
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Fig. 2.
SETA interacts with AIP1 and ALG-2 in
vitro and in cells. A, schematic describing
the SETA constructs used in this study. SETA NCcc is identical to the
sequence reported in GenBank accession no. AF131867, while SETA SH3-NC,
-N, and -C represent subregions of the molecule as indicated. Also
shown are the epitope tags used to identify recombinant SETA proteins;
SETA NCcc was tagged N-terminally with the Xpress tag, and SETA SH3
constructs were tagged C-terminally with V5. B, in
vitro confrontation between SETA or ALG-2 GST fusion proteins and
radiolabeled AIP1 or -galactosidase (lacZ) proteins
produced by in vitro coupled transcription and translation
(TNT). SETA SH3-NC and -N bind to AIP1, as does the positive control
ALG-2 protein, while SETA SH3-C does not. The interaction between SETA
SH3-N and AIP1 occurs in the absence of calcium. Neither SETA nor ALG-2
interact with -galactosidase (lacZ). The TNT proteins
alone are run on the right as a reference. C,
co-immunoprecipitation of SETA and AIP1 proteins from transiently
transfected cells. FLAG-tagged AIP1 and V5-tagged SETA SH3-NC,
Xpress-tagged SETA NCcc, or Xpress-tagged lacZ were
co-transfected into 293 cells. Immunoprecipitates were generated using
anti-V5 (lane 1), anti-Xpress (lanes
2-4), or anti-FLAG (lanes 5 and
6) antibodies or bacterially expressed GST ALG-2 fusion
protein (lanes 7 and 8) and analyzed
by Western blotting using anti-SETA (lanes 1 and
2), anti-FLAG (lanes 3, 4,
and 7), anti-V5 (lanes 5 and
8), anti-Xpress (lanes 6 and
9), or antibodies, as indicated. Molecular masses in kDa are
shown at the right. aa, amino acids.
/ Mouse Astrocytes and Glioma
Cells Express AIP1 and ALG-2--
The observation that SETA, AIP1, and
ALG-2 can interact raises the question of whether they are co-expressed
in cells. SETA was originally identified as a gene associated with
malignancy in astrocytes and is expressed in p53/
astrocytes derived from p53 knockout mice capable of forming tumors as
well as glioma-derived cell lines. Therefore, we analyzed these cells,
as well as normal rat astrocytes expressing various SETA protein
isoforms (see below), for AIP1 and ALG-2 mRNA expression. As shown
in Fig. 3, all of the cells tested
expressed AIP1 and ALG-2 mRNA. Two AIP1 mRNA species were
observed, with one just below the 28 S at about 4 kb and one above the
28 S at approximately 7 kb, as described previously (7). ALG-2 mRNA
ran as a single band below the 18 S at approximately 1 kb (8). AIP1 and
ALG-2 expression did not correlate with the ability of
p53/ astrocytes to form tumors, since similar levels of
expression were found in cells grown in DMEM plus 10% fetal calf serum
or in DMEM plus 20 ng/ml epidermal growth factor, which are capable of
forming tumors, and DMEM plus 10% basic fibroblast growth factor, which are not (3). Rat astrocytes with a normal p53 genotype also
expressed both AIP1 and ALG-2 mRNA, regardless of whether they had
been engineered to express SETA constructs or vector alone (see below).
Last, both genes were also expressed in a rat glioma cell line, GV2C8,
and two human glioma cell lines, LNZ308 and A172, all known to express
SETA mRNA (1). These data demonstrate that SETA, AIP1, and ALG-2
are co-expressed in a variety of cells relevant to the study of glial
cell transformation and so have the opportunity to interact.
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Fig. 3.
Northern analysis of AIP1 and ALG-2
expression. Varying quantities of poly(A)+ mRNA
were subjected to Northern blotting and hybridized with random primed
probes derived from the cDNAs for AIP1 or ALG-2 as indicated. Two
bands were seen in AIP1-probed Northern blots with one just below 28 S
at about 4 kb and one above the 28 S at approximately 7 kb (7). ALG-2
mRNA ran as a single band below 18 S at approximately 1 kb
(8)
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Fig. 4.
SETA Western blot of rat astrocytes.
Cell extracts derived from rat astrocytes infected with 1726/zeo
retroviruses encoding no additional gene (lanes 1 and 8), SETA SH3-N (lanes 2 and
9), SETA SH3-C (lanes 3 and
10), SETA SH3-NC (lanes 4 and
11), SETA NCcc (lanes 5, 7,
and 12), or SETA SH3-NC in the antisense orientation
(lanes 6 and 13) or extracts derived
from p53 / mouse astrocytes were subjected to Western
blotting with anti-SETA or V5 antibodies as indicated. Gels run in
parallel and stained with Coomassie Blue are shown below to
demonstrate similar loading of protein in all lanes. Endogenous SETA
appears as a series of bands as described under "Results," while
the introduced SETA SH3-N and -C proteins are revealed by V5 antibody
(lanes 2 and 3), the introduced SETA
SH3-NC is shown in lanes 4 and 11, and
SETA NCcc is shown in lanes 5, 7, and
12.
/ mouse astrocytes reported
previously (1) and shown here for comparison (Fig. 4, lane
14). While both cell types showed the triple of SETA
proteins near 50 kDa, primary rat astrocytes also expressed a much
larger form of SETA near 180 kDa and smaller forms at 35 kDa and below.
In addition, the band near 95 kDa in rat astrocytes appeared to migrate
faster in p53/ mouse astrocytes.
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Fig. 5.
Expression of SETA proteins encoding the
SH3-N domain sensitizes cells to apoptosis in response to UV
irradiation. Astrocytes expressing various SETA constructs were
challenged with 5 mJ/cm2 UV irradiation and prepared for
analysis by annexin V or TUNEL 24 h later. The percentage of
annexin V- or TUNEL-positive cells are shown. Cells expressing SETA
constructs that make a protein containing the SH3-N domain have
significantly higher rates of apoptosis than control cultures, with
the most dramatic increase seen in cultures expressing SETA SH3-N. Data
shown are mean ± S.E. from four or more independent experiments,
with three coverslips per experiment and over 200 cells counted per
coverslip.
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Fig. 6.
SETA proteins localize to the actin
cytoskeleton. Astrocytes harboring the control vector
(A-C) or expressing SETA NCcc (D), SH3-NC
(E, H-K), SH3-N (F), or SH3-C
(G) were analyzed by immunohistochemistry. Cells were
stained with anti-V5 antibody (A, E-G,
I, and K), polyclonal anti-SETA antibody
(B), polyclonal anti-AIP antibody (C), or Xpress
antibody (D). Cells in A were counterstained with
propidium iodide to reveal the nuclei. Cells expressing SETA
SH3-NC and stained with anti-V5 detected by secondary fluorescein
isothiocyanate-conjugated antibodies (I and K)
were counterstained with phalloidin-tetramethylrhodamine isothiocyanate
(H) or paxillin-detected with a tetramethylrhodamine
isothiocyanate-conjugated secondary antibody (J). The
arrows in H-K show areas that were stained in
both colors. Cells stained with secondary antibodies alone showed no
signal (not shown).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
astrocytes and in astrocytes in the adult brain is associated with the
tumorigenic state (1), suggesting that it may contribute to the process of glioma formation. The gene encodes SH3 domain-containing adapter molecules that are likely to exert their function by binding with high
affinity and specificity to other proteins. A yeast two-hybrid screen
of a rat CG4 glial progenitor cell cDNA library with the two SH3
domains of SETA NCcc as bait resulted in our isolation of the rat
homolog of the AIP1/Alix gene, which has been described as a binding
partner of ALG-2 by others (6-8). In vitro confrontation experiments and co-immunoprecipitation from transiently transfected 293 cells confirmed that SETA and AIP1 bind and showed that the SH3-N
domain of SETA and the proline-rich region of AIP1 are responsible for
this interaction. The ability to recover SETA-AIP1 complexes with ALG-2
suggests that SETA and ALG-2 may be able to bind to AIP1
simultaneously. Furthermore, SETA, AIP1, and ALG-2 are all co-expressed
in normal and transformed astrocytes, as well as rat and human glioma
cell lines, making their possible interaction plausible in cells
relevant to understanding glioma.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Luciano D'Adamio for generously providing AIP1 and ALG-2 cDNAs and constructs, experimental advice, and helpful discussions; Dr. Pasquale Vito for anti-AIP1 antibody; and Drs. Irene Newsham, Babette Fuss, and Shirley Taylor for helpful comments on the manuscript.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Brain Cancer Research Program of the James S. McDonnell Foundation, the Childhood Brain Tumor Foundation, the Rainbow Foundation, and the F. Norton Hord Foundation and an institutional research grant to Virginia Commonwealth University from the American Cancer Society.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/EMBL Data Bank with accession number(s) AF192757.
To whom correspondence should be addressed: Hermelin Brain Tumor
Center and Department of Neurosurgery, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202. Tel.: 313-916-7293; Fax: 425-732-8379;
E-mail: oliver@bogler.net.
Published, JBC Papers in Press, April 14, 2000, DOI 10.1074/jbc.M908994199
| |
ABBREVIATIONS |
|---|
The abbreviations used are: SH3, src homology 3; ALG-2, apoptosis-linked gene 2; AIP1, ALG-2-interacting protein 1; Alix, ALG-2-interacting protein X; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; kb, kilobase; SETA, SH3 domain-containing expressed in tumorigenic astrocytes; TUNEL, TdT dUTP nick end labeling.
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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|---|
| 1. | Bogler, O., Furnari, F. B., Kindler-Roehrborn, A., Sykes, V. W., Yung, R., Huang, H.-J. S., and Cavenee, W. K. (2000) Neuro-Oncology 2, 6-15 |
| 2. | Bogler, O., Huang, H. J., and Cavenee, W. K. (1995) Cancer Res. 55, 2746-2751 |
| 3. | Bogler, O., Nagane, M., Gillis, J., Huang, S. H.-J., and Cavenee, W. K. (1999) Cell Growth Differ. 10, 73-86 |
| 4. | Dustin, M. L., Olszowy, M. W., Holdorf, A. D., Li, J., Bromley, S., Desai, N., Widder, P., Rosenberger, F., van der Merwe, P. A., Allen, P. M., and Shaw, A. S. (1998) Cell 94, 667-677 |
| 5. | Kirsch, K. H., Georgescu, M.-M., Ishimura, S., and Hanafusa, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6211-6216 |
| 6. | Missotten, M., Nichols, A., Rieger, K., and Sadoul, R. (1999) Cell Death Differ. 6, 124-129 |
| 7. | Vito, P., Pellegrini, L., Guiet, C., and D'Adamio, L. (1999) J. Biol. Chem. 274, 1533-1540 |
| 8. | Vito, P., Lacana, E., and D'Adamio, L. (1996) Science 271, 521-525 |
| 9. | Sugimoto, Y., Aksentijevich, I., Gottesman, M. M., and Pastan, I. (1994) Bio/Technology 12, 694-698 |
| 10. | Bartz, S. R., and Vodicka, M. A. (1997) Methods 12, 337-342 |
| 11. | Mayer, B. J., and Eck, M. J. (1995) Curr. Biol. 5, 364-367 |
| 12. | Che, S., El-Hodiri, H. M., Wu, C. F., Nelman-Gonzalez, M., Weil, M. M., Etkin, L. D., Clark, R. B., and Kuang, J. (1999) J. Biol. Chem. 274, 5522-5531 |
| 13. | Lacana, E., Ganjei, J. K., Vito, P., and D'Adamio, L. (1997) J. Immunol. 158, 5129-5135 |
| 14. | Evan, G., and Littlewood, T. (1998) Science 281, 1317-1322 |
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