Previous Article | Next Article 
Molecular and Cellular Biology, March 1999, p. 1661-1672, Vol. 19, No. 3
Department of Physiology, Tufts University
School of Medicine, Boston, Massachusetts
02111,1 and Fox Chase Cancer Center,
Philadelphia, Pennsylvania 191112
Received 22 July 1998/Returned for modification 1 September
1998/Accepted 25 November 1998
Genetic screens in Drosophila have identified
p50cdc37 to be an essential component of the
sevenless receptor/mitogen-activated kinase protein (MAPK) signaling
pathway, but neither the function nor the target of
p50cdc37 in this pathway has been defined. In
this study, we examined the role of p50cdc37
and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling
biochemically. We found that coexpression of wild-type
p50cdc37 with Raf-1 resulted in robust and
dose-dependent activation of Raf-1 in Sf9 cells. In addition,
p50cdc37 greatly potentiated v-Src-mediated
Raf-1 activation. Moreover, we found that
p50cdc37 is the primary determinant of Hsp90
recruitment to Raf-1. Overexpression of a
p50cdc37 mutant which is unable to recruit
Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by
growth factors. Similarly, pretreatment with geldanamycin (GA), an
Hsp90-specific inhibitor, prevented both the association of Raf-1 with
the p50cdc37-Hsp90 heterodimer and Raf-1 kinase
activation by serum. Activation of Raf-1 via baculovirus coexpression
with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by
dominant negative p50cdc37 or by GA. Thus,
formation of a ternary Raf-1-p50cdc37-Hsp90
complex is crucial for Raf-1 activity and MAPK pathway signaling. These
results provide the first biochemical evidence for the requirement of
the p50cdc37-Hsp90 complex in protein kinase
regulation and for Raf-1 function in particular.
The mitogen-activated protein kinase
(MAPK) phosphorylation cascade, composed of Raf kinase, Mek (MAPK
kinase), and Erk (MAPK) itself, relays proliferative and
differentiative signals from the plasma membrane to the transcriptional
and cell cycle progression machinery (38). Although it is
established that Ras-GTP is required to tether Raf-1 to the plasma
membrane (reviewed in reference 1), the subsequent
events that lead to Raf-1 activation are poorly understood. The major
reasons for this are (i) only a small fraction (~3%) of the total
Raf-1 cytoplasmic pool needs to become activated for effective
signaling (23) and (ii) the entire process of Raf-1 plasma
membrane recruitment and activation is rapid and transient (for
reviews, see references 37 and
45). Thus, identification of both crucial
intermediates and the causative relationships in Raf-1 activation has
been difficult. However, it is clear that the N-terminal domain of
Raf-1 acts to repress the activity of the C-terminal kinase domain and
that its deletion results in constitutive activation of the kinase
(25, 68). Phosphorylation of Raf-1 and association with
other proteins in response to receptor activation most likely leads to
a conformational change in Raf-1 that relieves this repression
(37, 45).
Raf-1 fractionated from various cell types exists in large (300- to
500-kDa) multiprotein complexes (78). Known Raf-1-associated proteins include 14-3-3, Hsp90, and pp50, a 50-kDa Hsp90-associated protein (45, 78). 14-3-3 is required for Raf-1 function but probably is not directly involved in the Raf-1 activation process (37, 42, 44). The function of the pp50-Hsp90 complex in Raf-1 activation has yet to be addressed. pp50 had previously been
widely found in Hsp90-containing kinase complexes, notably involving
v-Src (reviewed in reference 4), and with both
cytoplasmic and membrane localized Raf-1 (66, 78).
Hsp90-associated pp50 has recently been identified immunologically and
by peptide mapping to be the 50-kDa gene product of the mammalian Cdc37
homologue p50cdc37 (51).
Cdc37 was originally identified in yeast as a cell cycle mutant that
gives a G1 cell cycle arrest phenotype (56).
Cutforth and Rubin (8) subsequently isolated an allele of
Drosophila Cdc37 (Dcdc37) that functioned as a dominant
enhancer of the sevenless phenotype in the
Drosophila eye. However, these genetic experiments have not
identified where and how Dcdc37 functions in the
sevenless mitogen-activated protein kinase (MAPK)
pathway. Vertebrate Cdc37 was cloned first from chicks (21,
27) and subsequently from mammals (20, 33, 50, 51,
69). The structure of Cdc37 reveals no significant homologies to
proteins of known function. The yeast protein is homologous to
mammalian and Dcdc37 through only the first 30 amino acids and diverges
significantly thereafter. Despite this limited homology, Dcdc37 will
complement the yeast gene (8). The cell cycle phenotype of
cdc37 appears to be due to a diminished capacity of
G1 cyclins and the cyclin-dependent kinase Cdc28 to
associate (19). Subsequent work by ourselves and others has
found that mammalian p50cdc37 interacts with
Cdk4 and accumulates Hsp90 to it (9, 20, 33, 69). Though
p50cdc37 has been found to interact with diverse
kinase families, its interactions are selective in that, for instance,
among cyclin-dependent kinases, it interacts with Cdk4 and the closely
related Cdk6 but not with Cdk2 (9, 28, 69). Thus, from
genetic studies, Cdc37 appears to operate in both the cell cycle and
the Ras/Raf/MAPK pathway in close cooperation with its Hsp90 chaperone
partner (28).
Hsp90 is an abundant and highly conserved protein (54) that
is essential in yeast and Drosophila (2, 8).
Unlike the more general Hsp70 and Hsp60 chaperones, Hsp90 appears to
have substrate-specific folding activity (30, 47, 54). It
has been best characterized for its essential role in steroid hormone receptor signaling, where it interacts with and modulates receptor function through a dynamic and regulated series of interactions with a
defined set of chaperone cofactors (54, 65). Hsp90's conformation and activity have been proposed to be regulated by nucleotide binding, and its associations and activity can be inhibited by geldanamycin (GA) an Hsp90-specific antibiotic which competes for
ATP binding to Hsp90 (22, 55). It has been further proposed that p50cdc37 may serve to target Hsp90 to a
subset of protein kinases and thereby help them achieve an active
conformation (28, 53). However, the distantly related yeast
Cdc37p by itself has been shown to have chaperone activity in vitro
(32).
The available mammalian association data (63, 66, 78),
although not informative about the functional significance of Raf-1
association with Hsp90 and p50cdc37,
nevertheless are complemented by genetic evidence from
Drosophila. Cutforth and Rubin (8) found that
Hsp90 mutations enhance the sevenless phenotype in the
Drosophila eye as does Dcdc37 and thus also functions in the
MAPK pathway. Subsequently, van der Straten et al. (76)
identified Hsp90 alleles that suppress the multiple R7 phenotype caused
by the constitutive high-level activation of a membrane-targeted D-Raf
kinase domain (RaftorY9). In fact, the two Hsp90 point
mutations recovered in this screen were the strongest dominant
suppressors of the multiple R7 photoreceptor cell phenotype caused by
the Ras-independent, activated Torso RTK-Raf chimeric protein.
Importantly, the mutant Hsp90 proteins identified in these genetic
screens exhibited reduced binding to D-Raf-1 and correlated with
diminished Raf kinase activity (76). Thus, neither deletion
of the N-terminal suppression domain nor membrane anchoring bypasses
the requirement of D-Raf-1 for Hsp90 association.
Here, we have addressed directly the biochemical role of
p50cdc37 and its partner, Hsp90, during Raf-1
activation and signaling to Mek and Erk. We found that
p50cdc37 and Hsp90 each interact directly with
Raf-1 but that p50cdc37 is the main determinant
of the assembly of heterotrimeric complex. Disruption of the
Raf-1-p50cdc37-Hsp90 ternary complex with the
Hsp90 inhibitor GA or with a dominant negative
p50cdc37 inhibits Raf-1 activity. Serum
stimulation promotes Raf-1-p50cdc37-Hsp90
complex formation and coexpression of p50cdc37
with Raf-1 in insect cells is sufficient to activate Raf-1. Moreover, p50cdc37 synergizes with Src for Raf-1
activation. Our data, coupled with the aforementioned genetic studies,
indicate that p50cdc37 and Hsp90 are critical
components of the MAPK cascade and of the Raf-1 activation complex in particular.
Cell culture and transfections.
Cos-1 cells were maintained
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum (FBS) and 0.1 µg of penicillin and streptomycin
per ml. Freshly plated cells were transfected at 70 to 80% confluence
with a total of 7.5 µg of DNAs per 100-mm-diameter dish, using
Lipofectamine (Life Technologies) or Targefect (Targetting Systems, San
Diego, Calif.). In experiments requiring replicate transfected
cultures, cells were split 24 h after the start of transfection
into appropriate smaller dishes so that 20 to 24 h later cultures
would have achieved confluence. At this point, cells were serum starved
for an additional 16 to 18 h. For stimulations, serum (at 20%) or
epidermal growth factor (EGF; 100 ng/ml) was directly added for 5 more
min before cells were lysed. A 2-mg/ml stock solution of geldanamycin
GA in dimethyl sulfoxide (DMSO) or DMSO alone was diluted 1:1,000 in
the culture media for the times indicated before cells were either
lysed directly or serum stimulated. Solubilized cell extracts were then
quantitated for protein content by the Bradford assay and analyzed by
direct Western blotting or by protein purification using antibodies or, for overexpressed glutathione S-transferase (GST) fusion
proteins, by glutathione (GSH)-Sepharose chromatography, followed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Baculovirus infection and culture of
Spodoptera frugiperda Sf9 cells was performed essentially as
described by Morrison (43). Unless otherwise indicated, all
baculoviruses were infected at comparable levels of multiplicity of
infection (MOI).
Antibody reagents.
The anti-p50cdc37
antibodies were raised in rabbits against the chick (pNG13 clone
[21]) or human GST-p50cdc37
protein. Anti-epitope tag antibodies obtained from Boehringer (antihemagglutinin [anti-HA] 12CA5 and anti-Myc 9E10) or from Kodak
(anti-FLAG M5). Santa Cruz Biotechnology was the supplier for
additional antibodies, including ones against Raf-1 (C-12) and GST
(Z-5). Monoclonal antibodies against Raf-1 and
p50cdc37, used in the experiment described in
Fig. 1B, were purchased from Transduction Laboratories. Anti-active
MAPK polyclonal antibody V6671 was obtained from Promega, and
antibodies directed against Hsp90 (SPA-830 and SPA-771) and recombinant
human Hsp90 purified from Escherichia coli (SPP-771) were
obtained from Stressgen.
Cloning and constructs.
For eukaryotic expression, the
complete open reading frame for the human
p50cdc37 cDNA was subcloned by PCR into the
EcoRI sites of pMT3 and pSG5 vectors and in frame with
N-terminal HA and FLAG, respectively, peptide epitopes. Similarly,
GST-p50cdc37 constructs were placed by PCR into
the BamHI-NotI sites of the pEBG eukaryotic
(57) and pGEX2T (Pharmacia) prokaryotic expression vectors.
For expression in insect (S. frugiperda Sf9) cells, the entire open reading frame for the FLAG-p50cdc37
fusion protein was subcloned from the pSG5 constructs into the EcoRI/NotI sites of the pFASTBAC1 (Life
Technologies) baculovirus vector. Deleted versions of the
FLAG-p50cdc37 fusion protein were produced by
using appropriate enzyme digestion of the full-length inserts in pSG5,
followed by agarose gel electrophoresis and DNA religation and further
subcloned into pFASTBAC1 by the same approach. Cloned inserts were
verified by DNA sequencing. Expression plasmids for Raf-1, Ras, and
v-Src used in this study have been described previously (14, 35,
46, 63).
In vitro synthesis of radiolabeled
p50cdc37.
Different full-length and deletion
forms of p50cdc37 were transcribed and
translated in vitro from the pSG5 expression constructs in the presence
of 20 µCi of [35S]methionine (EXPRESS protein labeling
mix; NEN), using the coupled rabbit reticulocyte lysate and T7 RNA
polymerase system (Promega).
Metabolic labeling.
Nontransfected or transfected cells 48 to 60 h posttransfection were initially incubated for 2 h in
methionine-free medium containing 2% dialyzed fetal serum and then
labeled for 4 h with [35S]methionine (NEN) in fresh
medium. Cells were then lysed, and equal amounts (counts per minute) of
labeled lysate were immunoprecipitated, as described below for
nonlabeled lysates, and analyzed by SDS-PAGE and fluorography.
Immunoprecipitation and immunoblotting.
Cells were harvested
48 to 60 h after transfection and extracted in Nonidet P-40 lysis
buffer (NP-40 LB; 0.5% NP-40, 20 mM HEPES [pH 7.5], 0.1 M NaCl, 2 mM
EGTA, 10% glycerol, 50 mM glycerophosphate, 2 mM dithiothreitol
[DTT]) containing protease and phosphatase inhibitors (2 mM sodium
vanadate, 1 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 10 µg each
of leupeptin and aprotinin per ml). For measuring Raf-1 kinase activity
in Sf9 cells in the experiments represented in Fig. 4, 5B, and C, and
6A, NP-40 LB was substituted with radioimmunoprecipitation assay (RIPA)
buffer (20 mM Tris [pH 8.0], 137 mM NaCl, 10% [vol/vol] glycerol,
1% [vol/vol] NP-40, 0.1% [wt/wt] SDS, 0.5% sodium deoxycholate, 2 mM EDTA). Cell lysates were cleared by centrifugation at 4°C for 15 min. The protein concentration was measured with a kit from Bio-Rad and
normalized for all samples in each individual total Western or
immunoprecipitation (IP) experiment. Equivalent aliquots of cleared
supernatants were mixed with Laemmli SDS-loading buffer (25 mm Tris
[pH 6.8], 1% SDS, 2.5% Protein purification and in vitro association assays.
GST
fusion proteins were produced and purified by GSH-Sepharose affinity
chromatography in NETN buffer (20 mM Tris, [pH 8.0], 0.1 M NaCl, 1 mM
EDTA, 0.5% NP-40) supplemented with proteinase and phosphatase
inhibitors as previously described (64). Kinase-defective bacterial His6-Mek-1 (K97M) was similarly prepared, using a
kit from Qiagen. FLAG-p50cdc37 was
immunoaffinity purified by agarose-cross-linked anti-FLAG monoclonal
antibody M2 (Kodak) according to the supplier's instructions. For
studying in vitro associations, GSH-Sepharose-bound GST fusion proteins
were then directly incubated with either purified or in
vitro-translated proteins in NETN buffer for 2 h at 4°C. Bound complexes were subsequently washed three times in 50 volumes of prechilled NETN buffer, and after SDS-PAGE they were either
immunoblotted or, for [35S]methionine-labeled proteins,
directly analyzed by fluorography.
Protein kinase assays.
For kinase reactions,
GSH-Sepharose-bound GST fusion proteins or immunocomplexes, prepared as
described above, were additionally washed in 50 volumes of kinase
buffer (25 mM HEPES [pH 7.5], 10 mM MgCl2, 10 mM
MnCl2, 1 mM DTT), drained, and incubated for 15 min at
30°C in 30 µl of fresh kinase buffer containing 20 µM ATP, 5 µCi of [ p50cdc37 bridges Hsp90 to Raf-1.
Previously Hsp90 and p50cdc37 were detected by
immunological methods in a complex with Raf-1 (51, 66, 78).
Here we have used cloned p50cdc37 and Raf-1
proteins to reconstitute and further characterize the precise
interactions among p50cdc37, Hsp90, and Raf-1.
Cos-1 cells express Raf-1, which is the principal Raf isoform
(16), and both Hsp90 and p50cdc37. In
accordance with previous findings for other tissues (11, 12, 34,
78), two proteins of approximately 90 and 50 kDa coprecipitate
with endogenous Raf-1 in Cos-1 cells (Fig.
1A). Subsequent disruption of the complex
and a second round of IP with anti-Hsp90 and
anti-p50cdc37 antisera indicates that these two
coprecipitating proteins are immunologically related to Hsp90 and
p50cdc37, respectively (Fig. 1A, lanes 1 to 5).
The converse experiment precipitating first with
anti-p50cdc37 antibodies shows stoichiometric
coimmunoprecipitation with Hsp90 but reveals only a faint Raf-1 band at
the expected 74-kDa range (lanes 6 to 8). This is probably due to the
fact that although a significant proportion of Raf-1 protein is bound
to p50cdc37 and Hsp90 (19a, 34, 60,
78), only a fraction of p50cdc37, which is
present in excess over Raf-1 (not shown) and Hsp90 (1 to 2% of total
cytosolic protein), is in a complex with the kinase. Our findings with
[35S]methionine-labeled proteins (Fig. 1A, lanes 6 to 8)
and by silver staining (not shown) indicate that Hsp90 copurifies in
approximately equimolar quantities with p50cdc37
and that the p50cdc37-Hsp90 interaction also
occurs in vitro in the absence of other proteins (63).
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
p50cdc37 Acting in Concert
with Hsp90 Is Required for Raf-1 Function
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-mercaptoethanol, 0.5 mg of bromophenol
blue per ml, 5% glycerol), separated by SDS-PAGE, and transferred to a
Hybond-ECL membrane (Amersham). Following preclearing, IP was performed
for 2 h at 4°C, using 0.5 µg of purified anti-FLAG,
anti-c-Myc, anti-HA monoclonal antibody or indicated purified rabbit
polyclonal antisera. Immune complexes were then recovered by binding to
GammaBind-Plus Sepharose (Pharmacia). Alternatively, GST fusion
proteins were purified using preequilibrated GSH-Sepharose (Pharmacia)
as described elsewhere (64). After three washes with 50 volumes lysis buffer, GSH-Sepharose-bound proteins and immunocomplexes
were processed for electrophoresis as described above. The entire
protein purification procedure was done at 4°C. Immunoblot detection
was performed with specified antibodies in 5% dried milk in
phosphate-buffered saline and developed as described by the
manufacturer of the enhanced chemiluminescence (ECL) system (Amersham).
For reblotting, membranes were incubated in 20 mM DTT-1% SDS in
phosphate-buffered saline for 10 min at ambient temperature.
-32P]ATP (6,000 Ci/mmol; NEN), and 0.5 µg
of recombinant kinase-defective His6-Mek-1(K97M). Assays
were terminated by the addition of Laemmli SDS-loading buffer, the
boiled samples were resolved by SDS-PAGE, and phosphorylated substrate
proteins were quantitated by phosphorimager analysis and autoradiography.
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
View larger version (20K):
[in a new window]
FIG. 1.
Association of p50cdc37, Hsp90,
and Raf-1 in vivo and in vitro. (A) Lane 1, anti-Raf-1 IP from
[35S]methionine-labeled Cos-1 cells. Lanes 2 to 5, after
the primary anti-Raf IP was boiled for 2 min in the presence of 0.5%
SDS, a second IP was carried out with anti-Hsp90 or control (c)
antibody (lanes 2 and 3) or with polyclonal
anti-p50cdc37 or nonimmune rabbit (c) antibody
(lanes 4 and 5, respectively). Lanes 6 and 7, anti-p50cdc37 primary IPs and nonimmune rabbit
serum IPs, respectively, from [35S]methionine-labeled
Cos-1 cells. A second IP with anti-Hsp90 antibody (lane 8) was
performed with a fraction of the anti-p50cdc37
primary immunoprecipitate identical to that run in lane 6. The relative
migration of molecular weight marker proteins is indicated. (B)
Plasmids pMT3-HA-p50cdc37 and pMT3-HA were
transiently transfected into Cos-1 cells, and extracts were
immunoprecipitated with anti-FLAG antibody (Ab) M5 as a control (lane
1) or anti-HA monoclonal antibody 12CA5 under either denaturing or mild
conditions (RIPA or NP-40 LB buffer; lanes 2 and 3, respectively) or,
to purify endogenous Raf-1 and p50cdc37
proteins, with anti-Raf-1 (lane 4) and
anti-p50cdc37 (lane 5) monoclonal antibodies.
Immunoprecipitated proteins were examined by Western blotting (WB) and
ECL for the presence of transfected HA-p50cdc37
with anti-HA antibody or for the presence of both transfected and
endogenous p50cdc37 with
anti-p50cdc37 rabbit antisera. Endogenous Raf-1
and Hsp90 proteins were detected with rabbit-anti-Raf-1 antibodies and
rat-anti-Hsp90, respectively (top to bottom panels). IgGH,
precipitating IgG antibody heavy chains. (C)
FLAG-p50cdc37 (immunoaffinity purified from
baculovirus-infected Sf9 cells) and Hsp90 (recombinant E. coli; Stressgen) were assayed in vitro for binding to bacterially
produced GST-Raf-1, GST-p50cdc37, or GST alone
as indicated by GSH-Sepharose pull-down assays and Western blotting
(WB) with the indicated antibodies as described in Materials and
Methods. Anti-Hsp90 immunoblotting performed with two distinct
Hsp90-specific antibodies (SPA-830 and SPA-771) is shown (bottom two
panels). The first two lanes indicate the input amounts of purified
proteins added. The arrowhead denotes the position of the full-length
GST-Raf-1 above the breakdown products.
|
N-Raf-1, a viral Raf form-like construct (3, 63), but not to GST alone (Fig. 2A) or to the N-terminal Raf-1 regulatory domain alone (not shown). This interaction of p50cdc37 with Raf-1 occurs via the N-terminal
half of p50cdc37, as a deletion mutant
(p50cdc37C) truncated at Met164 to half the
original size is sufficient to interact strongly with GST-N-Raf-1.
Interestingly, p50cdc37C is severely
compromised in its ability to associate with Hsp90 in transfected Cos-1
cells (Fig. 2B) compared with full-length p50cdc37 which readily associates with its
chaperone partner.
We then sought to determine whether this mutant could disrupt the
Hsp90-Raf-1 association in a dominant fashion. When
p50cdc37C was further coexpressed in Cos-1
cells with GST-tagged Raf-1, endogenous Hsp90 association to Raf-1 was
strongly inhibited in a dose-dependent manner, with increasing amounts
of p50cdc37C binding to the kinase (Fig. 2C).
In contrast, overexpressed wild-type p50cdc37
not only binds to Raf-1 but also recruits Hsp90 to the complex, in
agreement with results of the in vitro experiment shown in Fig. 1C. A
likely interpretation of this observation is that overexpressed p50cdc37C competes with endogenous
p50cdc37 for binding to Raf-1 and that the
subsequent Hsp90 association with GST-Raf-1, which largely depends on
intact p50cdc37, is prevented (Fig. 2C; compare
lanes 1, 3, and 5). Thus, although some direct Hsp90 binding to Raf-1
cannot be ruled out (Fig. 1C, lane 4), we conclude that the
p50cdc37 greatly potentiates Hsp90 accumulation
into the Raf-1 complex (Fig. 2D) most likely by bridging Hsp90 to
Raf-1. This result also suggests that
p50cdc37C might interfere with the function
of Hsp90 in the Raf-1 complex and potentially acts as a dominant
negative allele of p50cdc37 in functional assays
(described below).
Inability of Raf-1 to respond to serum activation correlates with
its inability to complex with p50cdc37-Hsp90
heterodimers.
GA, a benzoquinone ansamycin (10), was
originally described as a protein kinase inhibitor (74).
However, subsequent examination has shown that its effects on kinases
are indirect and that it specifically binds to and inhibits the action
of Hsp90 (80, 81). GA has been established to be a specific
reagent for assessing Hsp90's role in various signaling systems,
including v-Src (80), Raf-1 (60, 61), Lck
(24), heme-regulated eukaryotic initiation factor 2
kinase (75), and steroid nuclear receptors (31, 65) (reviewed in references 52 and
58). GA competitively displaces ATP and locks Hsp90
into its ADP-specific inactive conformation, disrupting a dynamic
equilibrium in which unliganded steroid receptor complexes alternate
among various chaperone heterocomplex intermediates (22, 31, 55,
65). GA-bound Hsp90 is then unable to form productive complexes
with its steroid receptor and kinase targets, which subsequently
results in their degradation upon prolonged in vivo GA treatment
(59, 60, 62, 80). In an attempt to define the roles of
p50cdc37 and Hsp90 in Raf-1 kinase heterocomplex
formation and activity, we used GA to abrogate Hsp90-Raf-1 association
and Raf-1 activation as has been shown by Schulte et al. (60,
61). However, to directly correlate Raf-1's ability to interact
with p50cdc37 and Hsp90 with its kinase
activity, we have designed our experiments to assess the effects of GA
on Raf-1 at a stage prior to the time when Raf-1 is depleted from the
cells due to prolonged GA treatment. In addition, to improve the
detection of associated proteins, we have alternatively used GST fusion
cDNAs of Raf-1 or p50cdc37 transiently
transfected in mammalian cells. GSH-Sepharose-purified GST-Raf-1 and
GST-p50cdc37 were then analyzed both for
associated proteins and for kinase activity (57, 64).
C further confirm the requirement for
Hsp90 association with Raf-1 independently of effects on Raf-1 protein
degradation (see below).
|
C does not bind Hsp90 but can
nevertheless still bind to Raf-1, this finding implies that the
GA-bound conformation of Hsp90 inhibits the ability of bound
p50cdc37 to associate with Raf-1 through either
steric hindrance, allosteric regulation, or an indirect mechanism.
p50cdc37 and Hsp90's respective associations
with endogenous Raf-1 also showed small but reproducible serum-mediated
enhancement and almost complete elimination by GA (Fig. 3B lanes 4 to
6). Thus, during serum activation of Raf-1, there is a stabilization of
p50cdc37-Hsp90-Raf-1 complex formation. A weak
associated MAPKKK activity could be detected in
GST-p50cdc37 pull-down-in vitro kinase assays
from cells coexpressing exogenous Raf-1 (not shown), consistent with
both our observation that the bulk of p50cdc37
is not Raf-1 associated (Fig. 1A) and the fact that only a small fraction of Raf-1 kinase actually becomes activated during signaling (23, 37, 45). A previous related study (78) using
standard antibody-based Raf-1 purification found no changes in
endogenous p50cdc37 and Hsp90 coprecipitating
with active and inactive transfected Raf-1. The availability of cloned
p50cdc37, including a new array of
Cdc37-specific antibodies, enabled us to perform reciprocal
GST-p50cdc37 and GST-Raf-1 pull-down assays.
Further, the antibody-free method of isolation allowed us to use
higher-stringency GST-protein purification for more accurate assessment
of changes in endogenous Raf-1 and p50cdc37
complexed with GST-p50cdc37 and GST-Raf-1,
respectively. This, especially in the case of p50cdc37, which on SDS-PAGE migrates closely
with immunoprecipitating antibodies, is, as we also find, technically
difficult. We have also observed that coexpression of one GST-tagged
protein with a non-GST-tagged version of the other improves further the
detection of an increase in Raf-1-p50cdc37
association during serum Raf-1 activation (not shown; see Fig. 3C).
In addition to its effects on serum activation of Raf-1, in experiments
similar to the one shown in Fig. 3A, we found that GA also inhibits
Raf-1 activity driven by cotransfected Ras(Q61L), a constitutively
active Ras mutant (not shown). This result indicates that inhibition of
Raf-1 by GA occurs downstream of Ras, in agreement with the original
observations of Schulte et al. (60, 61), who found that GA
had no effect on Ras levels and on Raf-1-Ras-GTP interaction. We have
further observed that as with serum induction, activated Ras
potentiates Raf-1 association with the p50cdc37
complex (Fig. 3C; compare lanes 3 and 4 and lanes 5 and 6), but in the
presence of GA, this association is entirely abolished (lanes 1 and 2)
although the p50cdc37-Hsp90 association again
remained unaffected (not shown). Altogether, the above results suggest
that Raf-1's ability to respond to upstream activating stimuli
correlates with its ability to form heterotrimeric complexes with
p50cdc37 and Hsp90.
Activation of Raf-1 by p50cdc37 overexpression. The Sf9 insect cell-baculovirus expression system is currently the most widely used in vivo system for evaluating potential Raf-1 activators (reviewed in references 43 and 44). Therefore, we used this system to further analyze the possible involvement of p50cdc37 in the Raf-1 activation process. Baculoviruses expressing full-length p50cdc37 and Raf-1, together or in triple combinations with v-Src- or v-Ras-expressing baculoviruses (Fig. 4A), were used to coinfect Sf9 cells. At 48 h postinfection, Raf-1 was immunoprecipitated from Sf9 cells in RIPA buffer and subsequently assayed for its ability to phosphorylate inactive recombinant Mek-1. Consistent with previous reports (reviewed in reference 44), v-Src and, to a lesser extent, v-Ras both activate Raf-1, an effect most prominent when the two oncoproteins are coexpressed (Fig. 4A, lanes 1 to 4). Surprisingly, p50cdc37, a unique protein with no apparent kinase or other recognizable enzymatic domain, by itself strongly activated Raf-1 to an even greater extent than v-Ras and almost as well as, although never better than, v-Src. In coinfected combinations, the p50cdc37-v-Src effect was synergistic (compare lanes 2, 5, and 6), but only modest cooperation was observed between p50cdc37 and v-Ras (lanes 3 and 7). The cooperation of p50cdc37 with v-Src and its dose-dependent activation of Raf-1 are shown even more clearly in the dose-response experiment shown in Fig. 4B.
|
Inhibition of Raf-1 activation by dominant negative
p50cdc37 and GA.
Since the deletion mutant
p50cdc37C fails to bind to both mammalian and
insect Hsp90, we sought to determine whether this mutant might
interfere with Raf-1 activity by displacing the wild-type insect
p50cdc37-Hsp90 complex from Raf-1 since it
retains the ability to bind to Raf-1 (Fig. 2). In the experiment shown
in Fig. 5A, we attempted to correlate the effects of
p50cdc37C on Raf-1 activity with its
aforementioned ability to displace the full-length
p50cdc37 protein upon overexpression (Fig. 2C).
Previously it has been found that endogenous insect Hsp90 and
p50cdc37 associate with overexpressed mammalian
Raf-1 in Sf9 cells (11, 12). However, since our
p50cdc37 antibodies fail to recognize
p50cdc37 from insect cells, Sf9 cells were
coinfected with baculoviruses expressing mammalian
p50cdc37 and Raf-1 alone or with increasing
amounts of a baculovirus expressing p50cdc37C. Extracts of infected cells were
then immunoprecipitated with anti-Raf-1 and analyzed for associated
mammalian p50cdc37 proteins and Hsp83, the
endogenous insect homologue of Hsp90 (8), as well as for
Raf-1 kinase activity. Figure 5A shows that, as we had previously observed in mammalian cells (Fig. 2C), p50cdc37C efficiently and in a dose-dependent
manner displaced its full-length counterpart from Raf-1 in coinfected
Sf9 cells and strongly reduced the association of insect Hsp90 with
Raf-1. The dissociation of p50cdc37 and Hsp90
from Raf-1 correlated closely with the reduction of Raf-1 activation to
basal levels (Fig. 5A, top). A control Western blot of total cellular
extracts from this experiment indicates that this effect was not due to
decreased expression of wild-type p50cdc37, endogenous Hsp90, or Raf-1
kinase (Fig. 5A). We conclude that p50cdc37C
functions as a dominant negative for the
p50cdc37-mediated
Raf-1-p50cdc37-Hsp90 complex formation
and subsequent Raf-1 kinase activation.
|
C could
inhibit Raf-1 activation by Ras and v-Src and again found that
overexpression of p50cdc37C in insect cells
abrogated Raf-1 activation by oncogenic Src and Ras (Fig. 5B). Thus,
activation of Raf-1 by both v-Src and v-Ras in Sf9 cells is dependent
on the ability of p50cdc37 and Hsp90 to form a
productive complex with Raf-1 kinase. To gain more insight into the
mechanism of p50cdc37-dependent Raf-1
activation, we assessed the effects of wild-type and dominant negative
p50cdc37 on the activity of Raf-1 catalytic
domain site mutants by coinfection of Sf9 cells. As expected,
Raf-1(K375M), which is kinase inactive (14), could not be
stimulated by p50cdc37 or Src (not shown).
Tyr340 and to a lesser extent Tyr341 have previously shown to be
important regulatory sites, whose phosphorylation by tyrosine kinases
presumably activates Raf-1 by interfering with negative regulation of
the catalytic domain by the amino terminus of the protein
(14). Since, as shown above, p50cdc37
binds both in vivo and in vitro to the catalytic half of the Raf-1
protein and interacts also both physically and functionally with Src
kinases (references 4 and 13 and
data not shown), we reasoned that p50cdc37's
role might be auxiliary to tyrosine kinase function, i.e., by
facilitating or promoting Raf-1 tyrosine phosphorylation or by
preserving the active Raf-1 conformation. To test this, we coexpressed
in Sf9 cells p50cdc37 along with Raf-1(Y340D), a
constitutively active mutant (14). Indeed,
p50cdc37's coexpression with Raf-1(Y340D) (Fig.
5C), even at the highest possible amounts (not shown), failed to
further superinduce the already high basal activity of this mutant,
consistent with the above-hypothesized role for
p50cdc37. However, when we also tested the
effect of p50cdc37C on Raf-1(Y340D), we found
again the previously noted strong inhibition of Raf-1 activity (Fig.
5C). Consistent with this, we have found that both
p50cdc37 and p50cdc37C
associate with Raf-1(Y340D), as judged by examination of the coexpressed proteins (not shown). The above results argue strongly for
a potential dual role of p50cdc37 and its Hsp90
chaperone cofactor in the Raf-1 activation process: one where
p50cdc37-Hsp90 might be involved both in the
efficient activation of Raf-1 and a second involving maintenance of the
active kinase conformation, once relief from repression by the
N-terminal domain is achieved either through tyrosine phosphorylation
by v-Src (Fig. 4) or by activation of amino acid mutations (Fig. 5C).
Using a complementary experimental approach, we then tested whether
GA-mediated inhibition of insect cell Hsp90 would abrogate baculovirus
Raf-1 activation as we had observed in Cos-1 cells. Indeed, GA
treatment of Sf9 cells coinfected with Raf-1 and viruses expressing
v-Src, v-Ras, or p50cdc37 resulted in dramatic
decreases in Raf-1 activity (Fig. 6A)
that correlated with a substantial loss of endogenous Hsp90 binding to
Raf-1 in all tested combinations (Fig. 6B and data not shown). It is of
note that under the conditions used, GA resulted in only slight
depletion in Raf-1 protein, which, interestingly, exhibited a
noticeable mobility up-shift during SDS-PAGE. Thus, the dramatic reduction in Raf-1 kinase activity cannot be accounted for by changes
in levels of Raf-1 protein expression (control anti-Raf-1 immunoblot in
Fig. 6A). As we have additionally observed, coexpression of Raf-1 with
Hsp90 deletion constructs also abrogate Raf-1 activation without
causing Raf-1 protein degradation (data not shown). Thus, Raf-1
activation by coexpression with p50cdc37, v-Src,
or v-Ras is dependent in each case on functional endogenous insect
Hsp90.
|
p50cdc37 contributes to the transduction of
EGF signals that activate the MAPK cascade via Raf-1.
Activated
Raf-1 transduces signals to multiple pathways. The best-studied of
these is the MAPK pathway. If, therefore, the association of the
p50cdc37-Hsp90 complex with Raf-1 contributes to
the activation of Raf-1, the dominant negative mutant
p50cdc37C, which disrupts this complex, would
be expected to interfere with the transduction of physiological signals
from Raf-1 to the MAPK cascade. To test this hypothesis, we
overexpressed p50cdc37C or its full-length
p50cdc37 counterpart in combination with Raf-1
in Cos-1 cells, using the Targefect high-efficiency transfection
system. Duplicate serum-starved cultures were harvested with or without
EGF stimulation, and solubilized cell extracts were then examined by
Western blotting with an antibody against activated phospho-MAPK or
with control antibodies against transfected Raf-1 or
p50cdc37 (Fig. 7).
The results revealed that in contrast to the wild-type protein (Fig. 7,
lanes 3 and 4), transfected p50cdc37C
inhibited EGF-stimulated Raf-1 activation as judged by Raf-1 kinase
assay (not shown) and subsequent MAPK activation as determined by
detection of dually phosphorylated endogenous Erk-2 with
anti-phospho-Erk antibodies (lanes 5 and 6). Thus, not only is the
p50cdc37 C-terminal mutant unable to support
Raf-1 activation, but it also prevents Raf-1-mediated downstream
signaling through the MAPK pathway. Thus, GA and
p50cdc37C, which target the Hsp90 and
p50cdc37 components of the Raf-1 activation
complex, respectively, produce similar adverse effects: disruption of
the native Raf-1 heterocomplex, inhibition of Raf-1 activation, and
interruption of signaling to downstream Raf-1 effectors. These findings
show that the p50cdc37-Hsp90 complex contributes
to the activation of Raf-1 by growth factors and plays a critical role
in the transduction of growth factor-generated Raf-1 signals to the
MAPK pathway.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
Based on observations that both Hsp90 and
p50cdc37 copurify with various protein kinases,
it has been proposed that these two proteins comprise a complex that
regulates kinase conformation and activity (4, 28, 53).
However, this hypothesis has yet to be examined biochemically. The
recent cloning of p50cdc37 has allowed us to
directly investigate the role of the
p50cdc37-Hsp90 complex in the regulation of the
Raf-1. We have found that coexpression of
p50cdc37 with Raf-1 leads to Raf-1 activation
and that disruption of the p50cdc37-Hsp90
heterodimer interaction with Raf-1 by either
p50cdc37C or GA inhibits Raf-1 activation and
signaling through Erk. These results indicate that the concerted action
of p50cdc37 and Hsp90 on Raf-1 plays a critical
role in cell signaling via the Raf-1/Mek/Erk pathway.
Although it has previously been hypothesized that Hsp90 brings
p50cdc37 into a complex with Raf and Src
(29, 67, 79), our analysis indicates that Raf-1-Hsp90
association is for the most part p50cdc37
dependent and that p50cdc37 is the factor which
primarily mediates the Raf-1-p50cdc37-Hsp90
complex formation. More specifically, p50cdc37
binds to the catalytic domain of Raf-1 through its N terminus and
tethers Hsp90 to Raf-1 through a second domain located at its
C-terminal half (Fig. 2D). This finding is consistent with the
observation of Stepanova et al. (69) that
p50cdc37 accumulates Hsp90 to Cdk4, although in
this case there were no clear effects on kinase activity. Further
support for our conclusion stated above is given by the observation
that p50cdc37C associates with Raf-1 even
though it cannot bind to Hsp90. Moreover, this mutant prevents the
accumulation of Hsp90 into the complex by displacing its endogenous
full-length counterpart from Raf-1.
Surprisingly, however, GA disrupts the association of the
Hsp90-p50cdc37 complex with Raf-1 even though it
is known to bind only Hsp90 and fails to dissociate the
Hsp90-p50cdc37 complex itself. This could be
explained in several ways. GA is known to competitively displace ATP
and, by binding tightly to Hsp90, to lock the chaperone into its
ADP-specific inactive conformation (7, 22, 55, 72). This
conformation may prevent complex binding by steric hindrance, since
most of p50cdc37 is bound to Hsp90.
p50cdc37C, in contrast, being unable to bind
Hsp90, would be free to associate with Raf-1. Alternatively,
p50cdc37 may bind to the GA-Hsp90 complex in
such a way that it is no longer able to bind to Raf-1. Thus, both Hsp90
and p50cdc37 must be in a functional complex in
order to form a productive heterotrimeric complex with Raf-1. In
general, however, these results validate experimentally the earlier
proposal that Hsp90's specific associations might be mediated through
Hsp90-associated cofactors and that pp50, in particular, might function
in targeting Hsp90 to v-Src and Raf-1 kinases (6, 52, 53).
It is notable that Hsp90 and p50cdc37 can
sometimes function independently of each other.
p50cdc37 has not been detected in steroid
receptor complexes (54), and we have found that Mek-1 forms
a tight complex with p50cdc37 that is
characteristically devoid of Hsp90 (19a).
Several lines of evidence indicate that
p50cdc37-Hsp90 association with Raf-1 is
necessary for the Raf-1 kinase activity. First, overexpressed
p50cdc37C reduces both Hsp90 association with
Raf-1 and Raf-1 kinase activity by competitively displacing wild-type
p50cdc37 from the Raf-1 complex. Second, GA, an
Hsp90-specific inhibitor, blunted Raf-1 activation by serum (Fig. 3),
and this inhibition correlated with a dramatic loss of
p50cdc37-Hsp90 heterodimers from the kinase.
That occupation of the ATP/ADP binding pocket of Hsp90 by GA results in
dissociation of the protein from Raf-1 is consistent with the notion
that alternating cycles of ATP and ADP binding regulate Hsp90
conformation and, in turn, its ability to mediate the formation of
productive signaling heterocomplexes (7, 22, 55, 72). The
inhibition by GA was also observed with BXB-Raf-1, a constitutively
active N-terminal Raf-1 deletion mutant (3), which
consistently binds to p50cdc37 and Hsp90 even
more strongly than its full-length counterpart (19a).
Coupled with our findings that the vast majority of cytoplasmic p50cdc37 is sequestered in heterodimeric
complexes by Hsp90 and that it is primarily responsible for bringing
Hsp90 into the Raf-1 complex, these results suggest that the interface
of p50cdc37-Raf-1 interaction is a target of GA
action and that GA-induced conformational alteration of the
Hsp90-p50cdc37 heterodimer either leads to the
release of the heterodimer as a whole from Raf-1 or prevents it from
rebinding to Raf-1. Freed Raf-1 then becomes subject to accelerated
degradation as previously observed by Schulte et al. (60).
Interestingly, p50cdc37C binding to Raf-1
excludes Hsp90 from the complex but does not lead, as GA treatment
does, to Raf-1 degradation. p50cdc37C further
inhibits Raf-1 activation, which also suggests that Hsp90 and
p50cdc37 play an active and positive role in
Raf-1 signaling rather than merely serving to stabilize the kinase.
Strikingly, we have found that p50cdc37 itself, upon coinfection in insect cells with Raf-1, results in strong dose-dependent Raf-1 catalytic activity. This activation is even stronger than that observed with v-Ras and only slightly weaker than v-Src-mediated Raf-1 activation. Moreover, p50cdc37 was able to enhance the weak v-Src-mediated activation of Raf-1(S621A), a well-characterized conformation-compromised, and thus inactive, Raf-1 mutant. Given that Hsp90, p50cdc37's partner, is a highly abundant protein, these results suggest that p50cdc37 may be a rate-limiting component under conditions of Raf-1 overexpression and may contribute to the formation or stabilization of the active Raf-1 conformational state. As with v-Src and v-Ras, this effect requires phosphorylatable Ser621 for function (46). In contrast, p50cdc37 failed to induce further the already high constitutive activity of Raf-1(Y340D), an N-terminal repression-relieved activated Raf-1 mutant (14). One possible interpretation of this result is that p50cdc37 enhances Src-mediated phosphorylation and activation of Raf-1, a notion supported by the observed physical and functional interactions between Src kinases and p50cdc37 (reference 4 and unpublished results), including their strong synergistic effect on activating Raf-1 activation (Fig. 4). However, our finding that the dominant negative p50cdc37 deletion also down-regulates Raf-1(Y340D) (Fig. 5) in a dose-dependent fashion (not shown) indicates that some of the effects of p50cdc37-Hsp90 complex are independent of tyrosine phosphorylation as well. Thus, it is likely that the p50cdc37-Hsp90 complex is further required to maintain the activated Raf-1 kinase in its active conformation. This latter interpretation would be consistent as well with the findings that activated Ras-independent Drosophila Raf alleles still require Hsp90 association for constitutive function at the membrane (76). It is not yet known whether the Drosophila cdc37 mutation can also suppress this activated Raf allele. This genetic result also indicates that Hsp90 affects Raf-1 activity independently of Raf-1 translocation to the plasma membrane.
Mere addition of purified p50cdc37 and Hsp90 to Raf-1 does not activate the kinase in vitro (unpublished observation). Furthermore, it is worth noting that under commonly used kinase assay conditions, Raf-1, precipitated in RIPA buffer and thus presumably stripped of bound p50cdc37 and Hsp90, remains active. This finding suggests that p50cdc37 and Hsp90 exert their activation role in vivo in conjunction with additional Raf-1 activation factors and do not need to stay associated with Raf-1 in vitro in order for the kinase to remain active; it also argues against a strictly structural role for the p50cdc37-Hsp90 complex in maintaining Raf-1 activity. This observation may also explain why we can detect only a relatively weak associated MAPKKK activity in p50cdc37 immunoprecipitates. As with other chaperone proteins, the p50cdc37-Hsp90 complex may interact with Raf-1 in a transient manner and release after catalyzing conformational changes in Raf-1.
Previous work in Raf-1 overexpression systems has suggested that there may be a limiting cytosolic factor which is required for maximal Raf-1 activation (5, 26, 36, 70, 78). Our results suggest that p50cdc37 could well be a component of this activity. However, since p50cdc37 is more abundant than Raf-1, the ability of p50cdc37 overexpression alone to activate endogenous Raf-1 is modest relative to its marked ability to activate coexpressed Raf-1. This finding suggests that in unstimulated cells there may be a stoichiometric inhibitor of Raf-1 signaling whose effects are partially overcome by overexpression of Raf-1. Conceivably, under these conditions, the p50cdc37-Hsp90 complex becomes limiting and overexpressed p50cdc37 complexes with the already abundant Hsp90 to reconstitute the Raf-1-p50cdc37-Hsp90 complex and allow activation of the kinase. That the Hsp90-p50cdc37 complex would be limiting in these experiments would also be consistent with a model in which the complex serves as a scaffold for Raf-1 oligomerization. There is evidence both that oligomerization can lead to Raf-1 activation (15, 35) and that Hsp90 forms homodimers and oligomers (40, 41, 48, 49). In further support, most of native Raf-1 is found in large (300- to 500-kDa) complexes with p50cdc37 and Hsp90, and it is this form of Raf-1 that becomes membrane activated (78).
An important remaining question is whether the associations or the activity of the p50cdc37-Hsp90 complex are subject to regulation. First, we have found increased formation of the Raf-1-p50cdc37-Hsp90 ternary complex after serum stimulation and in response to activated Ras. It is possible that this contributes to the activation of the small fraction of Raf-1 that is reportedly sufficient for effective signaling. This would be consistent as well with our finding that coexpression of p50cdc37 with Raf-1 accumulates Hsp90 and activates Raf-1 in a dose-dependent manner. Analogously, Garcia-Cardena et al. (18) have recently found that extracellular regulators of endothelial nitric oxide synthase induce the rapid recruitment of Hsp90 to the enzyme, resulting in its membrane activation. It is also possible that changes in protein association or modifications of preexisting Raf-1-Hsp90-p50cdc37 trimeric complexes are sufficient to cause Raf-1 activation or derepression during cell stimulation. Since both p50cdc37 and Hsp90 are phosphoproteins (4, 34, 78, 79), their protein associations within the Raf-1 signalsome could in turn be modulated by phosphorylation. Indeed, phosphorylation-dependent interactions appear to be involved in the regulatory interaction of other kinases with Hsp90, including v-Src (39), Lck (24), and HRI (73, 75). In addition, serum regulation of the phosphorylation state of the Hsp90-p50cdc37 complex could play an important role in Raf-1 activation. Alternatively, serum might regulate the nucleotide binding state and conformation of Hsp90 (22, 55, 72) that is associated with p50cdc37 and Raf-1 and thereby allosterically regulate its effects on Raf-1. This may occur either through assisting Raf-1 in the conformational transition to the activated state or by allowing it to achieve a configuration where it is competent to respond to upstream activators.
In summary, our findings complement and extend genetic data for Drosophila and indicate that the p50cdc37-Hsp90 chaperone complex is essential for signaling through the MAPK pathway at the level of Raf-1. Interestingly, the fact that Raf-1 (71, 77), Hsp90 (54), and, as verified by both mRNA and protein analyses (8, 19a), p50cdc37 all involve ubiquitously expressed proteins points to a potentially universal Raf-1-Hsp90-p50cdc37 signaling complex. Future experiments will address both the exact nature of Raf-1 regulation by the p50cdc37-Hsp90 heterodimer and whether additional kinases are similarly modulated.
| |
ACKNOWLEDGMENTS |
|---|
GA was provided by the Developmental Therapeutics Program of the NCI. We gratefully acknowledge J. Kyriakis, Z. Luo, J. Avruch, D. Morrison, and T. Roberts for supplying reagents, L. Feig, G. Mosialos, P. Dice and J. Kyriakis for reviewing the manuscript and for useful discussions, D.-W. Kim for assisting with p50cdc37 antibody preparation, and J. Lee for help with graphics.
This work was support by DOD Breast Cancer Research Program grants DAMD17-97-1-7990 and NIH grant GM51551 to B.H.C.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Physiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: ngrammat{at}earthlink.net.
N.G. dedicates this paper to John, George, and Bill.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
|
|---|
| 1. | Avruch, J., X. F. Zhang, and J. M. Kyriakis. 1994. Raf meets Ras: completing the framework of a signal transduction pathway. Trends Biochem. Sci. 19:279-283[Medline]. |
| 2. |
Borkovich, K. A.,
F. W. Farrelly,
D. B. Finkelstein,
J. Taulien, and S. Lindquist.
1989.
hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures.
Mol. Cell. Biol.
9:3919-3930 |
| 3. |
Bruder, J.,
G. Heidecker, and U. Rapp.
1992.
Serum-, TPA-, and ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase.
Genes Dev.
6:545-556 |
| 4. | Brugge, J. S. 1986. Interaction of the Rous sarcoma virus protein pp60src with the cellular proteins pp50 and pp90. Curr. Top. Microbiol. Immunol. 123:1-22[Medline]. |
| 5. |
Chow, Y. H.,
K. Pumiglia,
T. H. Jun,
P. Dent,
T. W. Sturgill, and R. Jove.
1995.
Functional mapping of the N-terminal regulatory domain in the human Raf-1 protein kinase.
J. Biol. Chem.
270:14100-14106 |
| 6. |
Courtneidge, S. A., and J. M. Bishop.
1982.
Transit of pp60v-src to the plasma membrane.
Proc. Natl. Acad. Sci. USA
79:7117-7121 |
| 7. |
Csermely, P., and C. R. Kahn.
1991.
The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity.
J. Biol. Chem.
266:4943-50 |
| 8. | Cutforth, T., and G. M. Rubin. 1994. Mutations in Hsp83 and cdc37 impair signaling by the sevenless receptor tyrosine kinase in Drosophila. Cell 77:1027-1036[Medline]. |
| 9. |
Dai, K.,
R. Kobayashi, and D. Beach.
1996.
Physical interaction of mammalian CDC37 with CDK4.
J. Biol. Chem.
271:22030-22034 |
| 10. | DeBoer, C., P. A. Meulman, R. J. Wnuk, and D. H. Peterson. 1970. Geldanamycin, a new antibiotic. J. Antibiot. (Tokyo) 23:442-447[Medline]. |
| 11. |
Dent, P.,
T. Jelinek,
D. K. Morrison,
M. J. Weber, and T. W. Sturgill.
1995.
Reversal of Raf-1 activation by purified and membrane-associated protein phosphatases.
Science
268:1902-1906 |
| 12. | Dent, P., D. B. Reardon, D. K. Morrison, and T. W. Sturgill. 1995. Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro. Mol. Cell. Biol. 15:4125-4135[Abstract]. |
| 13. | Dey, B., J. J. Lightbody, and F. Boschelli. 1996. CDC37 is required for p60v-src activity in yeast. Mol. Biol. Cell 7:1405-1417[Abstract]. |
| 14. |
Fabian, J. R.,
I. O. Daar, and D. K. Morrison.
1993.
Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
Mol. Cell. Biol.
13:7170-7179 |
| 15. | Farrar, M. A., I. Alberol, and R. M. Perlmutter. 1996. Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature 383:178-181[Medline]. |
| 16. | Faure, M., and H. R. Bourne. 1995. Differential effects on cAMP on the MAP kinase cascade: evidence for a cAMP-insensitive step that can bypass Raf-1. Mol. Biol. Cell 6:1025-1035[Abstract]. |
| 17. |
Ferrier, A. F.,
M. Lee,
W. B. Anderson,
G. Benvenuto,
D. K. Morrison,
D. R. Lowy, and J. E. DeClue.
1997.
Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility.
J. Biol. Chem.
272:2136-2142 |
| 18. | Garcia-Cardena, G., R. Fan, V. Shah, R. Sorrentino, G. Cirino, A. Papapetropoulos, and W. C. Sessa. 1998. Dynamic activation of endothelial nitric oxide synthase by Hsp90. Nature 392:821-824[Medline]. |
| 19. |
Gerber, M. R.,
A. Farrell,
R. J. Deshaies,
I. Herskowitz, and D. O. Morgan.
1995.
Cdc37 is required for association of the protein kinase Cdc28 with G1 and mitotic cyclins.
Proc. Natl. Acad. Sci. USA
92:4651-4655 |
| 19a. | Grammatikakis, N. Unpublished data. |
| 20. | Grammatikakis, N., A. Grammatikakis, H. Piwnica-Worms, B. P. Toole, and B. H. Cochran. 1996. The cell cycle, p. 72. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 21. |
Grammatikakis, N.,
A. Grammatikakis,
M. Yoneda,
Q. Yu,
S. D. Banerjee, and B. P. Toole.
1995.
A novel glycosaminoglycan-binding protein is the vertebrate homologue of the cell cycle control protein, Cdc37.
J. Biol. Chem.
270:16198-16205 |
| 22. |
Grenert, J. P.,
W. P. Sullivan,
P. Fadden,
T. A. J. Haystead,
J. Clark,
E. Mimnaugh,
H. Krutzsch,
H. J. Ochel,
T. W. Schulte,
E. Sausville,
L. M. Neckers, and D. O. Toft.
1997.
The amino-terminal domain of heat shock protein 90 (hsp90) that binds geldanamycin is an ATP/ADP switch domain that regulates hsp90 conformation.
J. Biol. Chem.
272:23843-2350 |
| 23. |
Hallberg, B.,
S. I. Rayter, and J. Downward.
1994.
Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.
J. Biol. Chem.
269:3913-3916 |
| 24. |
Hartson, S. D.,
E. A. Ottinger,
W. Huang,
G. Barany,
P. Burn, and R. L. Matts.
1998.
Modular folding and evidence for phosphorylation-induced stabilization of an hsp90-dependent kinase.
J. Biol. Chem.
273:8475-8482 |
| 25. |
Heidecker, G.,
M. Huleihel,
J. L. Cleveland,
W. Kolch,
T. W. Beck,
P. Lloyd,
T. Pawson, and U. R. Rapp.
1990.
Mutational activation of c-raf-1 and definition of the minimal transforming sequence.
Mol. Cell. Biol.
10:2503-2512 |
| 26. | Howe, L. R., S. J. Leevers, N. Gomez, S. Nakielny, P. Cohen, and C. J. Marshall. 1992. Activation of the MAP kinase pathway by the protein kinase raf. Cell 71:335-342[Medline]. |
| 27. |
Huang, L.,
N. Grammatikakis, and B. P. Toole.
1998.
Organization of the chick CDC37 gene.
J. Biol. Chem.
273:3598-3603 |
| 28. | Hunter, T., and R. Y. C. Poon. 1997. Cdc37: a protein kinase chaperone? Trends Cell Biol. 7:157-161. |
| 29. |
Hutchison, K. A.,
B. K. Brott,
J. H. De Leon,
G. H. Perdew,
R. Jove, and W. B. Pratt.
1992.
Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.
J. Biol. Chem.
267:2902-2908 |
| 30. | Jakob, U., and J. Buchner. 1994. Assisting spontaneity: the role of Hsp90 and small Hsps as molecular chaperones. Trends Biochem. Sci. 19:205-211[Medline]. |
| 31. |
Johnson, J. L., and D. O. Toft.
1995.
Binding of p23 and hsp90 during assembly with the progesterone receptor.
Mol. Endocrinol.
9:670-678 |
| 32. |
Kimura, Y.,
S. L. Rutherford,
Y. Miyata,
I. Yahara,
B. C. Freeman,
L. Yue,
R. I. Morimoto, and S. Lindquist.
1997.
Cdc37 is a molecular chaperone with specific functions in signal transduction.
Genes Dev.
11:1775-1785 |
| 33. | Lamphere, L., F. Fiore, X. Xu, L. Brizuela, S. Keezer, C. Sardet, G. F. Draetta, and J. Gyuris. 1997. Interaction between Cdc37 and Cdk4 in human cells. Oncogene 14:1999-2004[Medline]. |
| 34. | Lovric, J., O. Bischof, and K. Moelling. 1994. Cell cycle-dependent association of Gag-Mil and hsp90. FEBS Lett. 343:15-21[Medline]. |
| 35. | Luo, Z., G. Tzivion, P. J. Belshaw, D. Vavvas, M. Marshall, and J. Avruch. 1996. Oligomerization activates c-Raf-1 through a Ras-dependent mechanism. Nature 383:181-185[Medline]. |
| 36. |
Marais, R.,
Y. Light,
H. F. Paterson,
C. S. Mason, and C. J. Marshall.
1997.
Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.
J. Biol. Chem.
272:4378-4383 |
| 37. | Marais, R., and C. J. Marshall. 1996. Control of the ERK MAP kinase cascade by Ras and Raf. Cancer Surv. 27:101-125[Medline]. |
| 38. | Marshall, C. J. 1995. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179-185[Medline]. |
| 39. |
Mimnaugh, E. G.,
P. J. Worland,
L. Whitesell, and L. M. Neckers.
1995.
Possible role for serine/threonine phosphorylation in the regulation of the heteroprotein complex between the hsp90 stress protein and the pp60v-src tyrosine kinase.
J. Biol. Chem.
270:28654-28659 |
| 40. |
Minami, Y.,
H. Kawasaki,
Y. Miyata,
K. Suzuki, and I. Yahara.
1991.
Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90.
J. Biol. Chem.
266:10099-10103 |
| 41. |
Minami, Y.,
Y. Kimura,
H. Kawasaki,
K. Suzuki, and I. Yahara.
1994.
The carboxy-terminal region of the mammalian HSP90 is required for its dimerization and function in vivo.
Mol. Cell. Biol.
14:1459-1464 |
| 42. |
Morrison, D.
1994.
14-3-3: modulators of signaling proteins?
Science
266:56-57 |
| 43. | Morrison, D. K. 1995. Activation of Raf-1 by Ras in intact cells. Methods Enzymol. 255:301-310[Medline]. |
| 44. | Morrison, D. K. 1995. Mechanisms regulating Raf-1 activity in signal transduction pathways. Mol. Reprod. Dev. 42:507-514[Medline]. |
| 45. | Morrison, D. K., and R. E. Cutler. 1997. The complexity of Raf-1 regulation. Curr. Opin. Cell Biol. 9:174-179[Medline]. |
| 46. |
Morrison, D. K.,
G. Heidecker,
U. R. Rapp, and T. D. Copeland.
1993.
Identification of the major phosphorylation sites of the Raf-1 kinase.
J. Biol. Chem.
268:17309-17316 |
| 47. |
Nathan, D. F.,
M. H. Vos, and S. Lindquist.
1997.
In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone.
Proc. Natl. Acad. Sci. USA
94:12949-12956 |
| 48. | Nemoto, T., Y. Ohara-Nemoto, M. Ota, T. Takagi, and K. Yokoyama. 1995. Mechanism of dimer formation of the 90-kDa heat-shock protein. Eur. J. Biochem. 233:1-8[Medline]. |
| 49. | Nemoto, T., and N. Sato. 1998. Oligomeric forms of the 90-kDa heat shock protein. Biochem. J. 330:989-995. |
| 50. | Ozaki, T., K. Irie, and S. Sakiyama. 1995. Molecular cloning and cell cycle-dependent expression of a novel gene that is homologous to cdc37. DNA Cell Biol. 14:1017-1023[Medline]. |
| 51. | Perdew, G. H., H. Wiegand, J. P. Vanden Heuvel, C. Mitchell, and S. S. Singh. 1997. A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37. Biochemistry 36:3600-3607[Medline]. |
| 52. | Pratt, W. B. 1998. The hsp90-based chaperone system: involvement in signal transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp. Biol. Med. 217:420-434[Medline]. |
| 53. |
Pratt, W. B.
1993.
The role of heat-shock proteins in regulating the function, folding and trafficking of the glucocorticoid receptor.
J. Biol. Chem.
268:21455-21458 |
| 54. |
Pratt, W. B., and D. O. Toft.
1997.
Steroid receptor interactions with heat shock protein and immunophilin chaperones.
Endocrine Rev.
18:306-360 |
| 55. | Prodromou, C., S. M. Roe, R. O'Brien, J. E. Ladbury, P. W. Piper, and L. H. Pearl. 1997. Identification and structural characterization of the ATP/ADP-binding site in the Hsp90 molecular chaperone. Cell 90:65-75[Medline]. |
| 56. |
Reed, S. I.
1980.
The selection of S. cerevisiae mutants defective in the start event of cell division.
Genetics
95:561-577 |
| 57. | Sanchez, I., R. T. Hughes, B. J. Mayer, K. Yee, J. R. Woodgett, J. Avruch, J. M. Kyriakis, and L. I. Zon. 1994. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372:794-798[Medline]. |
| 58. |
Scheibel, T., and J. Buchner.
1998.
The Hsp90 complex a super-chaperone machine as a novel drug target.
Biochem. Pharmacol.
56:675-682[Medline].
|
| 59. |
Schneider, C.,
L. Sepp-Lorenzino,
E. Nimmesgern,
O. Ouerfelli,
S. Danishefsky,
N. Rosen, and F. U. Hartl.
1996.
Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90.
Proc. Natl. Acad. Sci. USA
93:14536-14541 |
| 60. |
Schulte, T. W.,
M. V. Blagosklonny,
C. Ingui, and L. Neckers.
1995.
Disruption of the Raf-1-Hsp90 molecular complex results in destabilization of Raf-1 and loss of Raf-1-Ras association.
J. Biol. Chem.
270:24585-24588 |
| 61. | Schulte, T. W., M. V. Blagosklonny, L. Romanova, J. F. Mushinski, B. P. Monia, J. F. Johnston, P. Nguyen, J. Trepel, and L. M. Neckers. 1996. Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway. Mol. Cell. Biol. 16:5839-5845[Abstract]. |
| 62. |
Segnitz, B., and U. Gehring.
1997.
The function of steroid hormone receptors is inhibited by the hsp90-specific compound geldanamycin.
J. Biol. Chem.
272:18694-18701 |
| 63. |
Silverstein, A. M.,
N. Grammatikakis,
B. H. Cochran,
M. Chinkers, and W. B. Pratt.
1998.
p50(cdc37) binds directly to the catalytic domain of Raf as well as to a site on hsp90 that is topologically adjacent to the tetratricopeptide repeat binding site.
J. Biol. Chem.
273:20090-20095 |
| 64. | Smith, D. B., and K. S. Johnson. 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67:31-40[Medline]. |
| 65. | Smith, D. F., L. Whitesell, S. C. Nair, S. Chen, V. Prapapanich, and R. A. Rimerman. 1995. Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent. Mol. Cell. Biol. 15:6804-6812[Abstract]. |
| 66. |
Stancato, L. F.,
Y. H. Chow,
K. A. Hutchison,
G. H. Perdew,
R. Jove, and W. B. Pratt.
1993.
Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.
J. Biol. Chem.
268:21711-21716 |
| 67. |
Stancato, L. F.,
Y. H. Chow,
J. K. Owens-Grillo,
A. W. Yem,
M. R. Deibel, Jr.,
R. Jove, and W. B. Pratt.
1994.
The native v-Raf.hsp90.p50 heterocomplex contains a novel immunophilin of the FK506 binding class.
J. Biol. Chem.
269:22157-22161 |
| 68. |
Stanton, V. P., Jr.,
D. W. Nichols,
A. P. Laudano, and G. M. Cooper.
1989.
Definition of the human Raf amino-terminal regulatory region by deletion mutagenesis.
Mol. Cell. Biol.
9:639-47 |
| 69. |
Stepanova, L.,
X. Leng,
S. Parker, and J. Harper.
1996.
Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4.
Genes Dev.
10:1491-1502 |
| 70. | Stokoe, D., and F. McCormick. 1997. Activation of c-Raf-1 by Ras and Src through different mechanisms: activation in vivo and in vitro. EMBO J. 16:2384-2396[Medline]. |
| 71. | Storm, S. M., J. L. Cleveland, and U. R. Rapp. 1990. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene 5:345-351[Medline]. |
| 72. |
Sullivan, W.,
B. Stensgard,
G. Caucutt,
B. Bartha,
N. McMahon,
E. S. Alnemri,
G. Litwack, and D. Toft.
1997.
Nucleotides and two functional states of hsp90.
J. Biol. Chem.
272:8007-8012 |
| 73. | Szyszka, R., G. Kramer, and B. Hardesty. 1989. The phosphorylation state of the reticulocyte 90-kDa heat shock protein affects its ability to increase phosphorylation of peptide initiation factor 2 alpha subunit by the heme-sensitive kinase. Biochemistry 28:1435-1438[Medline]. |
| 74. | Uehara, Y., Y. Murakami, K. Suzukake-Tsuchiya, Y. Moriya, H. Sano, K. Shibata, and S. Omura. 1988. Effects of herbimycin derivatives on src oncogene function in relation to antitumor activity. J. Antibiot. (Tokyo) 41:831-834[Medline]. |
| 75. |
Uma, S.,
S. D. Hartson,
J. J. Chen, and R. L. Matts.
1997.
Hsp90 is obligatory for the heme-regulated eIF-2alpha kinase to acquire and maintain an activable conformation.
J. Biol. Chem.
272:11648-11656 |
| 76. | van der Straten, A., C. Rommel, B. Dickson, and E. Hafen. 1997. The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila. EMBO J. 16:1961-1969[Medline]. |
| 77. | Wadewitz, A. G., M. A. Winer, and D. J. Wolgemuth. 1993. Developmental and cell lineage specificity of raf family gene expression in mouse testis. Oncogene 8:1055-1062[Medline]. |
| 78. |
Wartmann, M., and R. J. Davis.
1994.
The native structure of the activated Raf protein kinase is a membrane-bound multi-subunit complex.
J. Biol. Chem.
269:6695-6701 |
| 79. |
Whitelaw, M. L.,
K. Hutchison, and G. H. Perdew.
1991.
A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.
J. Biol. Chem.
266:16436-16440 |
| 80. |
Whitesell, L.,
E. G. Mimnaugh,
B. De Costa,
C. E. Myers, and L. M. Neckers.
1994.
Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.
Proc. Natl. Acad. Sci. USA
91:8324-8328 |
| 81. |
Whitesell, L.,
S. D. Shifrin,
G. Schwab, and L. M. Neckers.
1992.
Benzoquinonoid ansamycins possess selective tumoricidal activity unrelated to src kinase inhibition.
Cancer Res.
52:1721-1728 |
Molecular and Cellular Biology, March 1999, p. 1661-1672, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Romesser, P. B., Perlman, D. H., Faller, D. V., Costello, C. E., McComb, M. E., Denis, G. V.
(2009). Development of a Malignancy-Associated Proteomic Signature for Diffuse Large B-Cell Lymphoma. Am. J. Pathol.
175: 25-35
[Abstract] [Full Text] -
Hung, H.-C., Kay, S. A., Weber, F.
(2009). HSP90, a Capacitor of Behavioral Variation. J Biol Rhythms
24: 183-192
[Abstract] -
Lyu, J., Wesselschmidt, R. L., Lu, W.
(2009). Cdc37 Regulates Ryk Signaling by Stabilizing the Cleaved Ryk Intracellular Domain. J. Biol. Chem.
284: 12940-12948
[Abstract] [Full Text] -
Sreeramulu, S., Jonker, H. R. A., Langer, T., Richter, C., Lancaster, C. R. D., Schwalbe, H.
(2009). The Human Cdc37{middle dot}Hsp90 Complex Studied by Heteronuclear NMR Spectroscopy. J. Biol. Chem.
284: 3885-3896
[Abstract] [Full Text] -
Tucker, D. E., Gijon, M. A., Spencer, D. M., Qiu, Z.-H., Gelb, M. H., Leslie, C. C.
(2008). Regulation of cytosolic phospholipase A2{alpha} by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages. J. Leukoc. Biol.
84: 798-806
[Abstract] [Full Text] -
Babchia, N., Calipel, A., Mouriaux, F., Faussat, A.-M., Mascarelli, F.
(2008). 17-AAG and 17-DMAG-Induced Inhibition of Cell Proliferation through B-Raf Downregulation in WTB-Raf-Expressing Uveal Melanoma Cell Lines. IOVS
49: 2348-2356
[Abstract] [Full Text] -
Weihofen, A., Ostaszewski, B., Minami, Y., Selkoe, D. J.
(2008). Pink1 Parkinson mutations, the Cdc37/Hsp90 chaperones and Parkin all influence the maturation or subcellular distribution of Pink1. Hum Mol Genet
17: 602-616
[Abstract] [Full Text] -
Hinz, M., Broemer, M., Arslan, S. C., Otto, A., Mueller, E.-C., Dettmer, R., Scheidereit, C.
(2007). Signal Responsiveness of I{kappa}B Kinases Is Determined by Cdc37-assisted Transient Interaction with Hsp90. J. Biol. Chem.
282: 32311-32319
[Abstract] [Full Text] -
Ren, M., Santhanam, A., Lee, P., Caplan, A., Garrett, S.
(2007). Alteration of the Protein Kinase Binding Domain Enhances Function of the Saccharomyces cerevisiae Molecular Chaperone Cdc37. Eukaryot Cell
6: 1363-1372
[Abstract] [Full Text] -
Hawle, P., Horst, D., Bebelman, J. P., Yang, X. X., Siderius, M., van der Vies, S. M.
(2007). Cdc37p Is Required for Stress-Induced High-Osmolarity Glycerol and Protein Kinase C Mitogen-Activated Protein Kinase Pathway Functionality by Interaction with Hog1p and Slt2p (Mpk1p). Eukaryot Cell
6: 521-532
[Abstract] [Full Text] -
Mandal, A. K., Lee, P., Chen, J. A., Nillegoda, N., Heller, A., DiStasio, S., Oen, H., Victor, J., Nair, D. M., Brodsky, J. L., Caplan, A. J.
(2007). Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation. JCB
176: 319-328
[Abstract] [Full Text] -
Matei, D., Satpathy, M., Cao, L., Lai, Y.-C., Nakshatri, H., Donner, D. B.
(2007). The Platelet-derived Growth Factor Receptor {alpha} Is Destabilized by Geldanamycins in Cancer Cells. J. Biol. Chem.
282: 445-453
[Abstract] [Full Text] -
Catlett, M. G., Kaplan, K. B.
(2006). Sgt1p Is a Unique Co-chaperone That Acts as a Client Adaptor to Link Hsp90 to Skp1p. J. Biol. Chem.
281: 33739-33748
[Abstract] [Full Text] -
Beers, M., Kemphues, K.
(2006). Depletion of the co-chaperone CDC-37 reveals two modes of PAR-6 cortical association in C. elegans embryos. Development
133: 3745-3754
[Abstract] [Full Text] -
Shinozaki, F., Minami, M., Chiba, T., Suzuki, M., Yoshimatsu, K., Ichikawa, Y., Terasawa, K., Emori, Y., Matsumoto, K., Kurosaki, T., Nakai, A., Tanaka, K., Minami, Y.
(2006). Depletion of Hsp90beta Induces Multiple Defects in B Cell Receptor Signaling. J. Biol. Chem.
281: 16361-16369
[Abstract] [Full Text] -
Terasawa, K., Yoshimatsu, K., Iemura, S.-i., Natsume, T., Tanaka, K., Minami, Y.
(2006). Cdc37 interacts with the glycine-rich loop of hsp90 client kinases.. Mol. Cell. Biol.
26: 3378-3389
[Abstract] [Full Text] -
Shang, L., Tomasi, T. B.
(2006). The Heat Shock Protein 90-CDC37 Chaperone Complex Is Required for Signaling by Types I and II Interferons. J. Biol. Chem.
281: 1876-1884
[Abstract] [Full Text] -
Gloeckner, C. J., Kinkl, N., Schumacher, A., Braun, R. J., O'Neill, E., Meitinger, T., Kolch, W., Prokisch, H., Ueffing, M.
(2006). The Parkinson disease causing LRRK2 mutation I2020T is associated with increased kinase activity. Hum Mol Genet
15: 223-232
[Abstract] [Full Text] -
Turnbull, E. L., Martin, I. V., Fantes, P. A.
(2006). Activity of Cdc2 and its interaction with the cyclin Cdc13 depend on the molecular chaperone Cdc37 in Schizosaccharomyces pombe. J. Cell Sci.
119: 292-302
[Abstract] [Full Text] -
Grbovic, O. M., Basso, A. D., Sawai, A., Ye, Q., Friedlander, P., Solit, D., Rosen, N.
(2006). V600E B-Raf requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc. Natl. Acad. Sci. USA
103: 57-62
[Abstract] [Full Text] -
Misra, S., Ghatak, S., Toole, B. P.
(2005). Regulation of MDR1 Expression and Drug Resistance by a Positive Feedback Loop Involving Hyaluronan, Phosphoinositide 3-Kinase, and ErbB2. J. Biol. Chem.
280: 20310-20315
[Abstract] [Full Text] -
Terasawa, K., Minami, M., Minami, Y.
(2005). Constantly Updated Knowledge of Hsp90. J Biochem
137: 443-447
[Abstract] [Full Text] -
De Nardo, D., Masendycz, P., Ho, S., Cross, M., Fleetwood, A. J., Reynolds, E. C., Hamilton, J. A., Scholz, G. M.
(2005). A Central Role for the Hsp90{middle dot}Cdc37 Molecular Chaperone Module in Interleukin-1 Receptor-associated-kinase-dependent Signaling by Toll-like Receptors. J. Biol. Chem.
280: 9813-9822
[Abstract] [Full Text] -
Ghatak, S., Misra, S., Toole, B. P.
(2005). Hyaluronan Constitutively Regulates ErbB2 Phosphorylation and Signaling Complex Formation in Carcinoma Cells. J. Biol. Chem.
280: 8875-8883
[Abstract] [Full Text] -
Wang, X., Grammatikakis, N., Siganou, A., Stevenson, M. A., Calderwood, S. K.
(2004). Interactions between Extracellular Signal-regulated Protein Kinase 1, 14-3-3{epsilon}, and Heat Shock Factor 1 during Stress. J. Biol. Chem.
279: 49460-49469
[Abstract] [Full Text] -
Marsee, D. K., Venkateswaran, A., Tao, H., Vadysirisack, D., Zhang, Z., Vandre, D. D., Jhiang, S. M.
(2004). Inhibition of Heat Shock Protein 90, a Novel RET/PTC1-associated Protein, Increases Radioiodide Accumulation in Thyroid Cells. J. Biol. Chem.
279: 43990-43997
[Abstract] [Full Text] -
Prince, T., Matts, R. L.
(2004). Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37. J. Biol. Chem.
279: 39975-39981
[Abstract] [Full Text] -
Le Boeuf, F., Houle, F., Huot, J.
(2004). Regulation of Vascular Endothelial Growth Factor Receptor 2-mediated Phosphorylation of Focal Adhesion Kinase by Heat Shock Protein 90 and Src Kinase Activities. J. Biol. Chem.
279: 39175-39185
[Abstract] [Full Text] -
Chen, C., Sytkowski, A. J.
(2004). Erythropoietin regulation of Raf-1 and MEK: evidence for a Ras-independent mechanism. Blood
104: 73-80
[Abstract] [Full Text] -
Zhang, H., Wu, W., Du, Y., Santos, S. J., Conrad, S. E., Watson, J. T., Grammatikakis, N., Gallo, K. A.
(2004). Hsp90/p50cdc37 Is Required for Mixed-lineage Kinase (MLK) 3 Signaling. J. Biol. Chem.
279: 19457-19463
[Abstract] [Full Text] -
Miyata, Y., Nishida, E.
(2004). CK2 Controls Multiple Protein Kinases by Phosphorylating a Kinase-Targeting Molecular Chaperone, Cdc37. Mol. Cell. Biol.
24: 4065-4074
[Abstract] [Full Text] -
Lee, P., Shabbir, A., Cardozo, C., Caplan, A. J.
(2004). Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase. Mol. Biol. Cell
15: 1785-1792
[Abstract] [Full Text] -
Zhao, Q., Boschelli, F., Caplan, A. J., Arndt, K. T.
(2004). Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4. J. Biol. Chem.
279: 12560-12564
[Abstract] [Full Text] -
Arlander, S. J. H., Eapen, A. K., Vroman, B. T., McDonald, R. J., Toft, D. O., Karnitz, L. M.
(2003). Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress. J. Biol. Chem.
278: 52572-52577
[Abstract] [Full Text] -
Ikenoue, T., Hikiba, Y., Kanai, F., Tanaka, Y., Imamura, J., Imamura, T., Ohta, M., Ijichi, H., Tateishi, K., Kawakami, T., Aragaki, J., Matsumura, M., Kawabe, T., Omata, M.
(2003). Functional Analysis of Mutations within the Kinase Activation Segment of B-Raf in Human Colorectal Tumors. Cancer Res.
63: 8132-8137
[Abstract] [Full Text] -
Harding, A., Hsu, V., Kornfeld, K., Hancock, J. F.
(2003). Identification of Residues and Domains of Raf Important for Function in Vivo and in Vitro. J. Biol. Chem.
278: 45519-45527
[Abstract] [Full Text] -
Cotten, M., Stegmueller, K., Eickhoff, J., Hanke, M., Herzberger, K., Herget, T., Choidas, A., Daub, H., Godl, K.
(2003). Exploiting features of adenovirus replication to support mammalian kinase production. Nucleic Acids Res
31: e128-e128
[Abstract] [Full Text] -
Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., Mascarelli, F.
(2003). Mutation of B-Raf in Human Choroidal Melanoma Cells Mediates Cell Proliferation and Transformation through the MEK/ERK Pathway. J. Biol. Chem.
278: 42409-42418
[Abstract] [Full Text] -
Gober, M. D., Smith, C. C., Ueda, K., Toretsky, J. A., Aurelian, L.
(2003). Forced Expression of the H11 Heat Shock Protein Can Be Regulated by DNA Methylation and Trigger Apoptosis in Human Cells. J. Biol. Chem.
278: 37600-37609
[Abstract] [Full Text] -
Wang, X., Grammatikakis, N., Siganou, A., Calderwood, S. K.
(2003). Regulation of Molecular Chaperone Gene Transcription Involves the Serine Phosphorylation, 14-3-3{varepsilon} Binding, and Cytoplasmic Sequestration of Heat Shock Factor 1. Mol. Cell. Biol.
23: 6013-6026
[Abstract] [Full Text] -
Tatebe, H., Shiozaki, K.
(2003). Identification of Cdc37 as a Novel Regulator of the Stress-Responsive Mitogen-Activated Protein Kinase. Mol. Cell. Biol.
23: 5132-5142
[Abstract] [Full Text] -
Schwarze, S. R., Fu, V. X., Jarrard, D. F.
(2003). Cdc37 Enhances Proliferation and Is Necessary for Normal Human Prostate Epithelial Cell Survival. Cancer Res.
63: 4614-4619
[Abstract] [Full Text] -
Haughn, L., Hawley, R. G., Morrison, D. K., von Boehmer, H., Hockenbery, D. M.
(2003). BCL-2 and BCL-XL Restrict Lineage Choice during Hematopoietic Differentiation. J. Biol. Chem.
278: 25158-25165
[Abstract] [Full Text] -
Slavotinek, A., Biesecker, L. G.
(2003). Genetic modifiers in human development and malformation syndromes, including chaperone proteins. Hum Mol Genet
12: R45-50
[Abstract] [Full Text] -
Carystinos, G. D., Kandouz, M., Alaoui-Jamali, M. A., Batist, G.
(2003). Unexpected Induction of the Human Connexin 43 Promoter by the Ras Signaling Pathway Is Mediated by a Novel Putative Promoter Sequence. Mol. Pharmacol.
63: 821-831
[Abstract] [Full Text] -
Bandhakavi, S., McCann, R. O., Hanna, D. E., Glover, C. V. C.
(2003). A Positive Feedback Loop between Protein Kinase CKII and Cdc37 Promotes the Activity of Multiple Protein Kinases. J. Biol. Chem.
278: 2829-2836
[Abstract] [Full Text] -
Lee, P., Rao, J., Fliss, A., Yang, E., Garrett, S., Caplan, A. J.
(2002). The Cdc37 protein kinase-binding domain is sufficient for protein kinase activity and cell viability. JCB
159: 1051-1059
[Abstract] [Full Text] -
Basso, A. D., Solit, D. B., Chiosis, G., Giri, B., Tsichlis, P., Rosen, N.
(2002). Akt Forms an Intracellular Complex with Heat Shock Protein 90 (Hsp90) and Cdc37 and Is Destabilized by Inhibitors of Hsp90 Function. J. Biol. Chem.
277: 39858-39866
[Abstract] [Full Text] -
Isaacs, J. S., Jung, Y.-J., Mimnaugh, E. G., Martinez, A., Cuttitta, F., Neckers, L. M.
(2002). Hsp90 Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-1alpha -degradative Pathway. J. Biol. Chem.
277: 29936-29944
[Abstract] [Full Text] -
Nollen, E. A. A., Morimoto, R. I.
(2002). Chaperoning signaling pathways: molecular chaperones as stress-sensing `heat shock' proteins. J. Cell Sci.
115: 2809-2816
[Abstract] [Full Text] -
Wang, X., Grammatikakis, N., Hu, J.
(2002). Role of p50/CDC37 in Hepadnavirus Assembly and Replication. J. Biol. Chem.
277: 24361-24367
[Abstract] [Full Text] -
Siligardi, G., Panaretou, B., Meyer, P., Singh, S., Woolfson, D. N., Piper, P. W., Pearl, L. H., Prodromou, C.
(2002). Regulation of Hsp90 ATPase Activity by the Co-chaperone Cdc37p/p50cdc37. J. Biol. Chem.
277: 20151-20159
[Abstract] [Full Text] -
Wang, X., Hu, J.
(2002). Distinct Requirement for Two Stages of Protein-Primed Initiation of Reverse Transcription in Hepadnaviruses. J. Virol.
76: 5857-5865
[Abstract] [Full Text] -
Piatelli, M. J., Doughty, C., Chiles, T. C.
(2002). Requirement for a hsp90 Chaperone-dependent MEK1/2-ERK Pathway for B Cell Antigen Receptor-induced Cyclin D2 Expression in Mature B Lymphocytes. J. Biol. Chem.
277: 12144-12150
[Abstract] [Full Text] -
Grammatikakis, N., Vultur, A., Ramana, C. V., Siganou, A., Schweinfest, C. W., Watson, D. K., Raptis, L.
(2002). The Role of Hsp90N, a New Member of the Hsp90 Family, in Signal Transduction and Neoplastic Transformation. J. Biol. Chem.
277: 8312-8320
[Abstract] [Full Text] -
Hung, J.-J., Chung, C.-S., Chang, W.
(2002). Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells. J. Virol.
76: 1379-1390
[Abstract] [Full Text] -
Hu, J., Toft, D., Anselmo, D., Wang, X.
(2002). In Vitro Reconstitution of Functional Hepadnavirus Reverse Transcriptase with Cellular Chaperone Proteins. J. Virol.
76: 269-279
[Abstract] [Full Text] -
Scholz, G. M., Hartson, S. D., Cartledge, K., Volk, L., Matts, R. L., Dunn, A. R.
(2001). The Molecular Chaperone Hsp90 Is Required for Signal Transduction by Wild-Type Hck and Maintenance of Its Constitutively Active Counterpart. Cell Growth Differ.
12: 409-417
[Abstract] [Full Text] -
Kim, D.-W., Cochran, B. H.
(2001). JAK2 Activates TFII-I and Regulates Its Interaction with Extracellular Signal-Regulated Kinase. Mol. Cell. Biol.
21: 3387-3397
[Abstract] [Full Text] -
Hu, J., Anselmo, D.
(2000). In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90. J. Virol.
74: 11447-11455
[Abstract] [Full Text] -
Scholz, G., Hartson, S. D., Cartledge, K., Hall, N., Shao, J., Dunn, A. R., Matts, R. L.
(2000). p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck. Mol. Cell. Biol.
20: 6984-6995
[Abstract] [Full Text] -
Stepanova, L., Finegold, M., DeMayo, F., Schmidt, E. V., Harper, J. W.
(2000). The Oncoprotein Kinase Chaperone CDC37 Functions as an Oncogene in Mice and Collaborates with Both c-myc and Cyclin D1 in Transformation of Multiple Tissues. Mol. Cell. Biol.
20: 4462-4473
[Abstract] [Full Text] -
Kim, D.-W., Cochran, B. H.
(2000). Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fos Promoter. Mol. Cell. Biol.
20: 1140-1148
[Abstract] [Full Text] -
Farrell, A., Morgan, D. O.
(2000). Cdc37 Promotes the Stability of Protein Kinases Cdc28 and Cak1. Mol. Cell. Biol.
20: 749-754
[Abstract] [Full Text] -
O'Keeffe, B., Fong, Y., Chen, D., Zhou, S., Zhou, Q.
(2000). Requirement for a Kinase-specific Chaperone Pathway in the Production of a Cdk9/Cyclin T1 Heterodimer Responsible for P-TEFb-mediated Tat Stimulation of HIV-1 Transcription. J. Biol. Chem.
275: 279-287
[Abstract] [Full Text] -
Matsuda, S., Suzuki-Fujimoto, T., Minowa, A., Ueno, H., Katamura, K., Koyasu, S.
(1999). Temperature-sensitive ZAP70 Mutants Degrading through a Proteasome-independent Pathway. RESTORATION OF A KINASE DOMAIN MUTANT BY Cdc37. J. Biol. Chem.
274: 34515-34518
[Abstract] [Full Text] -
Lin, J.-H., Makris, A., McMahon, C., Bear, S. E., Patriotis, C., Prasad, V. R., Brent, R., Golemis, E. A., Tsichlis, P. N.
(1999). The Ankyrin Repeat-containing Adaptor Protein Tvl-1 Is a Novel Substrate and Regulator of Raf-1. J. Biol. Chem.
274: 14706-14715
[Abstract] [Full Text] -
Tikhomirov, O., Carpenter, G.
(2000). Geldanamycin Induces ErbB-2 Degradation by Proteolytic Fragmentation. J. Biol. Chem.
275: 26625-26631
[Abstract] [Full Text] -
Genevaux, P., Wawrzynow, A., Zylicz, M., Georgopoulos, C., Kelley, W. L.
(2001). DjlA Is a Third DnaK Co-chaperone of Escherichia coli, and DjlA-mediated Induction of Colanic Acid Capsule Requires DjlA-DnaK Interaction. J. Biol. Chem.
276: 7906-7912
[Abstract] [Full Text] -
Rao, J., Lee, P., Benzeno, S., Cardozo, C., Albertus, J., Robins, D. M., Caplan, A. J.
(2001). Functional Interaction of Human Cdc37 with the Androgen Receptor but Not with the Glucocorticoid Receptor. J. Biol. Chem.
276: 5814-5820
[Abstract] [Full Text] -
Shao, J., Grammatikakis, N., Scroggins, B. T., Uma, S., Huang, W., Chen, J.-J., Hartson, S. D., Matts, R. L.
(2001). Hsp90 Regulates p50cdc37 Function during the Biogenesis of the Active Conformation of the Heme-regulated eIF2alpha Kinase. J. Biol. Chem.
276: 206-214
[Abstract] [Full Text] -
Kumar, R., Grammatikakis, N., Chinkers, M.
(2001). Regulation of the Atrial Natriuretic Peptide Receptor by Heat Shock Protein 90 Complexes. J. Biol. Chem.
276: 11371-11375
[Abstract] [Full Text] -
Miyata, Y., Ikawa, Y., Shibuya, M., Nishida, E.
(2001). Specific Association of a Set of Molecular Chaperones Including HSP90 and Cdc37 with MOK, a Member of the Mitogen-activated Protein Kinase Superfamily. J. Biol. Chem.
276: 21841-21848
[Abstract] [Full Text] -
Scholz, G. M., Cartledge, K., Hall, N. E.
(2001). Identification and Characterization of Harc, a Novel Hsp90-associating Relative of Cdc37. J. Biol. Chem.
276: 30971-30979
[Abstract] [Full Text] -
Broustas, C. G., Grammatikakis, N., Eto, M., Dent, P., Brautigan, D. L., Kasid, U.
(2002). Phosphorylation of the Myosin-binding Subunit of Myosin Phosphatase by Raf-1 and Inhibition of Phosphatase Activity. J. Biol. Chem.
277: 3053-3059
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»