From the Faculty of Health Sciences and
Department of Pathology and Molecular
Medicine, McMaster University, Hamilton, Ontario, L8N 3Z5, Canada,
Department of Urology, Baylor College of Medicine, Houston,
Texas, 77035, ** Department of Biochemistry, McMaster University,
Hamilton, Ontario, L8N 3Z5, Canada and § Hamilton Regional
Cancer Centre, Hamilton, Ontario, L8V 5C2 Canada
Received for publication, October 11, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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p120 GTPase-activating protein (GAP)
down-regulates Ras by stimulating GTP hydrolysis of active Ras. In
addition to its association with Ras, GAP has been shown to bind to
several tyrosine-phosphorylated proteins in cells stimulated by growth
factors or expressing transforming tyrosine kinase variants. Here we
report the cloning and characterization of a novel GAP-binding protein,
mTid-1, a DnaJ chaperone protein that represents the murine homolog of
the Drosophila tumor suppressor l(2)tid gene.
Three alternatively spliced variants of mTid-1 were isolated, two of
which correspond to the recently identified hTid-1L and
hTid-1S forms of the human TID1 gene that
exhibit opposing effects on apoptosis. We demonstrate that both
cytoplasmic precursor and mitochondrial mature forms of mTid-1
associate with GAP in vivo. Interestingly, although mTid-1
is found tyrosine-phosphorylated in v-src-transformed fibroblast cells,
GAP selectively binds to the unphosphorylated form of mTid-1. In
immunofluorescence experiments, GAP and Tid-1 were shown to colocalize
at perinuclear mitochondrial membranes in response to epidermal growth
factor stimulation. These findings raise the possibility that Tid
chaperone proteins may play a role in governing the conformation,
activity, and/or subcellular distribution of GAP, thereby influencing
its biochemical and biological activity within cells.
As a guanine nucleotide-binding protein, Ras cycles between an
active GTP-bound and inactive GDP-bound conformation (1). Down-regulation of Ras activity is attributed to the cytosolic Ras
GTPase-activating Protein
(GAP)1 (2). GAP stimulates
the weak intrinsic GTPase activity of Ras, accelerating the hydrolysis
of bound GTP to GDP, thereby terminating mitogenic signals elicited by
Ras proteins. In addition to its role as a negative regulator of Ras,
it has been suggested that GAP may exert an effector function in the
control of cytoskeletal reorganization and cell migration (3, 4).
Although Ras binding and catalytic activity of GAP reside in its C
terminus, the N-terminal sequences consist of a number of conserved
protein modules including src-homology (SH) 2 and 3 protein interaction
domains that facilitate the formation of signaling complexes, which may
couple GAP to upstream regulators of Ras or downstream effector targets
(5). Indeed, activated tyrosine kinases that phosphorylate GAP also promote its association via its SH2 domains, with several
phosphotyrosine-containing proteins, including members of the
p62dok family of proteins
(6-12) and Rho/Rac GTPase-activating protein, p190 (13, 14).
p62dok codes for a pleckstrin
homology-containing adaptor protein first noted as one of the most
prominently tyrosine-phosphorylated proteins in v-src-, v-fps-, and
v-abl-transformed cells. Moreover, its hyperphosphorylation levels
correlate with the transformation phenotype of these oncogenic products
(7, 15). These observations led to the hypothesis that at least some of
the transforming capabilities of oncogenic tyrosine kinases might be
conferred by p62dok by virtue of its
constitutive occupancy of the SH2 domains of GAP, which may have
negative regulatory effects on GAP activity. Consistent with this
notion, the binding of phosphorylated p62dok to
GAP has been shown to significantly impair the GTPase-promoting activity of GAP towards Ras in vitro (16). By contrast, p190 functions as a Rho/Rac GTPase-activating protein and may serve as a
RasGAP effector, providing an interface between the Ras-signaling pathway and proteins of the Rho/Rac pathway that regulate the cytoskeletal architecture of the cell (13). Searching for
GAP-associated proteins is therefore of particular interest as it may
lead to the identification of novel cellular components which may play an important role in Ras regulation and also, in furthering our understanding of the mechanisms by which oncogenic tyrosine kinases may
effect transformation. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that
represents the murine homolog of the Drosophila tumor
suppressor l(2)tid gene (17) and the recently identified
human Tid (TID1) gene (18).
The ubiquitously expressed DnaJ family of proteins serve as regulatory
factors to the evolutionary conserved heat shock 70 (Hsp70) superfamily
of molecular chaperones (19, 20). This protein family is defined by a
highly conserved J-domain, which functions as the binding region for
Hsp70 chaperones and orchestrates their interaction with specific
substrates (21). Hsp70 proteins and their associated DnaJ co-chaperone
mediate a variety of cellular activities including the folding of newly
synthesized polypeptides, the translocation of proteins across
membranes, and assembly of multimeric protein complexes (22-25). More
recently, genetic and biochemical studies have implicated DnaJ and
Hsp70 proteins as important components of intracellular signaling
pathways linked to cell survival and growth regulation. In this
context, they regulate many facets of the signaling process that have
been described for protein modules such as pleckstrin homology SH2 and
SH3 domains, namely subcellular localization, regulation of enzymatic
activity, and enzyme/substrate recognition (5). For example, genetic studies of v-src toxicity in yeast indicate that the DnaJ protein, Ydj1, is necessary for the correct subcellular targeting and kinase activation of v-src (26, 27). Ydj1 has also been implicated as a
positive regulator of cell cycle progression essential for efficient
recognition and phosphorylation of cyclin CLN3 by cdc28, events that
signal CLN3 degradation (28). Additional biochemical evidence suggests
that members of the DnaJ and Hsp70 family interact with and modulate
the growth-suppressive properties of several tumor suppressor proteins,
including p53, Wilms' tumor suppressor (WT1), retinoblastoma (Rb) and
the double-stranded RNA-activated protein kinase PKR (29-36)
The Drosophila l(2)tid gene is the first member
of a DnaJ chaperone family to be classified as a tumor suppressor (17). Recessive mutations at the l(2)tid locus causes defects in
differentiation and morphogenesis of larval imaginal discs leading to
neoplastic growth of these cells into lethal tumors. hTid-1, the human
homologue of the l(2)tid-encoded protein Tid56, was recently
isolated in a screen for cellular substrates of the human
papillomavirus E7 oncoprotein (18). Two splice variants,
hTid-1L and hTid-1S, have been identified.
Interestingly, although both have been reported to localize to the
mitochondria, they seem to display opposing effects on apoptosis.
Notably, hTid-1S suppresses cytochrome c release
and caspase 3 activation in response to tumor necrosis factor In this study, we demonstrate that mTid-1, a novel member of the Tid
family of chaperone proteins, complexes with GAP both in
vitro and in vivo in a phosphorylation-independent
fashion. Immunofluorescence experiments reveal that Tid-1 proteins are localized to the cytosol, mitochondria, or nucleus, depending on the
particular cell type. Moreover, in response to epidermal growth factor
stimulation, GAP and Tid-1 colocalize to distinct perinuclear
subdomains resembling mitochondrial membranes. Furthermore, we present
evidence that Tid-1 can associate with both cytoplasmic and
mitochondrial Hsp70 chaperones. These findings suggest a possible novel
role for GAP in collaboration with Tid-1·Hsp 70 chaperone complexes
in the integration of mitogenic-signaling pathways at the plasma
membrane and control of apoptotic signal transduction at mitochondrial membranes.
Cell Lines--
NIH 3T3 mouse fibroblasts, Rat2 fibroblasts,
COS-1 monkey kidney, SAOS-2 human osteosarcoma, and MCF-7 breast cancer
cell lines were grown in Dulbecco's modified Eagle's medium
supplemented with 10% calf serum (Life Technologies). S7a cells (v-src
transformed Rat-2 fibroblasts) were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (Life
Technologies). All cells were maintained in a humidified incubator at
37 °C and 5% CO2.
Antibodies--
Mouse monoclonal antibodies to phosphotyrosine
(PY20) and p120 RasGAP (GAP) were purchased from Transduction
Laboratories (Lexington, KY); mitochondrial Hsp70 (Grp 75, MA3-028)
antibodies were from Affinity Bioreagents (Golden, CO). Rat monoclonal
Hsc70 (SPA-815) and rabbit polyclonal Hsp70 (SPA-812) antibodies were obtained from Stressgen Corp. (Victoria, BC). Mouse monoclonal 2G2
antibody (M00120) to an as yet uncharacterized integral membrane protein of the inner mitochondrial membrane was obtained from Bionostics (Toronto, ON). Rabbit antisera raised against a bacterial fusion protein TrpE-GAP (GAP amino acid residues 171-448) was kindly
provided by T. Pawson. Polyclonal rabbit antibodies to murine Tid-1
were generated using a glutathione S-transferase fusion
protein expressing the C-terminal 91 amino acid residues (389) of
mTid-1. Affinity purification of mouse Tid-1 antibodies for
immunofluorescence was carried out as described (38).
Plasmids and Transfections--
The cDNAs encoding
full-length mTid-1L (amino acid residues 1-480),
mTid-1I (residues 1-453), and mTid-1S
(residues 1-429) were subcloned into pcDNA3 and pcDNA3-MYC
expression vectors (Invitrogen). Transient transfections were performed
by calcium phosphate-DNA precipitation methods.
In Vitro Binding Assay--
M3.2 cDNA encoding mouse
Tid-1I sequences (amino acid residues 9-453) was in
vitro transcribed and translated using a T'N'T kit (Promega) with
T7 polymerase according to the manufacturer's recommendations.
Radiolabeled proteins (8 µl) derived from reactions either containing
or lacking M3.2 cDNA as template were incubated for 1 h at
4 °C with anti-mTid-1 antibodies. Labeled M3.2 proteins were also
mixed with 1 ml of S7a cell lysate, prepared in PLC buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol,
1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 100 µM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin), and incubated for 1 h at 4 °C with polyclonal antibodies to GAP or with normal rabbit
serum as a control. Immune complexes were washed three times with PLC
buffer and resolved by SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were visualized by autoradiography.
Immunoprecipitations and Western Blot Analysis--
Cells were
washed twice with PBS and lysed in ice-cold PLC buffer. Before lysis
and immunoprecipitation with Hsp70 antibody, NIH 3T3 cells were
heat-shocked for 1 h in a water bath heated to 43 °C and
returned to 37 °C for 6 h to allow for Hsp70 accumulation. For
immunoprecipitation experiments with Hsc70, cells were lysed in buffer
that either contained or lacked 10 mM ATP. All cell lysates
were normalized for protein content before immunoprecipitation with the
corresponding antibody. Typically, 1 mg of protein was used in
immunoprecipitations, and 50 µg of protein was used for whole cell
lysates. Proteins were separated by electrophoresis on an 8%
SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride
membrane (Roche Molecular Biochemicals). After incubation with the
respective primary antibody and subsequently with the corresponding
horseradish peroxidase-conjugated secondary antibody, proteins were
detected using ECL reagent (Amersham Pharmacia Biotech).
Northern Blot Analysis--
Total RNA was extracted from
FVB mouse tissues using TRIzol reagent (Life Technologies),
separated on a 1% formaldehyde-agarose gel, and blotted onto a
Immunofluorescence--
NIH 3T3, SAOS-2, and COS-1 cells were
seeded into six-well plates containing glass coverslips and allowed to
adhere overnight. COS-1 cells transiently transfected with mTid-1
expression plasmids were plated 24 h after transfection. For
mitochondrial staining with rhodamine 123 (Molecular Probes Inc.),
cells were incubated 10 min with a 1:4000 dilution of a 2.5 mM stock in PBS. Culture media was then added, and the
cells were incubated for an additional 30 min at 37 °C. Staining of
mitochondria with MitoTracker® mitochondrion-selective dye (Molecular
Probes Inc.) was done as per the manufacturer's instructions using a
working concentration of 50 nM. Slides were washed with PBS
then subjected to fixation in 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4, overnight at 4 °C. After three washes with
PBS, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min,
rinsed, and blocked in 5% normal goat serum (Vector Laboratories) in
PBS for 30 min at room temperature. Slides were then incubated for
1 h at room temperature in 5% goat serum containing
affinity-purified mTid-1 antibody at 15 µg/ml. For double-labeling, a
mixture of mTid-1 antibody and mitochondrial 2G2 antibody was used.
After three washes with PBS, cells were incubated for 1 h at room
temperature with a mixture of AlexaTM488 goat anti-rabbit IgG and
AlexaTM546 goat anti-mouse IgG (Molecular Probes Inc.), each diluted
1:200 in PBS. For nuclei staining, cells were incubated for 10 min with
a 1:100 dilution of 10 µg/ml Hoechst stain. Cells were viewed and
photographed using a Leitz Diaplan fluorescent microscope.
For GAP and Tid-1 colocalization experiments, cells were serum-starved
for 24 h and then stimulated for 5 min with 100 ng/ml epidermal
growth factor (EGF; Intergen) as described (40). Fixation, permeabilization, and incubation with secondary antibodies and Hoechst
were done as described above. Cells were incubated in 5% goat serum
containing a mixture of mTid-1 and mouse monoclonal RasGAP antibody for
1 h. To ensure no cross-reactivity between secondary antibodies
and fluorescein isothiocyanate and rhodamine channels, control
experiments were performed with cells stained for only one of mTid-1 or
RasGAP antibodies and illuminated with two different filter sets, respectively.
Identification of mTid-1 as a GAP-binding Protein--
In an
attempt to identify binding partners of GAP, we tested products of
cDNA clones that had been isolated from a mouse mammary cDNA
library in a separate screen for novel cellular targets of oncogenic
v-src tyrosine kinase (data not shown) for their ability to bind to GAP
in vitro. The product of one clone, M3.2, spanned an open
reading frame of 445 amino acids and exhibited a high degree of
sequence identity with the protein product of the Drosophila tumorous imaginal disc l(2)tid gene, Tid56 (17), and the
recently identified human TID1 gene, hTid-1 (18). We refer
to this murine homologue of hTid-1 as mTid-1. The ability of mTid-1 to
interact with GAP was tested in vitro by determining whether
products of in vitro transcribed and translated M3.2
cDNA could interact with GAP present in lysates from
v-src-transformed Rat2 fibroblasts (S7a). As shown in Fig.
1, a single radiolabeled translation
product of ~48 kDa was immunoprecipitated by mTid-1-specific
antibodies raised against the C-terminal residues of M3.2 expressed as
a glutathione S-transferase fusion protein from rabbit
reticulocyte lysates containing M3.2 cDNA (lane M3.2)
but not in the control lysate lacking a DNA template (lane
C). The 48-kDa protein was also observed in GAP immune complexes
after incubation of [35S]methionine-labeled M3.2
translation products with S7a lysates, suggesting that mTid-1 is
capable of associating with GAP proteins.
Deduced Amino Acid Sequence of Mouse Tid--
Full-length mouse
Tid-1 cDNA was obtained by further screening of a mouse thymus
cDNA library using M3.2 as a probe. As shown schematically in Fig.
2A, both mouse and human Tid-1
cDNAs encode proteins of 480 amino acids, with a predicted
molecular mass of 53 kDa. Overall, mouse and human Tid-1 proteins are
87.5% identical, with the lowest degree of similarity residing in the
first 88 amino acids. Analysis of the deduced protein sequence of
mTid-1 revealed typical modular features common to DnaJ chaperone
proteins, including an N-terminal J-domain, displaying ~95 and 69%
identity with that of hTid-1 and Drosophila Tid56,
respectively; a glycine/phenylalanine-rich hinge region, separating the
J-domain from a zinc finger-like region that contains four repeats of
the sequence CXXCXGXG (where X denotes any amino acid) and is thought to stabilize the
interaction of the target substrates with the Hsp70 chaperone machinery
(41); and a less conserved C-terminal domain, exhibiting ~92 and 39% identity with hTid-1 and Drosophila Tid56, respectively,
thought to be involved in substrate binding. Flanking the N terminus of the J-domain is a mitochondrial cleavage motif (LRP-GV) (42) common to
hTid-1 and other polypeptides targeted to the mitochondrial matrix.
This would suggest murine Tid-1 proteins, like their
Drosophila and human counterparts, may also be localized to
the mitochondria.
Mouse Tid-1 Gene Encodes Three Alternatively Spliced
Isoforms--
Three alternatively spliced variants of mouse Tid-1 were
isolated from a mouse thymus cDNA library (Fig. 2B). The
long form, mTid-1L, corresponds to the full-length
480-amino acid protein. In the intermediate form mTid-1I,
the C-terminal amino acids 447-480 of mTid-1L,
corresponding precisely to exon 11 of the human TID1 gene,
are spliced and replaced with six amino acids, KRSTGN, located within a
downstream exon.2 Notably,
the partial M3.2 cDNA clone spans amino acid residues 9-453 of
mTid-1I. Both the long and intermediate forms of mTid-1 agree with the previously reported human hTid-1L and
hTid-1S alternative splice variants (37), which we will
refer to herein as hTid-1L and hTid-1I,
respectively. The third and shortest variant, mTid-1S, contains an in-frame deletion of 50 amino acids (codons 211-260). This
deletion, which corresponds precisely to exon 5 of the human TID1 gene,2 results in a loss of two of the four
CXXCXGXG motifs. Although DnaJ
proteins containing only two cysteine-rich repeats have yet to be
identified, DnaJ homologs lacking the zinc finger motif altogether have
already been characterized and shown to exhibit some chaperone activity
(17, 21, 43). The expression pattern of mTid-1 mRNA in several
mouse tissues was determined by Northern blot analysis using a
mTid-1I cDNA fragment (nucleotides 690-1482) as a
probe. As depicted in Fig. 2C, a single transcript of ~2.6 kilobases is highly expressed in each tissue examined. This contrasts to the variable expression observed of hTid-1 mRNA in human
tissues, where strong signals are detected primarily in heart, liver,
and skeletal muscle (18). A prominent band of ~1.9 kilobases is also
evident in mouse testis. The identity of this mRNA species is not
known. Since transcripts encoding mTid-1L,
mTid-1I, and mTid-1S differ by less than 150 nucleotides, it is not possible to resolve individual mRNA species
by Northern blot analysis.
To detect endogenous mTid-1 proteins, cell lysates from Rat2 (R2)
fibroblasts were immunoprecipitated and immunoblotted with polyclonal
antibodies raised to mTid-1I. Two major forms of mTid-1, migrating as a doublet with apparent molecular sizes of 40 and 43 kDa,
were identified (Fig. 3A).
Interestingly, in several tumor cell lines screened by Western blot
analysis of whole cell lysates, an additional band was observed with an
approximate molecular mass of 46-48 kDa (Fig. 3B and data
not shown). Ectopic expression of mTid-1L and
mTid-1I in COS-1 cells gave rise to prominent bands that
comigrate with the 43- and 40-kDa endogenous forms of Tid-1, respectively (Fig. 3C). Although transfection of the
recombinant mTid-1S cDNA construct generated high
levels of mTid-1 proteins of the predicted 38-kDa size, the endogenous
short mTid-1S form was not readily observed in any of the
cell lysates examined. However, reverse transcription-polymerase chain
reaction analysis has revealed that mTid-1S transcripts are
selectively expressed in mouse thymus, kidney, and liver (data not
shown). Transient expression of the long, intermediate, and short
isoforms of mTid-1 also gives rise to additional higher molecular mass
polypeptides of apparent molecular sizes of 50, 48, and 46 kDa,
respectively (Fig. 3C). As noted above, the mTid-1 protein
is characterized by a mitochondrial cleavage signal situated between
residues 63 and 67. Because most mitochondrial proteins are
nuclear-encoded, they are synthesized as precursors in the cytosol and
processed to a mature form upon import into the mitochondria (42, 44). Therefore, the higher molecular mass proteins in the 46-50-kDa range
observed in tumor and transfected cells likely represent cytoplasmic
precursors of the mature mitochondrial forms. This suggestion is
supported by the identification of both cytoplasmic and mitochondrially
localized Drosophila l(2)tid-encoded proteins, believed to
represent the Tid56 precursor and mature Tid50 forms, respectively, as
revealed by immunoelectron microscopy of Drosophila Schneider 2 cells (45). The significance of the observed overexpression of endogenous precursor protein in tumor cell lines is currently being
investigated.
GAP Associates with mTid-1 in Vivo--
To assess possible
in vivo interactions between mTid-1 and GAP, lysates from
Rat2 (R2) and v-src-transformed Rat2 fibroblasts (S7a) cells were
immunoprecipitated with antibodies directed against p120 RasGAP (GAP)
and immunoblotted with anti-GAP and anti-mTid-1 serum. In both cell
lines examined, the endogenous 40-kDa mTid-1I and 43-kDa
mTid-1L forms of mTid-1 were found to coprecipitate with
GAP (Fig. 4A), suggesting that
the interaction between GAP and mTid-1 occurs in a kinase-independent
manner. However, analysis of mTid-1 immune complexes with anti-GAP
antibodies failed to demonstrate detectable binding between mTid-1 and
GAP (Fig. 4B, top panels). One possible
explanation is that antibodies to mTid-1 were raised to the C-terminal
substrate binding domain and, thus, may preclude binding of GAP to
mTid-1.
To determine if mTid-1 is an intracellular target of activated tyrosine
kinases, cell lysates prepared from R2 and S7a cells were probed with
anti-phosphotyrosine antibodies on Western blots. As shown in Fig.
4A, measurable tyrosine phosphorylation of
mTid-1L and mTid-1I proteins was observed in
S7a but not R2 cells, suggesting that mTid-1 may serve as a direct or
indirect substrate of v-src tyrosine kinase. Intriguingly, immunoblot
analysis of GAP immunoprecipitates with anti-phosphotyrosine antibodies
revealed that GAP associates predominantly with the unphosphorylated
forms of mTid-1 (Fig. 4B, bottom panels).
Phosphorylation of mTid-1 may impede its binding to GAP or,
alternatively, may function as a trigger for dissociating the
mTid-1·GAP complex, analogous to that described for the Hsp90·v-src heterocomplex (46).
The binding of each of the mTid-1 isoforms to GAP was further examined
using transient expression assays in COS-1 cells. Lysates from cells
transfected with mTid-1L, mTid-1I, and
mTid-1S were immunoprecipitated with anti-GAP serum
followed by immunoblotting with anti-mTid-1 antibodies. GAP was found
to associate with both full-length precursor and mature processed forms
of all three mTid-1 splice variants (Fig. 4C). Moreover, in
cells ectopically expressing mTid-1I, a significant
increase in the interaction of GAP with mTid-1I was
observed compared with GAP·mTid-1L or GAP·mTid-1S complexes, suggesting preferential binding of
GAP to the intermediate isoform of mTid-1I. Recently, it
was reported that hTid-1L and hTid-1I have
opposing effects on apoptosis, with hTid-1L-enhancing and
hTid-1I-suppressing apoptosis induced by tumor necrosis
factor mTid-1 Interacts with Multiple Members of the Hsp70 Family of
Molecular Chaperones--
The highly conserved J-domain of mouse Tid-1
suggests the protein may function as a cofactor of the Hsp70 chaperone
machinery. It has recently been demonstrated that hTid-1L
and hTid-1I interact specifically with mitochondrial Hsp70
(Grp75) (37). Given that our polyclonal antibodies are immunoreactive
to both cytoplasmic and mitochondrial mature forms of mTid-1, whereas
those used by Syken et al. (Ref. 37 and data not shown) only
recognize the mitochondrial-localized forms of hTid-1, we sought to
establish whether mTid-1-encoded proteins also bind nonmitochondrial
members of the Hsp70 family. To this end, coimmunoprecipitation assays were performed using antibodies to mTid-1 and antibodies specific to
three distinct members of the Hsp70 family, notably mitochondrial Grp75, cytoplasmic heat-shock-inducible Hsp70, and constitutively expressed Hsc70. As reported for hTid-1, endogenous 43-kDa
mTid-1L and 40-kDa mTid-1I proteins can form
complexes with mitochondrial Grp75 (Fig.
5A). To study the interaction
of mTid-1 with stress-inducible Hsp70, NIH 3T3 cells were heat-shocked
for 1 h at 43 °C before lysis and immunoprecipitation analysis
with anti-mTid-1 or anti-Hsp70 antibodies. A significant induction of
Hsp70 protein levels was observed in heat-shocked cell lysates, whereas
no concomitant increase in the expression of either endogenous
mTid-1L or mTid-1I proteins was observed. As
shown in Fig. 5B, cytosolic stress-associated Hsp70 was
recovered in association with mTid-1L and
mTid-1I upon heat treatment but not in control untreated
cells. Anti-Hsp70 antibodies were not as efficient as anti-Grp75
antibodies in coprecipitating mTid-1 proteins. Hsc/Hsp70 protein
complex assembly and dissociation is ATP-dependent.
Previous studies have shown that the binding of ATP to the ATPase
domain of Hsc70 induces conformational changes that modulate the
binding of cochaperone proteins (47-49). We therefore examined the ATP
dependence of Tid-1 interactions with Hsc70. As shown in Fig.
5C, the addition of 10 mM ATP to cell lysates before immunoprecipitation with anti-Hsc70 antibodies, was found to
enhance the association of Tid-1L and Tid-1I
with the constitutive cytosolic Hsc70. Taken together, these findings
implicate mTid-1 as a regulatory cofactor to members of Hsp70 chaperone
family. Furthermore, our observation that mTid-1 interacts with Hsc70 and Hsp70, which localize to nonmitochondrial, cytosolic, and nuclear
compartments of the cell, supports the idea that mTid-1 does not
function exclusively in mitochondria and that its biological activities
in mammalian cells may be dictated by subcellular location and its
Hsp70 partner. In this context, it is noteworthy that both hTid-1 and
Hsp/Hsc70 proteins have been implicated in cell death suppression (37,
50, 51), raising the possibility that Tid-1 proteins may modulate the
chaperone activity Hsp70 family members during the conformational
regulation of proteins involved in apoptotic signal transduction. In
any case, mTid-1 may be included among a number of
mitochondrial-targeted proteins that have been identified to function
at specific extramitochondrial locations (53).
We next reasoned that if Tid-1 is functioning as a Hsp/Hsc70 cofactor,
then conceivably some of the identified cellular partners of Tid-1 may
serve as substrates of Hsp/Hsc70 chaperones. To examine if GAP is
recruited to Hsp/Hsc70 chaperone complexes through its association with
mTid-1, we performed coimmunoprecipitation assays followed by
immunoblot analysis with anti-Hsp/Hsc70 and anti-GAP antibodies.
However, we were unable to observe an in vivo association of
Hsp/Hsc70 proteins with GAP, possibly due to the transient nature of
Hsp chaperone/substrate interactions (data not shown). This is
consistent with previous reports indicating that Hsp·substrate heterocomplexes are unstable and easily disrupted by standard cell
lysing and immunoprecipitation procedures (49, 52).
Immunolocalization of Endogenous and Ectopically Expressed
mTid-1--
To further explore the subcellular localization of Tid-1
in mammalian cells, the protein was visualized in several cell lines by
immunofluorescence microscopy. In mouse NIH 3T3 cells, mTid-1 was
detected both in the cytosol and in the nuclear compartment (Fig.
6A, panel i). The
fact that Tid-1 proteins do not possess sequences conforming to a
nuclear localization signal suggests that mTid-1 could gain entry into
the nucleus through its interactions with nuclear-bound proteins.
Indeed, hTid-1 has been reported to interact with the nuclear-localized
human papilloma virus E7 oncoprotein (18). Analysis of the
cytosolic-immunostaining pattern of mTid-1 at higher magnification
(Fig. 6A, panels iv-vi) revealed association of
mTid-1 with cytosolic organelles that morphologically resembled
mitochondria (Fig. 6A, panel vi).
Immunolocalization of Tid-1 in the human tumor cell line SAOS-2 (Fig.
6B, panel i) and primate-derived COS-1 cells
(Fig. 6C, panel i) displayed predominantly a
punctate distribution of signals throughout the cytosol. Two-color analysis using a mitochondrion-specific dye (Fig. 6B,
panels i and ii) or immunostaining with an as yet
uncharacterized integral mitochondrial membrane protein (Fig.
6C, panel i and ii) confirmed that a
large portion of Tid-1 proteins are concentrated within mitochondrial
structures. The mitochondrial localization of endogenous Tid-1 in COS-1
cells was in marked contrast to that of an ectopically expressed MYC
epitope-tagged form of mTid-1L, which exhibited diffuse
immunoreactivity throughout the cytosol (Fig. 6C,
panel iii). The presence of the MYC epitope upstream of the
mitochondrial signal sequence likely precludes signal recognition and,
thus, translocation and cleavage, resulting in a cytoplasmic-bound
protein. Indeed, Western blot analysis of lysates from COS-1 cells
expressing MYC-tagged mTid-1L with anti-mTid-1 serum
confirmed a predominance of the protein in its unprocessed, full-length
form (data not shown). Parallel immunolocalization assays of cells
expressing a nonepitope-tagged form of mTid-1L revealed a
combination of diffuse cytosolic staining and punctate foci, likely
representative of the unprocessed and mature forms of mTid-1 proteins,
respectively (Fig. 6C, panel v). The subcellular
distribution of the other two isoforms of mTid-1 transiently
transfected in COS-1 cells was indistinguishable from that of
mTid-1L-encoded proteins (data not shown). Taken together,
these data corroborate and further extend previous reports obtained
from subcellular fractionation experiments with endogenous hTid-1 and
Drosophila Tid56, demonstrating that Tid proteins are
predominantly distributed within mitochondrial fractions (37, 45).
Interestingly, immunogold electron microscopy with
Drosophila Schneider 2 cells indicated that the Tid56
protein can be found in the cytosol as well as associated with both
outer and inner mitochondrial compartments of the cell (45). Our data support the notion that the cellular background in which Tid-1 is
expressed can influence whether it resides in the cytosol, mitochondria, or is found also in the nucleus.
mTid-1 and GAP Colocalize in Response to EGF--
Based on the
evidence that mTid-1 and GAP associate in mammalian cells, we reasoned
that a portion of GAP proteins should be targeted to the same
membranous location where Tid-1 resides. To test this hypothesis, COS-1
cells were either serum-starved or treated with EGF, and the
subcellular location of Tid-1 and GAP was determined. Under serum
starvation, Tid-1 and GAP exhibited a punctate distribution throughout
the cytosolic compartment that showed some degree of overlap (Fig.
7A, panels i-iii).
Analysis of the immunofluorescent staining patterns after incubation
with EGF for 5 min (Fig. 7B, panels i and
ii) revealed extensive colocalization of GAP and Tid-1 to
common perinuclear subcellular domains (yellow stain, Fig.
7B, panel iii). The shift in GAP localization in
response to EGF is consistent with a previous study demonstrating GAP
translocates from the cytosol to perinuclear foci, suggestive of an
association with mitochondria upon EGF stimulation (40).
Conclusions--
We have identified the mouse homolog of a member
of the DnaJ family, hTid-1, as a novel p120 RasGAP-binding protein.
DnaJ proteins interact with Hsp70 chaperones to modulate their
chaperone activities in specific cellular compartments and cellular
protein processes. The variations in the intracellular localization of mTid-1 proteins and the apparent association with both cytosolic and
mitochondrial members of the Hsp70 family suggests that the in
vivo function of Tid-1 likely depends on cellular context. The
localization of mTid-1 to the mitochondria and its association with
mitochondrial Hsp70 suggests mTid-1 functions as a regulatory factor to
mitochondrial Hsp70. In this capacity, mTid-1 may cooperate with
mitochondrial Hsp70 to mediate the import and folding of proteins in
the mitochondrial matrix. Alternatively, given that both hTid-1 and
Hsp/Hsc70 proteins have been implicated as modulators of apoptosis, we
postulate that Tid-1 may cooperate with Hsp/Hsc70 chaperones to promote
changes in enzymatic activities, oligomerization states, binding
affinities, or intracellular targeting required for the activation of
signaling molecules involved in apoptotic signaling at the
mitochondrial surface.
The l(2)tid gene was originally classified as a tumor
suppressor in Drosophila in which recessive mutations lead
to malignant transformation of the imaginal discs of the larva (54).
Although TID1 has not previously been recognized as a tumor
suppressor in humans, it is tempting to speculate on its tumor
suppressor function in a mammalian setting as well. Importantly, our
data point to a potential role for mTid-1 in GAP-mediated
regulation of cell growth. In this capacity,
mTid-1·Hsp/Hsc70 heterocomplexes may govern the
conformational maturation and/or activity of GAP required for its role
as a negative regulator of Ras or as a regulator of cytoskeletal
organization (4). mTid-1 may also assist in the assembly of complexes
consisting of GAP and other signaling proteins such as
p62dok and p190 involved in GAP-directed
activities. Alternatively, the association of mTid-1 with GAP may
function to sequester GAP from the cytosol to the mitochondria, thereby
modulating its interaction with and GTPase-promoting activity towards
Ras in response to growth factor receptor activation. At any rate, one
may envision that in the absence of functional Tid proteins, GAP may
escape the regulation imposed by Hsp70 association and consequently
affect the ability of GAP to effectively down-regulate Ras, which may contribute to a hyperproliferative phenotype. Significantly, it has
been shown that loss of expression of another Ras GTPase-activating protein, neurofibromatosis-1, in neurofibroma tumors correlates with
increased levels of activated Ras (55). Additional investigations of
the biochemical function of Tid-1 and GAP complexes may improve our
understanding of how Tid-1 DnaJ proteins may exert their effects on
cell survival and cell growth.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
stimulation, whereas hTid-1L enhances the apoptogenic effects of the tumor necrosis factor
receptor (37).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-probe membrane (Bio-Rad). A 733-base pair fragment corresponding to
nucleotides 690-1482 of mouse Tid-lI cDNA labeled with
[32P]dCTP (PerkinElmer Life Sciences) was used to
probe the blot. Mouse glyceraldehyde-3-phosphate dehydrogenase served
as a quantitative control. Membranes were stripped and reprobed for
mouse glyceraldehyde-3-phosphate dehydrogenase as control.
Hybridizations were carried out according to the method of Church and
Gilbert (39).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
In vitro association between GAP
and mTid-1. M3.2 cDNA encoding mouse Tid-1 (mTid-1)
sequences was in vitro transcribed/translated using a
reticulocyte lysate-based system. [35S]Methionine-labeled
proteins derived from reactions containing M3.2 as template (M3.2) or a
control reaction (C) lacking this DNA were
immunoprecipitated (IP) with anti-mTid-1 antibodies raised
to C-terminal 91 amino acids of M3.2 open reading frame
(anti-mTid-1) or mixed with S7a lysates and
immunoprecipitated with anti-GAP antibodies (anti-GAP) or
with normal rabbit serum (NRS) as control. Immunocomplexes
were analyzed by SDS-polyacrylamide gel electrophoresis. The major
radiolabeled product corresponding to mTid-1 protein is indicated by an
arrow.
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Fig. 2.
Sequence alignments and schematic
representation of three isoforms of mouse Tid-1. A,
amino acid sequence of mouse Tid-1 (mTid-1;
GenBankTM accession number AY009320) in comparison with
Drosophila Tid56 and human Tid-1 (hTid-1).
Shaded areas mark identical amino acid residues.
Boxes highlight the mitochondrial signal sequence and the
CXXCXGXG repeats. B,
structural organization of the three isoforms of mouse Tid-1 isolated
from a mouse thymus cDNA library. Boxes highlight the
structural domains of Tid-1. The positions of amino acid residues
flanking the boundaries of each region are indicated. C,
Northern blot analysis of mTid-1 mRNA expression in various mouse
tissues. A single transcript of ~2.6 kilobases was highly expressed
in each tissue examined. Mouse glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as a loading control.
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Fig. 3.
Two major isoforms of mTid-1 are recognized
in cell lysates. A, cell lysates from Rat2 (R2) were
immunoprecipitated (IP) with either pre-immune serum
or anti-mouse Tid-1 antibodies raised against the C terminus of
mTid-1I, separated by SDS-polyacrylamide gel
electrophoresis, and immunoblotted with anti-mTid-1 serum. The
arrows indicate the 43- and 40-kDa isoforms of murine Tid-1
recognized in cells. B, additional bands of 46 and 48 kDa
were detected in osteosarcoma (SaOS-2) and MCF7 breast tumor cell
lines, respectively, by Western blot analysis. C, cell
lysates prepared from COS-1 cells transfected with long
(mTid-1L), intermediate (mTid-1I), and short
(mTid-1S) isoforms of mouse Tid-1 cDNA were also
subjected to Western blot analysis with anti-mTid-1 antibodies. The
apparent molecular size of the resulting polypeptides representing
precursor and processed forms of mTid-1 are indicated in the
margin.
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Fig. 4.
GAP associates with precursor and processed
forms mTid-1 in vivo. A, cell lysates
from Rat2 (R2) and S7a cells were immunoprecipitated (IP)
with antiserum to GAP and immunoblotted with anti-GAP and anti-mTid-1
antibodies (right panel). mTid-1 and GAP levels were
analyzed by probing aliquots (50 µg) of whole cell lysates
(WCL) with antibodies to mTid-1 and GAP, respectively
(left panel). Tyrosine phosphorylation levels of mTid-1 and
GAP were determined by Western blot analysis of R2 and S7a lysates with
antibodies to phosphotyrosine (pTyr; middle
panel). B, anti-GAP and anti-mTid-1
immunoprecipitates (IP) from S7a cell lysates were
immunoblotted with anti-phosphotyrosine antibodies (left
panel). The corresponding anti-GAP and anti-mTid-1 immunoblots of
the same membrane are shown in the right panel. C, COS-1
cells ectopically expressing mTid-1L, mTid-1I,
and mTid-1S were subjected to immunoprecipitation
(IP) and immunoblot analysis with antibodies to GAP and
mTid-1 respectively. Aliquots (50 µg) of whole cell lysates
(WCL) were probed with anti-mTid-1 antibodies to confirm
comparable expression of mTid-1L, mTid-1I, and
mTid-1S (bottom panel).
(37). Interestingly, the relative level of endogenous
mTid-1I is elevated in v-src-transformed S7a cells (Figs.
3B, 4A), consistent with the idea that v-src
might induce expression of the anti-apoptotic form of
mTid-1I. Although the biochemical function of the
mTid-1·GAP complex is not known, enhanced binding of
mTid-1I to GAP may conceivably exert inhibitory effects on
GAP activity. In this context, it is noteworthy that the persistent association of a Ydj1 mutant with v-src severely compromises the in vivo activity of the kinase without affecting the levels
of v-src in the cell (26). Alternatively, increased binding of mTid-1
with GAP may serve to sequester GAP away from Ras. Either of these
possibilities could contribute to aberrant cell growth through
dysregulation of the Ras-signaling pathway.
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Fig. 5.
Both mTid-1L and
mTid-1I interact with cytoplasmic and mitochondrial members
of the Hsp70 chaperone family. A, immunoprecipitation
(IP) and Western blotting analysis was performed on COS-1
cells using antibodies to mTid-1 and mitochondrial Hsp70 (Grp75).
B, NIH 3T3 cells were heat shock-treated at 43 °C
(+) for 1 h to induce expression of stress-inducible
cytosolic Hsp70. Cell lysates were subjected to immunoprecipitation
followed by SDS-polyacrylamide gel electrophoresis and immunoblotting
using either inducible-specific Hsp70 or mTid-1 antibodies as
indicated. C, COS-1 cell lysates prepared with or without
the addition of 10 mM ATP were immunoprecipitated with
Hsc70 antibodies and immunoblotted with anti-mTid-1 antibodies.
WCL, whole cell lysates.
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Fig. 6.
Immunofluorescence analysis of intracellular
distribution of Tid-1 proteins demonstrates cytosolic,
mitochondrial and nuclear location. A, endogenous
mTid-1 proteins were localized in NIH 3T3 cells by
immunofluorescence using affinity-purified mTid-1 antibodies and
fluorescein isothiocyanate-conjugated secondary antibody (panel
i and iv). Magnification is either 25× (panels
i-iii) or 100× (panel iv-vi) for different fields.
The arrowheads denote extensive nuclear localization of
mTid-1. Mitochondria were visualized by staining cells with
MitoTracker® (panel iii). Representative mitochondrial
staining in NIH cells with rhodamine 123 is shown in panel
vi. Similar localization studies were carried out in SAOS-2
(B) and COS-1 (C) cells. The punctated
distribution of signals within the cytosol corresponds to the staining
obtained with mitochondrion-selective dyes (A, panel
vi, and B, panel ii) or with a monoclonal
antibody (2G2) to an integral membrane protein of the inner
mitochondrial membrane and rhodamine-conjugated secondary antibody
(C, panel ii). Subcellular localization of the
precursor form of mTid-1L (C, panel
iii) or processed mTid-1L isoform (C,
panel v) was examined using COS-1 transfectants expressing
MYC epitope-tagged or untagged mTid-1L, respectively.
Transfected cells are indicated by arrowheads. Nuclei were
identified by Hoechst staining.
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Fig. 7.
Tid-1 and GAP colocalize to
perinuclear mitochondrial membranes in response to EGF. The
colocalization of endogenous Tid-1 proteins and GAP was examined in
COS-1 cells by immunofluorescence using anti-mTid-1 antibodies and
fluorescein isothiocyanate-conjugated anti-rabbit IgG
(green) or mouse monoclonal anti-GAP antibodies followed by
rhodamine-conjugated anti-mouse IgG (red). COS-1 cells were
either serum-starved for 24 h (A) or treated with 100 ng/ml EGF at 37 °C for 5 min (B). Colocalization of Tid-1
and GAP appears as yellow (A and B,
panels iii).
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ACKNOWLEDGEMENT |
---|
We thank V. Lhoták for critical reading of this manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the National Cancer Institute of Canada (to M. R-A.).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.
¶ Recipient of an Ontario graduate scholarship and a Cancer Research Society studentship.
§§ A Scholar of the Medical Research Council of Canada. To whom correspondence should be addressed: Hamilton Regional Cancer Centre, 699 Concession St., Hamilton, Ontario, L8V 5C2 Canada. Tel.: 905-387-9711 (ext. 67148); Fax: 905-575-6330; E-mail: maria. rozakis@hrcc.on.ca.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M009267200
2 X. Yin, and M. Rozakis-Adcock, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GAP, GTPase-activating protein; SH, src homology; Hsp, heat shock protein; EGF, epidermal growth factor; PBS, phosphate-buffered saline.
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