CAIR-1/BAG-3 Abrogates Heat Shock Protein-70 Chaperone Complex-mediated Protein Degradation
ACCUMULATION OF POLY-UBIQUITINATED Hsp90 CLIENT PROTEINS*
Howard Doong
,
Kathryn Rizzo
,
Shengyun Fang ¶,
Vyta Kulpa
,
Allan M. Weissman ¶ and
Elise C. Kohn
From the
Molecular Signaling Section, Laboratory
of Pathology and ¶Regulation of Protein Function
Laboratory, Center for Cancer Research, NCI, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, September 20, 2002
, and in revised form, May 1, 2003.
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ABSTRACT
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BAG family proteins are regulatory co-chaperones for heat shock protein
(Hsp) 70. Hsp70 facilitates the removal of injured proteins by
ubiquitin-mediated proteasomal degradation. This process can be driven by
geldanamycin, an irreversible blocker of Hsp90. We hypothesize that
CAIR-1/BAG-3 inhibits Hsp-mediated proteasomal degradation. Human breast
cancer cells were engineered to overexpress either full-length CAIR-1 (FL),
which binds Hsp70, or a BAG domain-deletion mutant (dBAG) that cannot bind
Hsp70. FL overexpression prevented geldanamycin-mediated loss of total and
phospho-Akt and other Hsp client proteins. dBAG provided no protection,
indicating a requirement for Hsp70 binding. Ubiquitinated Akt accumulated in
FL-expressing cells, mimicking the effect of lactacystin proteasomal
inhibition, indicating that CAIR-1 inhibits proteasomal degradation distal to
protein ubiquitination in a BAG domain-dependent manner. Protein protection in
FL cells was generalizable to downstream Akt targets, GSK3
, P70S6
kinase, CREB, and other Hsp client proteins, including Raf-1, cyclin-dependent
kinase 4, and epidermal growth factor receptor. These findings suggest that
Hsp70 is a chaperone driving a multiprotein degradation complex and that the
inhibitory co-chaperone CAIR-1 functions distal to client ubiquitination.
Furthermore, poly-ubiquitination is not sufficient for efficient proteasomal
targeting of Hsp client proteins.
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INTRODUCTION
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BAG family proteins interact with and regulate heat shock protein
(Hsp)1-70 activity in
protein folding and activation. Hsp-70 has a dual function wherein it
complexes with nascent or injured proteins and chaperones them for refolding
and resumption of function, or through which proteins are removed by
poly-ubiquitination and proteasomal degradation
(1,
2). Co-chaperone proteins that
augment or inhibit refolding and reconstitution have been identified
(35).
The BAG family proteins are Hsp70 co-chaperones and are characterized by a
common C-terminal BAG domain that interacts with the ATP-binding site of
Hsp-70 (6,
7). It is through this
interaction and by competing for binding to the ATPase domain that the BAG
family proteins, including CAIR-1/BAG-3/bis, down-regulate the refolding
chaperone properties of Hsp70
(1,
2,
5,
8). Chaperoned client proteins
are instead targeted for proteasomal degradation when the folding/re-folding
activity of Hsp proteins is interrupted. Hsp70 and Hsp90 have both been
implicated in the proteasomal degradation of chaperoned client proteins
(913).
Hsp90, in particular, has been characterized as the driver for Hsp-mediated
proteasomal degradation (9,
10). Hsp90 is a protective
chaperone when complexed with p50/immunophilin, p23, and ATP but drives client
proteins to poly-ubiquitination and proteasomal degradation when ATP-depleted
and bound in complex with Hsp70 and P60HOP
(10). The removal of client
proteins by proteasomal degradation is a potential mechanism of action for the
regulation of cell survival, proliferation, and transforming activities in
cancer and other cellular stresses and is thus a logical target for molecular
therapeutics. BAG family proteins, including CAIR-1, have been reported to
have anti-apoptotic and pro-survival activities in cultured cells
(8,
1418).
We hypothesized that CAIR-1 would function as an inhibitory co-chaperone in
Hsp70/Hsp90-mediated proteasomal protein degradation.
Geldanamycin (GA), an ansamycin antibiotic now in cancer clinical trials,
is a selective and specific inhibitor of Hsp90
(19,
20). It binds irreversibly to
the ATP-binding site of Hsp90 driving Hsp90-bound client proteins to the
degradation pathway and preventing Hsp90 from recycling into a constructive
protein chaperone mode. The current model of proteasomal degradation mediated
through Hsp interaction places the client protein initially in complex with
Hsp90, which then recruits Hsp70 and P60HOP (see Ref.
1 and reviewed in
Fig. 10). Client proteins are
ubiquitinated by ubiquitin ligases
(21), and these
poly-ubiquitinated proteins are then targeted to the proteasome for
destruction.

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FIG. 10. Model to describe CAIR-1 blockade of proteasomal degradation through
binding to Hsp70. Client protein is bound initially to Hsp90. This may be
a multimeric complex containing Hsp70 as some binding to Hsp70 is observed in
the co-precipitation time course (Fig.
3). GA treatment results in a shift of the client protein to
Hsp70, followed by its poly-ubiquitination. Poly-ubiquitinated client protein
is degraded by the proteasome in control and dBAG cells. CAIR-1 bound Hsp70
abrogates proteasomal degradation of the poly-ubiquitinated client protein and
results in retained function.
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FIG. 3. Akt shifts chaperone binding during GA exposure from Hsp90 to Hsp70.
Neo or FL cells were subjected to a time course of GA exposure (2
µM), lysed, and lysates immunoprecipitated (IP) with
anti-Akt. Parallel immunoprecipitations were blotted (IB) for Hsp90
(A) and Hsp70 (B; 1:40,000 primary with 1:100,000
anti-rabbit secondary antibody). A CAIR-1-independent shift of Akt client
protein binding from Hsp90 to Hsp70 is shown. C, Akt/chaperone
binding was quantitated and normalized against total immunoprecipitated Akt
(not shown) to correct for Akt loss in the face of GA exposure. Akt binding to
Hsp90 increased transiently to 2-fold over control, whereas binding to
Hsp70 increased over 20-fold (p = 0.024). D, FL cells had
net increase in Akt/Hsp70 binding beginning after 6 h compared with neo
controls (20-versus 12-fold, respectively, p < 0.0001).
E, Akt does not bind to Hsc70. No co-immunoprecipitation of Akt to
Hsc70 (1:10,000 primary with 1:50,000 anti-rabbit secondary antibody) was
identified under control or geldanamycin-treated conditions. Binding of Akt to
Hsp70 was confirmed.
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We evaluated the effect of CAIR-1 on the fate of a client protein known to
be degraded in response to GA to explore the role of CAIR-1 in Hsp70-Hsp90
complex-mediated proteasomal degradation. The serine/threonine kinase
Akt/protein kinase B is one of several proven Hsp90 client proteins
(22,
23). Akt propagates
anti-apoptotic and pro-survival cellular signals through phosphorylation of
its downstream partners (24,
25). We now report that CAIR-1
abrogates the GA-driven degradation of Akt, allowing for continued Akt
signaling. Whereas proteasomal degradation of Akt is markedly inhibited by
CAIR-1, the capacity of Akt to be ubiquitinated in response to GA remains.
Overexpression of CAIR-1 mimics the effect of the proteasomal inhibitor
lactacystin, preventing the loss of poly-ubiquitinated proteins, without
altering the capacity of the cell for ubiquitination. Inhibition of
proteasomal degradation, similar to Akt, was observed for other Hsp90 client
proteins. This suggests the negative co-chaperone function of CAIR-1 is a
generalizable function. Protection of ubiquitinated client proteins is
dependent upon the presence of the BAG domain, placing Hsp70 in a central
position in this multimolecular degradation complex. These findings provide a
possible explanation for the anti-apoptotic effects of CAIR-1 and suggest that
targeting CAIR-1/Hsp70 interactions may be a novel molecular target for
intervention.
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EXPERIMENTAL PROCEDURES
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MaterialsAnti-Hsp70 (catalog SPA-811), -Hsc-70 (catalog
SPA-816), -Hsp90, and -ubiquitin antibodies and Hsp70 and Hsp90 proteins were
purchased from StressGen (Victoria, British Columbia, Canada). Antibodies to
Akt, phospho (p)-Akt, p-GSK-3
, P70S6 kinase, p-P70S6 kinase, cleaved
caspase-3, CREB, and p-CREB were obtained from Cell Signaling (Beverly, MA).
Antibodies to GSK-3
, EGFR, CDK-4, Raf-1, PARP, and tubulin were from
Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA).
Monoclonal anti-His6 antibody was obtained from Clontech (Palo
Alto, CA). pGEX-4T3 was from Amersham Biosciences. The mammalian expression
vector PCIneo was from Promega (Madison, WI). Secondary antibodies, protein
A/G beads, and BCA protein quantitation kits were purchased from Pierce.
LipofectAMINE PLUS and other molecular reagents were from Invitrogen. FuGENE 6
transfection reagent was from Roche Applied Science. All other products were
of molecular or analytical grade. Geldanamycin (GA) was generously provided by
the Developmental Therapeutics Program, NCI, National Institutes of
Health.
Preparation of CAIR-1 Mutants and Stable TransfectantsThe
full-length human CAIR-1 (FL) and the BAG-domain deleted CAIR-1 (dBAG)
sequences were generated by PCR from plasmid containing the full-length CAIR-1
message, previously verified and published sequence
(26). Amplified sequence was
confirmed prior to subcloning into pCIneo for stable transfection. The
50-amino acid BAG domain deletion mutant (dBAG; deleted amino acids
445494) (14) was
engineered with an novel internal HindIII site. Both FL and dBAG
constructs are tagged with His6 at the N terminus. FL CAIR-1 was
generated using primers 1 and 2 and dBAG by ligating the N terminus (primers 1
and 3; below) to the C terminus (primers 4 and 2). Primer 1, sense,
5'-GGT CCT CGA GCC ACC ATG CAT CAC CAT CAC CAT CAC ATG AGC GCC GCC ACC
CAC-3'; primer 2, antisense, 5'-ACC GAA TTC CTA CTA CGG TGC TGC
TGG GTT ACC-3'; primer 3 antisense, 5'-ACC AAG CTT GCC TTC AAA GTT
GTC TAC AGC CTG-3'; primer 4, sense, 5'-GGT AAG CTT GAA CAG AAA
GCC ATT GAT GTC CCA-3'.
Stable clonal cell lines were established with G418 selection. Cells were
maintained in G418 400 µg/ml until split for treatment and lysis.
Transient transfection was done to confirm the findings of the single cell
clonal sublines. Briefly, MDA-435 cells were subjected to transfection using
FuGENE 6 reagent according to the manufacturer's instructions. At 24 h, cells
were washed, incubated with 2 µM GA for 18 h, and then lysed for
study. Cells were transfected with His6-CAIR-1. CAIR-1 expression
was confirmed by its His6 tag.
Lysate Production and Immunoprecipitation and GST Pull-down
ExperimentsCells were exposed to 10 µM lactacystin
and/or 2 µM GA unless otherwise indicated, with a 0.1%
Me2SO vehicle control. Total cell lysates were prepared from
subconfluent cells using modified RIPA buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 10 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 mM
Na3VO4,4mM EDTA, 10 mM NaF, 10
mM sodium pyrophosphate, 1% Nonidet P-40, and 0.1% sodium
deoxycholate) and subjected to electrophoresis, electrical transfer to
membrane, block and immunoblot, or immunoprecipitation followed by
electrophoresis and immunoblot as described
(26). 10 mM
iodoacetamide was added to the modified RIPA lysis buffer for studies of
ubiquitination, and 4 M urea was included in the sample buffer for
PARP immunoblots.
The BAG domain (amino acids 445494) was amplified,
sequence-verified, and subcloned into pGEX-4T3 for expression according to
manufacturer's instructions. Fusion proteins were immobilized on
glutathione-Sepharose 4B beads for pull downs as described
(26) using 30 µg of lysate
in 400 µl of binding buffer (20 mM Tris, pH 7.5, 100
mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40, 2 mM
dithiothreitol, 0.05% bovine serum albumin, and 5% glycerol). Complexes were
rocked at 4 °C for 2 h and then washed 5 times in 1 ml of TENNS buffer
(2.5 mM Tris, pH 7.5, 2.5 mM EDTA, 250 mM
NaCl, 1% Nonidet P-40, and 2.5% sucrose)
(26). Beads were resuspended
in sample buffer, and bound proteins were analyzed by electrophoresis and
immunoblot. All data shown represent n
3 replicates.
Heat Shock InjuryCells were plated in serum-containing
conditions and cultured overnight prior to heat shock. Culture dishes were
placed in a 44 °C water bath for 40 min after which the cells were
returned to the culture incubator. Cells were lysed at the indicated times and
subjected to immunoblot analysis for cleaved caspase-3 and PARP as described
above.
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RESULTS
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CAIR-1 Abrogates GA-induced Loss of p-Akt and Total Akt in an
Hsp70-dependent FashionAkt is a client protein of Hsp90 and is
subject to proteasomal degradation in cells exposed to GA
(22). We hypothesized that
CAIR-1 would alter the balance of the chaperone degradation system through its
binding to Hsp70. Stably transfected cell lines expressing either full-length
CAIR-1 (FL) or a BAG domain deletion mutant (dBAG), both with an engineered
His6 tag or empty vector (neo), were generated to investigate this
hypothesis (Fig. 1A).
Transfection conditions did not result in changes in expression of CAIR-1,
Hsp90, or Hsc70 (constitutive Hsp) protein (not shown) nor with 2
µM GA treatment of wild type cells for up to 18 h
(Fig. 1B). In
contrast, the previously reported GA-mediated induction of Hsp70 was
confirmed. Stable transfectants were next subjected to GA treatment, resulting
in loss of both p-Akt and total Akt in neo and dBAG cells
(Fig. 1C). Cells
overexpressing FL CAIR-1 showed a marked attenuation of GA-mediated loss.

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FIG. 1. CAIR-1 expression abrogates loss of p-Akt and Akt in GA-treated
cells. A, MDA-435 cells were constructed to clonally express
His6-tagged full-length (FL) or BAG deletion mutant
(dBAG). The presence of expressed protein is shown by
immunoprecipitation (IP) with anti-CAIR-1 and confirmation with
anti-His6. IB, immunoblot. B, GA treatment
induces Hsp70 production. Wild type MDA-435 cells treated with 2
µM GA over an 18-h time course were assessed for quantity of
Hsc70, Hsp90, endogenous CAIR-1, and Hsp70. Only Hsp70 is induced under GA
treatment conditions. C, expression of FL protects both p-Akt and Akt
from GA-mediated loss. Cells were exposed to 2 µM GA for 18 h
prior to lysis, and lysates were subjected to immunoblot as indicated.
Transient transfection with FL and dBAG versus neo confirmed these
findings.
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Hsp90, the specific molecular target for GA
(19,
20), has been proposed to be
the primary chaperone for Hsp-mediated protein degradation
(10). For this reason, the
ability of CAIR-1 to bind to Hsp90 was assessed
(Fig. 2). As no CAIR-1/Hsp90
binding was observed in immunoprecipitations of whole cell lysates (not
shown), GST pull-down constructs were engineered and tested. The CAIR-1 BAG
domain, when expressed as a GST fusion protein, selectively bound native
(Fig. 2A, left
panel) and recombinant Hsp70 (right panel) in pull-down
experiments. It did not bind endogenous or purified Hsp90 (left and
right panels, respectively; Fig.
2A, bottom). Furthermore, Hsp70
co-immunoprecipitated with FL but not dBAG from cell lysates, confirming the
requirement for the BAG domain for association of CAIR-1 with Hsp70. Finally,
commensurate with the increased quantity of Hsp70 with GA treatment
(Fig. 1B), GA
treatment also resulted in increased CAIR-1/Hsp70 immunocomplex formation
(Fig. 2C). The
dependence of the BAG domain, the site of interaction with Hsp70, and the lack
of CAIR-1 binding to Hsp90 suggest a primary role for Hsp70 in the protection
of Akt by CAIR-1.

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FIG. 2. CAIR-1 interacts selectively with Hsp70 through its BAG domain.
A, the CAIR-1 BAG domain selectively pulls down Hsp70 but does not
recognize Hsp90. Pull-down assays were done using the engineered GST-BAG
domain. GST alone was used as the negative control. Assays used either MDA-435
cell lysate (left panel) or recombinant Hsp70 (upper right
panel) or commercial purified Hsp90 (lower right panel). The
presence of Hsp70 or Hsp90 in cell lysate is confirmed in the lysate
immunoblot lane. GA exposure (2 µM, 18 h) induces Hsp70
production providing more substrate for GST-BAG domain binding, shown in the
4th lane. B, the dBAG mutant cannot co-precipitate Hsp70 from lysate.
Cells were lysed, and the lysate was subjected to immunoprecipitation
(IP) with anti-His6 and blotted for bound Hsp70. A lysate
immunoblot (IB) control is shown in the far right lane to
confirm Hsp70. C, GA treatment increases binding of Hsp70 to CAIR-1.
Wild type MDA-435 cells were treated with 2 µM GA for the
indicated times, lysed, and subjected to co-immunoprecipitation. Commensurate
with the increase in Hsp70 production (Fig.
1), increased CAIR-1-Hsp70 complex is observed with GA
exposure.
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Akt Shifts Chaperone Binding from Hsp90 to Hsp70 during GA
ExposureHsp90 client proteins have been shown to be subjected to
proteasomal degradation (22).
We confirm Akt client binding to Hsp90 prior to and during early GA exposure
(Fig. 3). Parallel time course
experiments demonstrate that Akt shifts primary chaperone from Hsp90 to Hsp70
in the same time course where Hsp70 is induced by GA. A 20-fold increase in
Akt-Hsp70 complex is seen in the face of a stable 1.52-fold increase in
Akt/Hsp90, when corrected for the loss of Akt over time (p = 0.024,
Mann-Whitney; Fig.
3C). FL cells accumulated more Akt-Hsp70 complex than neo
control cells beginning 6 h into incubation with GA (12 versus
>20-fold, p = <0.0001). This indicates that the chaperone shift
of Akt from Hsp90 to Hsp70 proceeds also in the control setting where
substantial loss of Akt is occurring. No co-precipitation of Hsp70 and Hps90
could be demonstrated, suggesting that there is a shuttling of the Akt client
protein from Hsp90 to Hsp70. Furthermore, Akt does not bind to Hsc-70 in
control or geldanamycin-treated cells (Fig.
3E), and geldanamycin does not induce Hsc70 production
(data not shown).
CAIR-1 Expression Prevents Degradation of Poly-ubiquitinated
AktCurrent models for degradation of proteins chaperoned by the
Hsp90 complex include poly-ubiquitination through Hsp70-associated ubiquitin
ligases followed by proteasomal degradation
(9). The effect of CAIR-1
expression on Akt ubiquitination in response to GA exposure was next evaluated
(Fig. 4). No notable
accumulation of poly-ubiquitinated forms of Akt were observed in the absence
of proteasome inhibition or CAIR-1 overexpression in neo vector control cells
(Fig. 4A, upper
panel). In contrast, substantial accumulation of ubiquitinated Akt was
observed in GA-treated cells expressing FL
(Fig. 4A, lower
panel). Fig. 4B
shows dBAG cells have a wild type phenotype, no accumulation of
poly-ubiquitinated Akt (Fig.
4B, lanes 37 compared with lanes
8 and 9, neo and FL, respectively). The loss in total
Akt in neo and dBAG cells compared with FL transfectants is shown in the
lower panel. No difference in total protein ubiquitination was seen
in the three cell lines arguing that there was no fundamental difference in
the general function of the ubiquitin system (data not shown). A quantitative
comparison of ubiquitinated Akt in the three cells is shown in
Fig. 4C. The increase
in ubiquitinated forms is dis-proportionate to the greater total Akt level in
GA-treated FL cells compared with those expressing either neo or dBAG. These
data suggest that GA is stimulating Akt ubiquitination and that CAIR-1 is
preventing subsequent proteasomal degradation of the ubiquitinated forms.

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FIG. 4. CAIR-1 expression permits accumulation of poly-ubiquitinated Akt.
A, accumulation of poly-ubiquitinated Akt is seen only in FL cells.
Neo (upper panel) and FL (lower panel) cells were exposed to
GA 2 µM over an 18-h time course, lysed, and immunoprecipitated
(IP) with anti-Akt and blotted (IB) for ubiquitin
(Ub). Little coprecipitate is seen in neo cells. B, BAG
domain deletion mutants do not accumulate ubiquitinated Akt. Neo (lanes
1 and 8), dBAG cells (lanes 37), and FL cells
(lanes 2 and 9) were exposed to GA 2 µM as
indicated, lysed, and evaluated for ubiquitinated Akt. No accumulation of
ubiquitinated Akt is observed in neo and dBAG cells. In contrast, FL cells
have a robust poly-ubiquitinated Akt smear at 18 h. The reduction of total Akt
is shown in the bottom panel. C, FL cells accumulate a 7-fold higher
quantity of ubiquitinated Akt. Replicate blots were scanned, and ubiquitinated
Akt was analyzed. Results are normalized to Akt ubiquitination at time 0 in
control vector neo cells. , neo; , FL; and , dBAG.
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To evaluate this further, cells were treated with the irreversible
proteasome inhibitor, lactacystin
(27). Cells were pretreated
with lactacystin, and exposure to lactacystin continued during the GA
treatment. This resulted in the accumulation of ubiquitinated forms of Akt, as
might be expected for a ubiquitination substrate, in response to GA in neo and
dBAG cells as well as in those expressing FL
(Fig. 5, A and
B). A quantitation of replicate experiments is shown in
Fig. 5C, demonstrating
the similar accumulation of ubiquitinated Akt in neo, dBAG, and FL cells.
Lactacystin prevented the GA-induced loss of Akt in neo and dBAG cells
consistent with a proteasomal route of degradation. It is possible that CAIR-1
initiates its protection of client proteins prior to interaction with the
proteasome or that it occurs within that macromolecular complex. No evidence
was found by co-immunoprecipitation studies that either CAIR-1 or Hsp70
interact directly with the regulatory or catalytic components of the
proteasome (data not shown). Thus, CAIR-1 functions to prevent degradation of
poly-ubiquitinated Akt in a BAG domain and Hsp70-dependent fashion.

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FIG. 5. Proteasomal inhibition mimics CAIR-1 overexpression. A,
cell pretreatment with lactacystin protects poly-ubiquitinated Akt from
GA-mediated loss. Cells were exposed to lactacystin for 4 h prior to and
during GA incubation. Neo (upper panel) and FL (lower panel)
cells were exposed to 2 µM GA over an 18-h time course, lysed,
and evaluated for ubiquitinated Akt. Little difference is seen between neo and
FL cells in the presence of lactacystin. IP, immunoprecipitation;
IB, immunoblot; Ub, ubiquitin. B, lactacystin
protects Akt in dBAG cells. Neo (lanes 1 and 8), dBAG cells
(lanes 37), and FL cells (lanes 2 and 9)
were exposed to lactacystin for 4 h prior to and continued during 2
µM GA exposure as indicated. Cell lysates were evaluated for
ubiquitinated Akt. Ubiquitinated Akt is observed in all cell types in the
presence of lactacystin. The total Akt control is shown in the bottom
panel. C, accumulation of ubiquitinated Akt in all cell types in the
presence of lactacystin. Replicate blots were scanned and ubiquitinated Akt
analyzed. Results are normalized to Akt ubiquitination at time 0 in control
vector neo cells. , neo; , FL; and , dBAG.
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Akt Is a Substrate for Ubiquitination in the Absence of GA
Akt has been described to be a caspase substrate
(28). It has not been shown
previously to be ubiquitinated or degraded by the proteasome under
nonpharmacologic conditions. Cells were thus exposed to lactacystin in the
absence of GA and Akt protein, and ubiquitination was assessed
(Fig. 6). Cells were exposed to
lactacystin for a total of 22 h to mimic the duration of exposure used in the
GA-driven ubiquitination experiments in Figs.
4 and
5. No difference in relative
amounts of poly-ubiquitination or total Akt was detected in neo, dBAG, and FL
cells. When exposed to GA, degradation of Akt is driven in the neo and dBAG
cells but is attenuated markedly by CAIR-1 expression in FL cells. These
studies demonstrate that Akt is a substrate for ubiquitination under
physiologic culture conditions.

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FIG. 6. Akt is a substrate for ubiquitination in the absence of GA. Cells
were incubated with lactacystin alone for 4 and 22 h to evaluate the
ubiquitination of Akt at the equivalents of time 0 and 18 h in the GA
experiments. Equivalent ubiquitination of Akt is seen in neo, dBAG, and FL
cells in the absence of GA exposure. These data indicate that Akt is a
substrate for poly-ubiquitination in the absence of the pharmacologic drive of
geldanamycin. IP, immunoprecipitation; IB, immunoblot;
Ub, ubiquitin.
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CAIR-1 Protects Akt-mediated Phosphorylation of Akt Partner
ProteinsAkt regulates the activation of multiple downstream
partners (25). Protection of
phosphorylation of the Akt downstream partners would be expected if the
function of Akt was intact. Treatment with GA in neo control and dBAG cells
resulted in loss of phosphorylated forms of the Akt downstream partner
proteins glycogen synthase kinase-3
(GSK-3
), P70S6 kinase, and
cAMP-response element-binding protein (CREB;
Fig. 7). In contrast,
phosphorylated forms were observed at basal or near basal levels in FL cells.
Some loss of total GSK-3
and P70S6 kinase protein was observed in
GA-treated cells, suggesting that these proteins may themselves be clients for
Hsp90/Hsp70 chaperoned degradation. Consistent with this possibility,
ubiquitinated GSK-3
and P70S6 kinase was detected in GA-treated FL cells
but not in neo control cells (Fig.
7B). These data show that Akt function is maintained by
overexpression of FL CAIR-1 in the GA-treated cells.
CAIR-1 Protects Other Hsp90 Client ProteinsThe Hsp70-Hsp90
multimolecular chaperone complex is a general mechanism for signal pathway
regulation through protein degradation. A number of Hsp90 client proteins have
been identified (10). To
confirm that the effect of CAIR-1 may be a general mechanism, not limited to
the Akt pathway, other well described Hsp90 client proteins were examined.
Raf-1, cyclin-dependent kinase 4 (CDK-4), and epidermal growth factor receptor
(EGFR) have been reported to be degraded upon GA treatment
(20,
22,
29,
30). Their sensitivity to GA
was confirmed in neo control cells and also observed in dBAG-expressing cells
(Fig. 8). Similar to the
findings shown for Akt and its downstream pathway partners, protection from
GA-driven degradation of all three client proteins tested was observed in FL
cells. Commensurate with this is the accumulation of poly-ubiquitinated forms
of each client protein in FL cells in the absence of lactacystin
(Fig. 8B). Therefore,
the protective effect of CAIR-1 on poly-ubiquitinated chaperone client
proteins appears to be a general means by which the chaperone degradation
pathway can be regulated.

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FIG. 8. CAIR-1 protects multiple Hsp90 client proteins. A, the
protective effect of CAIR-1 is generalizable to other Hsp90 client proteins.
Relative quantity of the well described Hsp90 client proteins, CDK-4, Raf-1,
and EGFR, was evaluated in lysates from cells exposed to 2 µM GA
for 18 h. FL expression protects all clients, whereas dBAG is ineffective.
B, FL expression results in accumulation of poly-ubiquitinated client
protein. GA-treated cells were lysed and subjected to analysis for
poly-ubiquitinated client protein as described for Akt. Accumulation of client
protein is observed only in the FL transfectants. IP,
immunoprecipitation; IB, immunoblot; Ub, ubiquitin.
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Overexpression of CAIR-1 Is Not a Global Protectant for Cell
InjuryWe next examined the effect of CAIR-1 on heat shock and
staurosporine in order to evaluate whether its protective effects are seen in
other forms of injury. Heat shock was selected as it induces Hsp70 but has no
effect on Hsp90 (31) and
staurosporine because it prevents induction of Hsp70 in response to injury and
does not involve Hsp90 (32,
33). Cells were exposed at 44
°C, returned to normal culture conditions for increasing periods of time,
and then assayed for activation of caspase-3 and cleavage of PARP as molecular
markers of injury (Fig. 9).
Overexpression of CAIR-1 protects cells from heat shock injury. Neo control
and dBAG cells had demonstrable evidence of heat shock injury as observed by a
greater number of floating cells and by a lower net total cellular protein
content in the cultures. This apoptotic injury was confirmed by the presence
of cleaved caspase-3 and cleavage of PARP selectively in neo control and dBAG
cells. All cells had induction of Hsp70 production by 6 h post-heat shock. In
contrast, treatment with 4 µM staurosporine caused visible cell
injury in all cells by 3 h into exposure. Expression of dBAG and FL CAIR-1 may
sensitize the cell to injury, as cleavage of PARP and caspase-3 appears in
those cells as early as 3 h of exposure. Akt is uniformly reduced in neo,
dBAG, and FL cells through the staurosporine exposure, and there is little if
any effect on Hsp70 and Hsp90 quantities. No binding to Hsp70 of Akt was seen
at early time points, before Akt degradation was complete. These data suggest
that CAIR-1 may be selective to forms of injury in which Hsp70 is induced
and/or involved.

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FIG. 9. CAIR-1 overexpression protects cells from heat shock injury but not from
staurosporine. A, CAIR-1 protects cells from heat shock. FL,
dBAG, and neo control cells were exposed to heat shock (44 °C for 40 min)
and then returned to culture conditions for the indicated hours. Cell lysates
were examined for the presence of cleaved caspase-3, PARP cleavage products,
induction of Hsp70, and expression of the His-tagged CAIR-1 or dBAG proteins
in respective blots. dBAG cells and neo control cells underwent apoptosis in
response to heat shock injury as demonstrated by the production of cleaved
caspase-3 and PARP cleavage. No significant difference was seen in the ability
of heat shock to induce Hsp70 production in neo, dBAG, or FL cells. Neo
control, lanes 1, 4, 7, and 10; dBAG, lanes 2, 5,
8, and 11; FL, 3, 6, 9, and 12. B, Akt
complexes with Hsp-70 after heat shock. Cells exposed to heat shock and then
allowed to recover for 18 h were lysed, and lysates subjected to
immunoprecipitation (IP) for Akt. IB, immunoblot. Akt is
bound to Hsp-70 only in FL heat-shocked cells. C, no protection from
staurosporine exposure. Cells were exposed to staurosporine 4 µM
for up to 12 h as indicated, lysed, and lysates analyzed. Progressive PARP
cleavage with commensurate loss of total PARP is seen in all cells, starting
as early as 3 h in the dBAG and FL cells. A progressive increase in caspase-3
cleavage is seen. Loss of Akt progressed equally in all cell types in the
absence of notable Hsp-70 and Hsp-90. Neo control, lanes 1, 4, 7, and
10; dBAG, lanes 2, 5, 8, and 11; FL, lanes 3,
6, 9, and 12. D, Akt/Hsp70 binding is not detectable in
staurosporine-treated cells. Co-precipitation of Akt and Hsp70 was done to
evaluate complex formation under the stress of staurosporine. Control
(lanes 13), 3-(lanes 46), and 6-h (lanes
79) exposures were tested without demonstration of the
immunocomplex. GA-treated neo (lane 11) controls confirm that the
Akt-Hsp70 complex forms in cells treated with GA. Neo control, lanes 1, 4,
7, and 11; dBAG, lanes 2, 5, and 8; FL,
lanes 3, 6, and 9.
|
|
 |
DISCUSSION
|
---|
BAG family proteins bind to and regulate Hsp70 function in protein folding
and activation (4,
7,
34). Hsp70-chaperoned
degradation protein clients are subject to ubiquitin-mediated proteasomal
degradation. Chaperoned proteasomal degradation involves a multimolecular
complex including Hsps70 and 90 and P60HOP in the chaperone component, a
client protein, and access to the ubiquitination system and the proteasome
(11,
12,
21). How and where the BAG
proteins fit into this complex and what regulatory role they have has been
unknown. We demonstrate that overexpression of CAIR-1 results in inhibition of
the degradation of poly-ubiquitinated heat shock client proteins. It is a
generalizable function under the geldanamycin exposure, neither client- nor
pathway-specific. Furthermore, protection of the index client protein, Akt,
resulted in protection of its function as shown by downstream pathway partner
phosphorylation. The CAIR-1 BAG domain is required for this protection,
indicating that the inhibitory action occurs through BAG domain-dependent
binding to Hsp70. This is similar to the reported BAG domain-dependent
inhibition of Hsp70 protein folding shown for BAG-1 and BAG-3
(4,
35). The abrogation of client
protein degradation by CAIR-1 binding to Hsp70 indicates that Hsp70 has a
prominent role in the degradation chaperone complex. This role for Hsp70 is
underscored by the demonstration of the shift in chaperone protein binding of
the Akt client protein from Hsp90 to Hsp70 occurring during the time course of
poly-ubiquitination and subsequent protein degradation. The protective effect
of CAIR-1 occurs late in the process leading to protein degradation, after
poly-ubiquitination but prior to proteasomal degradation. A schematic model
demonstrating how CAIR-1 might function in this manner is presented in
Fig. 10. Thus, CAIR-1 plays an
inhibitory function in post-ubiquitination proteasomal degradation of Hsp70
client proteins.
The findings that CAIR-1 can modulate outcome of poly-ubiquitinated
proteins further supports a previously unappreciated degree of complexity in
proteasomal targeting through Hsp70, suggesting that CAIR-1 and possibly other
members of this family disrupt proteasomal signaling. The disruption occurs
distal to protein ubiquitination and is in accord with accumulating evidence
(36,
37) that poly-ubiquitination
itself may not be sufficient to trigger proteasomal degradation.
Interestingly, another chaperone protein involved in regulation of the outcome
of poly-ubiquitinated proteins, the valosin-containing protein (VCP)/p97, is
also an ATPase. Loss of VCP/p97 function is associated with an inhibition of
ubiquitin-proteasome-mediated degradation and accumulation of ubiquitinated
proteins (37). The abrogation
of protein degradation may provide a possible explanation for the
anti-apoptotic effects of CAIR-1 that have been described. Furthermore, data
suggest that the native function of CAIR-1 may be to regulate signal protein
quantity and function under basal and stressed conditions. Finally, they
suggest that targeting CAIR-1 interactions may have utility as a pro-apoptotic
intervention in treatment of malignancy and other pathology because of
dysregulated signaling events. The demonstration that overexpression of CAIR-1
had protective effects against apoptosis due to heat shock, but not to
staurosporine, further supports its potential utility as a molecular target to
negate protection pathways in which Hsp70 is induced and/or active.
BAG domain-containing proteins act as negative regulatory co-chaperones for
folding of nascent proteins. Expression of intact and truncated BAG
domain-containing isoforms of BAG-1 have been shown to abrogate protein
folding in unfolded protein response assays and in folding of nascent proteins
expressed in vivo (4,
6,
35). Further studies indicate
that the C-terminal region of CAIR-1 containing the BAG domain, like BAG-1 and
its BAG domain, inhibited nascent client protein refolding assays in cell-free
systems and where driven by exogenous introduction of client protein, as in
luciferase reconstitution assays
(5). A regulatory role for
CAIR-1 has not been shown in protein folding experiments in intact cells, such
has been demonstrated for BAG-1 in presentation of the glucocorticoid receptor
(34,
38). These findings coupled
with our results suggest that CAIR-1 functions as a general negative
regulatory co-chaperone for Hsp70.
Hsp70 also functions as a chaperone for protein degradation, in complex
with Hsp90, P60Hop, the C terminus of Hsc70-interacting protein, CHIP, and now
CAIR-1. Hsp90 had been described as a central degradation chaperone. Our
hypothesis and subsequent findings that CAIR-1 negatively regulates this event
through a Hsp70-specific step now places Hsp70 in that central complex. The
demonstration of a client protein-chaperone protein binding shift from Hsp90
to Hsp70 during the exposure to GA supports the Hsp70-driven model and is
consistent with the described Hsp70/CHIP interaction
(12,
13). The protective effects of
CAIR-1 and its activity with a broad array of client proteins coupled with its
BAG domain-requiring activity identifies CAIR-1 as a general negative
regulator of the Hsp70-client protein complex. That the same events occurred
with heat shock, also an inducer of Hsp70 but not staurosporine which blocks
Hsp70 transcription, supports this conclusion. The BAG domain requirement and
lack of direct interaction of CAIR-1 with Hsp90 suggests either that this is a
loosely held Hsp70-Hsp90 multiprotein complex or that Hsp90 may initiate the
complex and focus the client protein down the pathway to Hsp70, where it is
subsequently ubiquitinated and degraded.
BAG domain-containing proteins have been shown to promote or be a marker of
malignant behaviors including proliferation
(15,
39),2
experimental metastasis (40,
41), inhibition of apoptosis
(8,
16,
17,
42), and to be associated with
a poor outcome in breast cancer
(4345).
Overexpression of CAIR-1 may both protect cells and support transformed cells
through its inhibition of protein degradation. The removal of client proteins
by ubiquitination and proteasomal degradation is a potential mechanism of
action for the demonstrated BAG protein enhancement of cell survival,
proliferation, and transforming activities in cancer
(15,
17,
39,
42,
46,
47), and is thus a logical
target for molecular therapeutics. We demonstrate protection of proteins key
in proliferation and survival pathways in cancer cells, including Akt and its
downstream substrates (24),
and EGFR, CDK-4, and Raf-1. The protection of Akt has broader implications in
that we now show that ubiquitination and proteasomal degradation is a
physiologic mechanism for removal of Akt. Therapeutic modulation of the
inhibitory activity of CAIR-1 may interrupt the Akt survival pathway under
selected stresses. CAIR-1 has been shown to function as an anti-apoptotic
protein in heat shock (Fig. 9)
and in a BAX-driven background
(8) but is not a general
protectant as shown by the sensitivity of the FL cells to staurosporine. In
the BAX studies, transient expression of CAIR-1 on the BAX background was
sufficient to reduce apoptosis in a fashion similar to that seen with
co-expression of BAX and BCL-2. Co-expression of sub-threshold quantities of
BCL-2 and CAIR-1 in that study similarly abrogated BAX-induced apoptosis,
showing additivity of these two proteins.
We3 and others
(5,
8) have shown that CAIR-1 and
BCL-2 are binding partners, which may account for the synergism between CAIR-1
and BCL-2 shown in the BAX model. It also has been reported that CAIR-1
provides a survival signal in serum-deprived cells
(42).
The data presented herein coupled with the heat shock and BAX/BCL-2 results
argue that CAIR-1 may function in more than one pathway to regulate cellular
response to pro-apoptotic events. The lack of effect in the
staurosporine-treated cells shows the selectivity of the CAIR-1 activity to
the Hsp70-associated pathways and indicates that it is not a global
protectant. The degradation promoting tool used in this study, GA, is now in
clinical trials as an anti-cancer agent. The demonstrated blockade of
GA-mediated protein degradation identifies CAIR-1 as a putative
chemoresistance protein that may block the anti-cancer effect of GA in
vivo. For these collective reasons, CAIR-1 is a logical molecular target
to interrupt for cancer treatment.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence may be addressed: Molecular Signaling Section,
Laboratory of Pathology, 10 Center Dr., MSC 1500, Bethesda, MD 20892-1500.
Tel.: 301-402-2726; Fax: 301-480-5142; E-mail:
ek1b{at}nih.gov.
1 The abbreviations used are: Hsp, heat shock protein; GA, geldanamycin; FL,
full-length CAIR-1; dBAG, BAG domain deleted CAIR-1; CREB, cAMP-response
element-binding protein; PARP, poly-ADP ribose polymerase; GSK3
,
glycogen synthase kinase 3
; GST, glutathione S-transferase;
p-Akt, phospho-Akt; EGFR, epidermal growth factor receptor; CDK-4,
cyclin-dependent kinase 4. 
2 E. C. Kohn and H. Doong, unpublished results. 
3 H. Doong and E. C. Kohn, unpublished data. 
 |
ACKNOWLEDGMENTS
|
---|
We thank A. Vrailas for technical assistance and Drs. L. Liotta and G.
Mills for suggestions and critical review.
 |
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