From the Department of Biochemistry and Molecular
Biology, Oklahoma State University, Stillwater, Oklahoma 74078, the
§ Kimmel Cancer Institute, Thomas Jefferson University,
Philadelphia, Philadelphia 19107, and the ¶ Harvard-Massachusetts
Institute of Technology, Division of Health Sciences and Technology,
Cambridge, Massachusetts 02139
Received for publication, August 19, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Recent studies indicate that p50cdc37
facilitates Hsp90-mediated biogenesis of certain protein kinases. In
this report, we examined whether p50cdc37 is required for the
biogenesis of the heme-regulated eIF2 The heme-regulated inhibitor
(HRI)1 of protein synthesis
is a protein-serine kinase which coordinates the synthesis of globin chains with the availability of heme in reticulocytes (reviewed in
Refs. 1 and 2). Under heme-deficient conditions, HRI phosphorylates the
The biogenesis and activation of HRI into an active heme-regulatable
eIF2 Under heme-deficient conditions, however, a portion of the population
of HRI molecules "transform" to produce kinase populations with
enhanced auto-kinase and eIF2 Hsp90 binds numerous other protein kinases, primarily when they are in
relatively inactive conformations (reviewed in Refs. 8 and 9). However,
Hsp90's association with inactive kinases reflects its essential
positive role in facilitating kinase folding, maturation, and
activation rather than a recognition of repressed kinase molecules
per se. Consistent with this model for Hsp90 function, the
Hsp90 chaperone machine does not interact with previously transformed
HRI molecules when their kinase activity is subsequently inhibited by
hemin addition, nor do such "repressed" HRI molecules require Hsp90
support to maintain their ability to reactivate in response to
heme-deficiency or stress (3). Consistent with the specific role of
Hsp90 in kinase biogenesis, both repressed HRI and transformed HRI
exhibit the same slow electrophoretic mobility on SDS-PAGE, indicating
that repressed HRI retains the hsp90-independent "transformed"
conformation (3).
Hsp90 functions in concert with a number of co-chaperones and cohorts
to facilitate their clients' acquisition of functional conformations
(reviewed in Ref. 10). Early studies detected a 50-kDa protein present
in heterocomplexes formed between Hsp90 and viral members of the Src
family of protein tyrosine kinases (reviewed in Ref. 11). This 50-kDa
protein has recently been identified as a product of the vertebrate
homolog of the yeast cell division cycle gene CDC37
(12-16). In vitro, the yeast p50cdc37 homolog
exhibits chaperone activity (17). Genetic (17-20) and biochemical (14,
21) studies indicate that p50cdc37 plays an essential positive
role in supporting a number of protein kinases. Therefore, we examined
whether p50cdc37 is a component of the Hsp90 chaperone complex
which is required for the maturation and transformation of HRI. In this
report, we present evidence that p50cdc37 acts in concert with
Hsp90 and facilitates the transformation and activation of HRI. We
further demonstrate that nucleotide-mediated conformational switching
of Hsp90 regulates the kinase binding activity of p50cdc37.
Construction of Plasmids for Expression of Wild Type and Mutant
p50cdc37 Proteins--
Two cDNA clones were obtained from
Genome Systems, Inc.: one lacking sequences encoding the N-terminal 8 amino acids (MVDYSVWD) of human p50cdc37 (dbEST number 810394, GenBankTM accession number AA172101 (designated here as
p50cdc37/N8aa)); and another containing sequence encoding the
full-length human Cdc37 gene product (dbEST number 321710, GenBankTM accession number R87892 (designated here
p50cdc37)). The coding sequence for p50cdc37/N8aa was
ligated into the bacterial expression vector pET-30a(+). Recombinant
His-tagged p50cdc37/N8aa was purified on Ni2+-NTA
resin (Ni2+-nitrilotriacetic acid coupled to agarose,
Qiagen), and used as antigen to produce polyclonal mouse ascites
anti-p50cdc37 antibody. For expression of full-length
recombinant p50cdc37 in Escherichia coli, the
sequence encoding full-length p50cdc37 was cloned into the
pQE-32 expression vector (Qiagen). (His6)-p50cdc37
was expressed in M15[pREP4] cells and purified on
Ni2+-NTA-agarose (Qiagen). Coomassie Blue staining of the
purified proteins separated by SDS-PAGE indicated that the proteins
were greater than 90% pure.
For in vitro translation, sequences encoding
p50cdc37/N8aa or full-length p50cdc37 were cloned into
a modified version of pSP64T (22), and translated by coupled
transcription/translation in RRL (23). The mutant p50cdc37/N8aa
protein produced includes 11 N-terminal amino acids (MADIGSEFGST) encoded by sequences carried over from the pET-30a(+) vector, followed
by the coding sequence of the human Cdc37 gene product from
His9 to Val378. The previously described
N-terminal deletion mutant of human p50cdc37 lacking 163 amino
acids from its N terminus (21) (designated here as
p50cdc37/ Expression and Purification of p50cdc37 Protein
Constructs from Eukaryotic Cells--
Human FLAG-tagged
p50cdc37 and FLAG-tagged p50cdc37/ Analysis of the Association of p50cdc37 with HRI in
Cultured Mammalian Cells--
Human K562 erythroleukemia cells were
cultured for 48 h at 37 °C and 5% CO2 in RPMI
medium supplemented with 10% fetal bovine serum (Life Technologies,
Inc.). Approximately 1 × 107 geldanamycin-treated (1 µg/ml for 2 h) or untreated (equivalent volume of
Me2SO for 2 h) cells were lysed in buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% Nonidet
P-40, 1% Triton X-100, 10% glycerol, 1 mM
Na3VO4, 2 mM EGTA, 1 mM
dithiothreitol, 50 mM glycerophosphate, 1 mM
NaF, 0.4 mM phenylmethylsulfonyl fluoride, and 10 µg/ml
each of leupeptin and aprotinin, and cell extracts were clarified.
Endogenous p50cdc37 was immunoadsorbed from cell extracts via a
mixture of rabbit, and mouse anti-p50cdc37 (Transduction
Laboratories) antibodies bound to GammaBind-Plus Sepharose (Amersham
Pharmacia Biotech), and the immunopellets were washed as described
previously (21). Half of the recovered p50cdc37 immunocomplexes
were analyzed by Western/ECL (Amersham Pharmacia Biotech) for the
presence of associated HRI, using guinea pig antibody raised against
the N-terminal 154 amino acids of rabbit HRI. Blots were then stripped,
and rabbit anti-p50cdc37 antibody was used to verify that
equivalent amounts of p50cdc37 were adsorbed from each extract.
The second half of the anti-p50cdc37 immunocomplexes were
analyzed by Western blotting with rat anti-Hsp90 monoclonal antibody
(SPA-830 from Stressgen).
De Novo Synthesis and Maturation of HRI in Rabbit Reticulocyte
Lysate--
Coupled transcription/translation (TnT) of plasmids
encoding HRI or (His7)-HRI in rabbit reticulocyte lysate
(TnT RRL) and the subsequent maturation of the expressed HRI or
(His7)-HRI in hemin-supplemented or heme-deficient RRL was
carried out as described previously (3, 4).
Co-translational Interaction of Hsp90 and p50cdc37 with
HRI--
HRI and luciferase were synthesized with concomitant
35S-radiolabeling in separate TnT RRL for 15 min at
30 °C. TnT RRL was either treated, or not treated with 1 mM puromycin for 5 min at 30 °C to release the nascent
polypeptide chains from polyribosome. The polyribsomes were then
isolated by centrifugation and analyzed by Western blotting for Hsp90
and p50cdc37 as described previously (3, 4).
Immunoadsorption of Protein Complexes--
Chaperone/cochaperone
and chaperone/kinase heterocomplexes were analyzed by reciprocal
immunoadsorptions utilizing resin-bound anti-p50cdc37 and
anti-His-tag antibodies as described previously (23). Nonimmune mouse
IgG (MOPC 21 from Sigma) was used as a control for nonspecific binding.
As an additional control for nonspecific binding,
[35S]HRI lacking the histidine tag was assessed in
parallel with reactions containing
(His7)-[35S]HRI. Relative amounts of
immunoadsorbed proteins were quantified by scanning densitometry of
autoradiograms or Western blots. Comparisons of changes in the relative
protein levels made in the text reflect corrections made for levels of
nonspecific binding.
Assay of the Effect of p50cdc37 and p50cdc37/
Alternatively, [35S]HRI or
(His7)-[35S]HRI were chased into 7 volumes of
heme-deficient protein synthesizing RRL containing purified (His6)-p50cdc37 (~1.5 µg/µl),
GST-p50cdc37 (~1 µg/µl), or an equivalent volume of the
appropriate control buffer. After 1 h of maturation at 30 °C,
samples were adsorbed to Ni2+-NTA resin (Fig.
6B) or GaG-agarose containing bound mouse monoclonal anti-(His5)-IgG (Fig. 7). Resins were subsequently
washed and analyzed for kinase activity (3, 4).
Assay of the Kinase Activity of
(His7)-[35S]HRI--
To quantify HRI kinase
activity, (His7)-[35S]HRI or
[35S]HRI (control for nonspecific binding) was captured
from RRL reaction mixtures on Ni2+-NTA-agarose (Qiagen)
that had been pre-equilibrated with 10 mM Tris-HCl, pH 7.4, 10 mM imidazole, and 50 mM NaF, or by
immunoadsorption with anti-(His5) monoclonal antibody.
Assays for eIF2 p50cdc37 Interacts with HRI in Concert with Hsp90--
To
determine whether p50cdc37 was a component of the chaperone
complex that Hsp90 forms with HRI, we examined the ability of
anti-His-tag antibodies to co-adsorb Hsp90 and p50cdc37 upon
immunoadsorption of newly synthesized
(His7)-[35S]HRI from RRL (Fig.
1A). As previously shown (3),
immunoadsorption of (His7)-[35S]HRI folding
intermediates immediately after their synthesis (8 min
post-translation) specifically co-adsorbed Hsp90. Immunoadsorption of
newly synthesized HRI also co-adsorbed p50cdc37, demonstrating
the existence of a heterocomplex containing p50cdc37 and these
newly synthesized HRI molecules. The amount of Hsp90 and
p50cdc37 co-adsorbed with HRI was similar whether the newly
synthesized HRI was immunoadsorbed from heme-replete or heme-deficient
RRL. Thus, heme did not directly regulate the polypeptide binding
activity of Hsp90 (3, 7) or p50cdc37.
Previously, we have demonstrated that Hsp90 interacts with nascent HRI
co-translationally (3). To determine whether p50cdc37 similarly
binds to HRI during its synthesis on ribosomes, polyribosomes were
isolated from RRL that was programmed with HRI template. For a negative
control, ribosomes were also prepared from RRL that was programmed with
luciferase template. Western blotting indicated that both Hsp90 and
p50cdc37 were present in the ribosome pellet containing bound
nascent HRI polypeptide chains (Fig. 1B), but were absent
when the nascent polypeptide chains had been released by incubation
with puromycin prior to isolation of the polyribosomes. Thus,
p50cdc37, like Hsp90, becomes specifically associated with HRI
prior to the completion of kinase synthesis and the release of newly
synthesized HRI from the ribosome.
The interaction of Hsp90 with HRI persists after release of newly
synthesized HRI from polyribosomes in heme-replete RRL (3). To assess
if the interaction between p50cdc37 and HRI was similarly
maintained, the newly synthesized
(His7)-[35S]HRI population was subjected to
maturational incubations in hemin-supplemented RRL where the
transformation of HRI into an active kinase is suppressed. After a
60-min incubation in the presence of hemin, neither the level of Hsp90
nor p50cdc37 associated with HRI declined significantly
relative to the level observed immediately after translation (Fig.
1A, lane 4 versus 6 and 8). Thus, in heme-replete
RRL, HRI continued to interact with p50cdc37 for prolonged
periods after its synthesis.
In heme-deficient RRL, HRI autophosphorylates and transforms into an
active kinase that no longer interacts with Hsp90 (3). To determine
whether transformation of HRI similarly terminated its interaction with
p50cdc37, the newly synthesized HRI kinase population was
incubated in hemin-deficient RRL for 60 min. Under these conditions,
~50% of the pulse-labeled (His7)-[35S]HRI
exhibited the slower electrophoretic mobility associated with
"transformation" (3). The amount of Hsp90 and p50cdc37 that
was co-adsorbed with (His7)-HRI from heme-deficient RRL decrease by ~67 and 75%, respectively, relative to that co-adsorbed with the "mature-competent/untransformed" HRI population present in
RRL held under heme-replete conditions (Fig. 1A, lane 2 versus 4). Thus, the transformation of HRI that was induced by hemin deficiency correlated with a reduction in the interaction of HRI with
Hsp90 and p50cdc37.
Previously, the interaction between Hsp90 and HRI was demonstrated to
be specific to the "untransformed" (fast electrophoretic mobility)
form of HRI (3). To determine whether p50cdc37, like Hsp90,
recognized a specific component of the HRI population, the kinase
populations associated with p50cdc37 were captured by
immunoadsorption with anti-p50cdc37 antibodies after incubation
of newly synthesized HRI in heme-replete or heme-deficient RRL for 8 or
60 min (Fig. 2). Only untransformed forms
of [35S]HRI, consisting of early folding intermediates (8 min chase) or mature-competent HRI (60 min chase), were specifically
coimmunoadsorbed with p50cdc37 from either heme-replete or
heme-deficient RRL (Fig. 2A, PEL). The 65% decrease in the
amount of untransformed HRI that was co-adsorbed with p50cdc37
from heme-deficient RRL correlated with the approximate 50%
transformation of the [35S]HRI population after 60 min of
incubation in heme-deficient RRL. The observation that transformed
(slow electrophoretic mobility form) HRI was not coimmunoadsorbed by
anti-p50cdc37 antibodies is consistent with the data presented
in Fig. 1, and supports the conclusion that p50cdc37, like
Hsp90, does not interact with transformed HRI.
Previously, we have demonstrated that the kinase activity of
transformed HRI is inhibited upon addition of hemin to heme-deficient maturation mixes, and that this population of "repressed HRI" molecules does not interact with Hsp90 (3). Similarly, we have observed
that repressed HRI did not co-adsorb with p50cdc37 from RRL
(not shown). Thus, p50cdc37 did not bind to transformed HRI
regardless of whether the kinase was active or repressed.
The N-terminal Domain of p50cdc37 Binds HRI Independent of
Hsp90--
Hsp90 and p50cdc37 both recognize untransformed
populations of HRI molecules (Fig. 1 and 2). To differentiate between
p50cdc37 associating with HRI directly or binding HRI
indirectly via its interaction with Hsp90, we compared the kinase
binding activity of affinity purified FLAG-tagged p50cdc37 with
that of a C-terminal truncated FLAG-tagged Cdc37 gene product (p50cdc37/
The above result implied that the C-terminal truncated Cdc37 gene
products could bind kinase folding intermediates independent of Hsp90
function. To test this, the effects of poisoning RRL with the
Hsp90-specific antagonist geldanamycin (25, 26) were assessed.
Consistent with previous reports (14, 21), the ability of full-length
FLAG-p50cdc37 gene product to bind untransformed kinase
molecules was markedly reduced (67%) in the presence of geldanamycin
(Fig. 3A, upper panel), but the presence of geldanamycin had
no inhibitory effect on the interaction of FLAG-p50cdc37 with
Hsp90 (Fig. 3A, middle panel). In contrast, inhibition of Hsp90 function by geldanamycin did not abrogate the ability of the
truncated Cdc37 gene product p50cdc37/
The effects of geldanamycin on chaperone binding to untransformed HRI
intermediates was confirmed by the reciprocal co-adsorption assays
(Fig. 3B). (His7)-HRI was immunoadsorbed
from control or geldanamycin-treated RRL and analyzed for the presence
of co-adsorbed Hsp90 and p50cdc37. While the association of
Hsp90 and p50cdc37 with (His7)-HRI in control RRL
was readily detected, treatment of RRL with geldanamycin resulted in
the near quantitative disruption of the association of Hsp90 and
p50cdc37 with (His7)-HRI (Fig. 3B), thus
confirming that inhibition of Hsp90 disrupted the direct recognition of
kinase molecules by p50cdc37.
The failure of C-terminal deleted p50cdc37 to bind Hsp90 (21)
suggested that the elements of p50cdc37 which mediated its
interaction with Hsp90 resided in the C-terminal domain of
p50cdc37. To further differentiate whether the C-terminal
region of p50cdc37 contained the Hsp90-binding site or whether
it was simply required to maintain the structure of an Hsp90-binding
site elsewhere in the protein, we constructed the corresponding
N-terminal deletion mutant of p50cdc37 lacking the first 163 amino acids from its N terminus (p50cdc37/ Mutation of the N Terminus of p50cdc37 Inhibits Its Binding
to HRI--
To further characterize the domains and motifs of
p50cdc37 required for kinase binding, we utilized a
p50cdc37 mutant whose first 8 N-terminal amino acids were
replaced by 11 residues encoded by irrelevant plasmid sequence
(p50cdc37/N8aa). Two observations indicated that replacement of
the first 8 residues of p50cdc37 did not cause global
disruption of p50cdc37 structure. (i) This mutation did not
compromise the ability of [35S]p50cdc37/N8aa to
associate with Hsp90 (Fig.
5A). (ii) This mutation did not compromise the structural integrity of p50cdc37/N8aa as
determined by mild proteolytic knicking: the sensitivity to protease
digestion and the pattern of proteolytic fragments generated were
essentially the same for the p50cdc37/N8aa and the wild type
p50cdc37 proteins (Fig. 5B). However,
p50cdc37/N8aa was not co-adsorbed with epitope-tagged
(His7)-HRI despite the efficient co-adsorption of HRI by
wild-type p50cdc37 and the equivalent amounts of each Cdc37
gene product present in these assays (Fig. 5C). These
observations demonstrated that the N-terminal 8 amino acids of
p50cdc37 were essential for its interaction with HRI.
p50cdc37 Enhances HRI Activity in Heme-deficient RRL in an
Hsp90-dependent Fashion--
To further characterize the
role of p50cdc37 in the biogenesis of HRI, we examined the
effect of affinity purified (His6)-p50cdc37 on the
transformation and activation of newly synthesized
[35S]HRI in heme-deficient RRL (Fig.
6). Aliquots of the HRI transformation reactions were also taken at the indicated times and analyzed by
SDS-PAGE for transformation-specific shifts in HRI's electrophoretic mobility (Fig. 6A). Maturation of HRI in heme-deficient RRL
containing (His6)-p50cdc37 produced transformed HRI
molecules equivalent to the storage buffer control. However, inclusion
of (His6)-p50cdc37 in HRI activation reactions
increased the proportion of transformed HRI that became
hyperphosphorylated at all times points examined relative to the
control. Results similar to those presented in Fig. 6 were also
obtained upon supplementing RRL with GST-tagged p50cdc37 (not
shown: e.g. see Fig. 7).
The effect of p50cdc37 on the eIF2
The data presented in Fig. 3 indicated that p50cdc37 required
geldanamycin-inhibitable Hsp90 function for it to form a stable complex with untransformed HRI intermediates. To determine whether endogenous Hsp90 function was also required for the stimulatory effects of recombinant p50cdc37 on HRI transformation and activity, the
effect of including geldanamycin in the transformation-activation
assays was assessed (Fig. 7). Similar to the results obtained with
(His6)-p50cdc37, supplementation of RRL with
recombinant GST-p50cdc37 enhanced the eIF2
To further test the hypothesis that the positive effect which
p50cdc37 has on HRI activity is modulated through its
interaction with Hsp90, we examined the effect of the
p50cdc37/
The effects of the p50cdc37 and p50cdc37/ p50cdc37 Does Not Enhance HRI Activity after Its
Transformation--
HRI can become further activated in RRL after its
transformation and release from Hsp90. This further activation of
transformed HRI is negatively attenuated by the interaction of Hsc70
with HRI, and is also accompanied by HRI's hyperphosphorylation (4). To verify that p50cdc37 functions are specific to untransformed
HRI, HRI was transformed in hemin-deficient RRL prior to the addition
of recombinant p50cdc37 (Fig. 9).
As an additional control, denatured (reduced carboxymethylated) bovine
serum albumin was similarly added to heme-deficient RRL after the
completion of HRI's transformation to block the ability of Hsc70 to
negatively attenuate HRI's activation (4, 27, 28). Addition of reduced
carboxymethylated bovine serum albumin to heme-deficient RRL stimulated
the eIF2 Interaction of p50cdc37 with HRI in Vivo--
Interactions
between endogenously expressed p50cdc37 and HRI were examined
in the chronic myelogenous leukemic K562 cell line to determine whether
p50cdc37 also interacted with HRI in vivo. K562
cells, cultured in the presence or absence of geldanamycin for 2 h, were lysed and endogenous p50cdc37 was immunoadsorbed (Fig.
10). Immunoblotting with HRI-specific antibodies indicated that HRI was co-adsorbed with p50cdc37
from extracts of control cells (Fig. 10, upper panel).
However, treatment of K562 cells with geldanamycin abolished the
interaction of p50cdc37 with HRI. In contrast, geldanamycin
treatment of K562 cells had no effect on the avid interaction between
p50cdc37 and Hsp90 in vivo (Fig. 10, middle
panel). Stripping and reprobing with anti-p50cdc37
antisera verified that equivalent amounts of p50cdc37 were
immunoprecipitated from the extracts of the untreated and geldanamycin-treated cells (Fig. 10, lower panel). These
results indicated that HRI existed in geldanamycin-sensitive native
complexes with p50cdc37 in cultured K562 cells.
p50cdc37 plays a positive role in facilitating activation
of HRI in response to heme-deficiency. Supplementation of
heme-deficient RRL with recombinant p50cdc37 during the HRI
activation process stimulates the production of HRI populations with
enhanced kinase activity and stimulates the production of HRI
populations with retarded electrophoretic mobilities which are
diagnostic of HRI activation/transformation. While our present work is
the first to directly address the role of p50cdc37 in kinase
biogenesis in an in vitro model system, these observations are consistent with biochemical and genetic data which indicate that
p50cdc37 plays an essential positive role in supporting the
function of numerous protein kinases (14, 17, 19-21, 29, 30). This positive function is consistent with a primary role for
p50cdc37 in a striking partnership with Hsp90 as a
kinase-specific cohort.
The stimulatory effect of p50cdc37 is specific to the HRI
activation process: p50cdc37 has no effect on the kinase
activity or electrophoretic mobility of HRI when added subsequent to
HRI's transformation. Consistent with this specificity,
p50cdc37 interacts with newly synthesized HRI and with the
inactive HRI population maintained in the presence of hemin, but does
not interact with HRI that has been transformed in response to heme
deficiency or with transformed HRI whose activity has been repressed by
hemin. Therefore, like Hsp90's association with inactive kinases, the interaction of p50cdc37 with HRI appears to reflect its role in
facilitating kinase folding, maturation, and activation, and not its
association with kinase molecules whose activity is repressed per
se. Similarly, the fast electrophoretic form of Raf-1 has been
found to be preferentially co-adsorbed with p50cdc37 from
cultured cells.2 The observed
specificity of p50cdc37 for untransformed HRI intermediates is
also consistent with other studies demonstrating that p50cdc37
and Hsp90 recognize kinase molecules which represent specific maturation or activation intermediates (11, 14, 20, 29).
p50cdc37 has at least two semi-independent units that mediate
its biological activity. The N-terminal half of p50cdc37
contains an autonomous kinase-binding unit, since it binds HRI (Fig. 3)
and Raf-1 (21) when its C-terminal half was deleted. Consistent with
this localization, the first 8 amino acids at the N terminus of
p50cdc37 were found to be essential to its kinase binding
activity (Fig. 5). Thus, these residues either participate directly in
kinase binding or provide essential support to the structure of an
N-terminal kinase-binding domain. Additionally, complexes formed
between p50cdc37/ In contrast to the N-terminal region, the C-terminal half of
p50cdc37 contains one or more motifs or domains which mediate
its interaction with Hsp90. Deletion of the C-terminal half of
p50cdc37 abolishes its interaction with Hsp90 (Fig. 3), a
finding consistent with previous in vivo characterizations
of the truncated Cdc37 gene product (21). However, it was not
previously clear if such truncations directly deleted a discrete
Hsp90-binding domain or motif, or if they indirectly compromised Hsp90
binding by disrupting essential support of other protein structures
present within the N-terminal half of p50cdc37. We demonstrate
here that the C-terminal half of p50cdc37 contains
semi-autonomous Hsp90 binding activity (Fig. 4). Thus, sequences or
structures present in the C-terminal region of p50cdc37
directly mediate its binding to Hsp90. However, despite the observation that the N-terminal domain of p50cdc37 does not stably interact
with Hsp90, we cannot rule out the possibility that motifs in regions
outside of the C terminus of p50cdc37 may also contribute
directly or indirectly to the interaction of p50cdc37 with
Hsp90.
Our data indicate that p50cdc37 exerts its biochemical effects
in cooperation with Hsp90. This conclusion is indicated by the
observation that the Hsp90-specific inhibitor geldanamycin inhibits the
ability of recombinant p50cdc37 to stimulate HRI transformation
(Fig. 7). Geldanamycin similarly inhibits the ability of
p50cdc37 overexpression to stimulate Raf activation in cultured
cells (21), indicating that Hsp90 is required for p50cdc37 to
exert its positive effect on Raf-1 activation in vivo.
Furthermore, these results do not reflect a direct disruption of the
Hsp90-p50cdc37 interaction (Figs. 3 and 10) (21). Instead,
geldanamycin disrupts the normal association of p50cdc37 with
HRI in RRL and in cultured K562 cells (Figs. 3 and 10). This finding is
consistent with previous characterization of the effects of
geldanamycin on p50cdc37 interactions with CDK4, Raf-1, and KSR
in vivo (14, 21, 32).
In contrast, geldanamycin does not inhibit the ability of C-terminal
truncated Cdc37 gene products to bind kinase folding intermediates;
inhibition of Hsp90 actually increases the availability of HRI
molecules for binding to truncated cdc37 proteins (Fig. 3). This
observation has 3 important implications. 1) Geldanamycin's disruption
of p50cdc37-kinase heterocomplexes does not result from
sequestration or masking of kinase-folding intermediates by
geldanamycin-poisoned Hsp90 machinery. 2) The physical interaction of
full-length p50cdc37 with Hsp90 determines geldanamycin's
ability to inhibit the binding of p50cdc37 to kinase molecules.
3) Geldanamycin-inhibitable Hsp90 function is necessary to assemble
heterocomplexes formed directly between kinase molecules and
full-length p50cdc37 and to chaperone the kinase substrate
toward its activation-specific conformation.
Geldanamycin is known to act as a specific inhibitor of Hsp90 through
its ability to bind avidly within Hsp90's nucleotide-binding pocket,
thus blocking the binding of ATP (26, 33, 34). This binding prevents
nucleotide-mediated switching between alternative Hsp90 conformations
(33, 35-37) and abolishes Hsp90's ability to establish high affinity
interactions with its substrates (23, 37), thus inhibiting
Hsp90-supported kinase function. Therefore, geldanamycin's ability to
inhibit the binding of wild type p50cdc37 to kinase-folding
intermediates demonstrates that Hsp90's nucleotide-mediated conformational switching regulates the direct binding of
p50cdc37 to Hsp90's kinase clients. Additionally, our data
illuminate potential mechanisms underlying the chemotherapeutic
potential of benzoquinonoid ansamycins.
kinase (HRI) in reticulocyte
lysate. p50cdc37 interacted with nascent HRI co-translationally
and this interaction persisted during the maturation and activation of
HRI. p50cdc37 stimulated HRI's activation in response to heme
deficiency, but did not activate HRI per se.
p50cdc37 function was specific to immature and inactive forms
of the kinase. Analysis of mutant Cdc37 gene products indicated
that the N-terminal portion of p50cdc37 interacted with
immature HRI, but not with Hsp90, while the C-terminal portion of
p50cdc37 interacted with Hsp90. The Hsp90-specific inhibitor
geldanamycin disrupted the ability of both Hsp90 and p50cdc37
to bind HRI and promote its activation, but did not disrupt the native
association of p50cdc37 with Hsp90. A C-terminal truncated
mutant of p50cdc37 inhibited HRI's activation, prevented the
interaction of Hsp90 with HRI, and bound to HRI irrespective of
geldanamycin treatment. Additionally, native complexes of HRI with
p50cdc37 were detected in cultured K562 erythroleukemia cells.
These results suggest that p50cdc37 provides an activity
essential to HRI biogenesis via a process regulated by
nucleotide-mediated conformational switching of its partner
Hsp90.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of eukaryotic translational initiation factor eIF2.
Phosphorylation of eIF2
causes an inhibition of polypeptide chain
initiation and the arrest of protein synthesis, preventing the
synthesis of apo-globin chains in the absence of heme. HRI is also
activated under heme-replete conditions in response to a host of other
adverse environmental stimuli, such as heat shock, agents that generate
oxidative stress, and the presence of denatured proteins (1, 2).
kinase requires its functional interaction with the chaperone
machinery containing the 90-kDa heat shock protein (Hsp90) and the
70-kDa heat shock cognate protein (Hsc70) (3, 4). During HRI biogenesis
and its subsequent transformation and activation, several discrete HRI
intermediates are generated; these intermediates can be distinguished
on the basis of their competence to become an active kinase in response
to heme deficiency or upon treatment with sulfhydryl reactive compounds
such as N-ethylmaleimide. Immediately after their
synthesis, HRI molecules are not active in heme-replete or
heme-deficient rabbit reticulocyte lysate (RRL) and cannot be activated
by N-ethylmaleimide treatment. This immature population
interacts with Hsp90 and Hsc70 (3-7). Subsequent to this immature
phase, a "mature-competent" HRI population appears. The
mature-competent population can be activated by heme-deficiency or
treatment with N-ethylmaleimide, but remains quiescent in
the absence of such "stimuli." The mature-competent HRI population continues to interact with Hsp90 machinery, and this interaction is
required to maintain HRI's ability to respond to heme deficiency (3).
kinase activities. Transformation of
HRI requires Hsp90 function and autophosphorylation of HRI, and
correlates with the production of a population of HRI molecules which
exhibit retarded electrophoretic mobility on SDS-PAGE (3). This
transformation frees HRI from its functional requirements for Hsp90 and
terminates its physical association with Hsp90 machinery (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N) was similarly cloned into and expressed from
pSP64T. Tryptic fingerprints were generated from wild type
[35S]p50cdc37 and
[35S]p50cdc37/N8aa proteins as described
previously for de novo synthesized [35S]p56lck kinase (24).
C were expressed
in and purified from Sf9 cell as described previously (21). FLAG-p50cdc37/
C lacks 214 amino acids from the C terminus of
p50cdc37. GST-tagged p50cdc37 was also expressed in and
purified from COS-1 cells as described previously (21).
C
on HRI Transformation--
[35S]HRI or
(His7)-[35S]HRI was synthesized in TnT RRL
(3, 4). Subsequently, 4 µl of the TnT RRL was transferred to
heme-deficient RRL (30 µl) which contained 10 µl of immunoresin (M2
anti-FLAG-agarose) that had been previously saturated with
FLAG-peptide, FLAG-p50cdc37, or FLAG-p50cdc37/
C. The
reaction mixtures were incubated at 30 °C for 1 h. After washing of the immunoresins, the immunoresins were analyzed for bound
[35S]HRI, HRI kinase activity, and/or associated
chaperones, as specified in the figure legends.
kinase activity were performed as described (3, 4).
The kinase activity of HRI was quantified by scanning densitometry of
the 32P-labeled eIF2
band visualized by autoradiography
and expressed as optical density (O.D.) × mm2.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of endogenous p50cdc37
and Hsp90 with newly synthesized
(His7)-[35S]HRI in RRL. A,
(His7)-[35S]HRI (lanes 2, 4, 6, and 8) and [35S]HRI lacking the
(His7)-tag (lanes 1, 3, 5, and
7) were synthesized and then matured in normal
heme-deficient (lanes 1, 2, 5, and 6) or
hemin-supplemented (lanes 3, 4, 7, and 8) RRL as
described under "Experimental Procedures." Aliquots (30 µl) of
the reaction mixtures were taken after 8-min (lanes 5-8) or
60-min (lanes 1-4) of maturation and mixed with GaG-agarose
pre-coupled with anti-(His5) monoclonal antibody as
described under "Experimental Procedures." After washing the immune
pellets, samples were analyzed by SDS-PAGE, followed by transfer to
polyvinylidene difluoride membrane.
(His7)-[35S]HRI was visualized by
autoradiography (upper panel: TR, transformed HRI
with slower electrophoretic mobility; MC, mature competent
form of HRI with faster electrophoretic mobility). Endogenous RRL Hsp90
and p50cdc37 that was specifically co-adsorbed with
(His7)-[35S]HRI were detected by Western
blotting membranes with anti-Hsp90 (middle panel) or
anti-p50cdc37 (lower panel) antibodies.
HC, antibody heavy chain. Band densities were quantified by
scanning densitometry and expressed as optical density × mm2 (numbers above each panel). Densitometry
indicated that equivalent amounts of total
(His7)-[35S]HRI were specifically
immunoadsorbed (even lanes). B, co-translational
interaction of HRI with Hsp90 and p50cdc37 in RRL: TnT RRLs
were programmed with HRI (lanes 3 and 4) or
luciferase template (lanes 1 and 2) for 15 min at
30 °C. Translation mixes were then either treated (lanes
2 and 4) or not treated (lanes 1 and
3) with 1 mM puromycin for 5 min at 30 °C to
release the nascent chains, followed by separation on 15 to 40%
sucrose gradients as described under "Experimental Procedures."
Isolated polyribosomes were analyzed by SDS-PAGE, followed by Western
blot detection for Hsp90 and p50cdc37.
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Fig. 2.
Interaction of newly synthesized HRI with
endogenous p50cdc37 in RRL. [35S]HRI was
synthesized in TnT RRL and subsequently matured in normal
hemin-supplemented (10 µM hemin) (lanes 1, 2, 5, and 6) or heme-deficient (lanes 3, 4, 7, and 8) RRL protein synthesis mixtures as described under
"Experimental Procedures." Aliquots (30 µl) of the reaction
mixtures were taken after 8 (lanes 1-4) or 60 min
(lanes 5-8) of maturation and mixed with GaG-agarose
pre-coupled with anti-p50cdc37 polyclonal antibodies
(lanes 2, 4, 6, and 8) or nonimmune control mouse
IgG (lanes 1, 3, 5, and 7) as described under
"Experimental Procedures." A 2-µl aliquot of each reaction was
also taken at 8 or 60 min for analysis of the forms of
[35S]HRI which were present in the RRL maturation
mixtures prior to immunoadsorption. After washing the immune pellets,
samples were analyzed by SDS-PAGE, followed by transfer to
polyvinylidene difluoride membrane and autoradiography. A,
[35S]HRI co-adsorbed with p50cdc37 (A,
PEL); [35S]HRI present in the RRL maturation
mixes prior to immunoadsorption (A, UF)
[35S]HRI*, transformed HRI with slower electrophoretic
mobility. Band densities were quantified by scanning densitometry and
expressed as optical density × mm2 (numbers
above each panel). B, p50cdc37 that was
specifically immunoadsorbed was detected by probing the polyvinylidene
difluoride membrane of PEL panel with anti-p50cdc37 polyclonal
antibodies. HC, antibody heavy chain. Densitometry indicated
that equivalent amounts of total [35S]HRI were present
during the immunoadsorption (UF), and that equivalent
amounts of p50cdc37 were specifically immunoadsorbed.
C). p50cdc37/
C had previously been
shown to bind the Raf-1 kinase, but not Hsp90 (21). HRI was readily
captured by resins containing the full-length wild-type Cdc37 gene
product and by resins containing the truncated Cdc37 gene product
p50cdc37/
C, but was not captured by anti-FLAG control resins
(Fig. 3A, upper panel). In
contrast, Hsp90 was adsorbed by resins containing the wild-type Cdc37
gene product, but only barely detectable amounts of Hsp90 were bound to
resins containing p50cdc37/
C truncated Cdc37 gene product
(Fig. 3A, middle panel). These findings suggested that Cdc37
gene products did not bind to kinase folding intermediates indirectly
via an interaction with kinase-bound Hsp90.
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Fig. 3.
Effect of geldanamycin on the association of
HRI with p50cdc37 and the
p50cdc37/ C mutant. A,
[35S]HRI was synthesized in TnT RRL in the presence of 10 µg/ml geldanamycin (lanes 2, 4, and 6) or an
equivalent amount of Me2SO (lanes 1, 3, and
5), followed by maturation in normal heme-deficient RRL
containing M2-agarose pre-saturated with control FLAG-peptide
(lanes 1 and 2), or Sf9 cell-expressed
FLAG-tagged p50cdc37 (lanes 3 and 4), or
FLAG-tagged p50cdc37/
C (lanes 5 and 6)
for 60 min at 30 °C as described under "Experimental
Procedures," followed by the addition of 20 mM sodium
molybdate. After washing the M2-agarose pellets with buffer containing
20 mM sodium molybdate, samples were analyzed by SDS-PAGE,
followed by transfer to polyvinylidene difluoride membrane. Co-adsorbed
[35S]HRI was detected by autoradiography (upper
panel). Co-adsorbed Hsp90 was visualized by Western blot with
anti-Hsp90 antibodies (middle panel). Band densities were
quantified by scanning densitometry and expressed as optical
density × mm2 (numbers above each panel).
M2 resin-immobilized FLAG-tagged proteins were visualized by Coomassie
Blue staining (lower panel,
C, p50cdc37/
C).
B, (His7)-[35S]HRI (lanes
2 and 4) and [35S]HRI lacking
(His7)-tag (lanes 1 and 3) were
synthesized for 25 min in TnT RRL in the presence of 10 µg/ml
geldanamycin (lanes 3 and 4) or an equivalent
amount of Me2SO (lanes 1 and 2).
20-µl aliquots were immunoadsorbed with GaG-agarose containing bound
anti-(His5) monoclonal antibody as described under
"Experimental Procedures." After washing the immune pellets,
samples were analyzed by SDS-PAGE, followed by transfer to
polyvinylidene difluoride membrane.
(His7)-[35S]HRI was visualized by
autoradiography (upper panel). Hsp90 (middle
panel) and p50cdc37 (lower panel) were detected
by Western blot with anti-Hsp90 and anti-p50cdc37 antibodies,
respectively. HC, antibody heavy chain.
C to bind
untransformed HRI. In fact, geldanamycin treatment appeared to
potentiate the kinase binding activity of truncated Cdc37 gene
products. Thus, the resistance of the interaction between p50cdc37/
C and HRI to inhibition by geldanamycin indicated
that the N-terminal half of the Cdc37 gene product was capable of
binding kinase folding intermediates independent of Hsp90.
N).
p50cdc37/
N was synthesized in TnT RRL and assayed for Hsp90
binding. Anti-Hsp90 antibody specifically co-adsorbed
[35S]p50cdc37/
N in conjunction with Hsp90
(Fig. 4A). Furthermore,
anti-His antibodies specifically co-adsorbed Hsp90 with His-tagged
p50cdc37/
N (Fig. 4B). These findings indicated
that the C-terminal domain of p50cdc37 contained elements
adequate for Hsp90 binding, independent of the presence of
p50cdc37's N-terminal domain.
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Fig. 4.
Interaction of the N-terminal domain deletion
mutant of p50cdc37,
p50cdc37/ N, with endogenous Hsp90 in
RRL. A, [35S]p50cdc37/
N was
synthesized in TnT RRL at 30 °C for 25 min. Aliquots of (20 µl)
TnT RRL containing [35S]p50cdc37/
N were then
immunoadsorbed with goat anti-mouse IgM cross-linked to agarose
pre-coupled with mouse monoclonal anti-Hsp90 IgM, 8D3 (lane
2), or equivalent amount of nonimmune mouse IgM (lane
1). Samples were analyzed by SDS-PAGE, followed by transfer to
polyvinylidene difluoride membrane. Immunoadsorbed Hsp90 was visualized
by Western blot with anti-Hsp90 antibody (upper panel), and
co-adsorbed [35S]p50cdc37/
N was detected by
autoradiography (lower panel). B,
(His6)-[35S]p50cdc37/
N was
synthesized in TnT RRL at 30 °C for 25 min. Aliquots of (20 µl)
TnT RRL containing
(His6)-[35S]p50cdc37/
N were then
immunoadsorbed with GaG-agarose containing bound
anti-(His5)-antibody (lane 2) or an equivalent
amount of nonimmune mouse IgG (lane 1). Samples were
analyzed by SDS-PAGE, followed by transfer to polyvinylidene difluoride
membrane. Immunoprecipitated
(His6)-[35S]p50cdc37/
N was
visualized by autoradiography (lower panel) and co-adsorbed
Hsp90 was detected by Western blot with anti-Hsp90 antibody
(upper panel).
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Fig. 5.
Effect of mutation of the N-terminal amino
acids of p50cdc37 on the interaction of p50cdc37 with
HRI and Hsp90. A, co-adsorption of
p50cdc37/N8aa with endogenous Hsp90:
[35S]p50cdc37/N8aa was synthesized in TnT RRL at
30 °C for 40 min, followed by a 40-min maturation in the presence of
ATA (60 µM). An aliquot of the TnT RRL mixture (30 µl)
was then immunoadsorbed with GaG-agarose containing bound mouse
polyclonal anti-Hsp90 antibodies (lane 2) or nonimmune mouse
IgG (lane 1) as described under "Experimental
Procedures." Samples were separated by SDS-PAGE, transferred to
polyvinylidene difluoride membrane, and co-adsorbed
[35S]p50cdc37/N8aa was detected by
autoradiography (lower panel) whereas the immunoprecipitated
Hsp90 was visualized by Western blot analysis with anti-Hsp90 antibody
(upper panel). B, comparison of proteolytic
peptide mapping between wild type p50cdc37 and
p50cdc37/N8aa mutant: wild type p50cdc37
(WT) and p50cdc37/N8aa (N8aa) were synthesized with
concomitant 35S-radiolabeling in separate TnT RRL at
30 °C for 40 min, followed by a 1-h maturation in the presence of
ATA (60 µM). TnT RRL containing
[35S]p50cdc37 or
[35S]p50cdc37/N8aa were then diluted into 3 volumes of proteolysis assay buffer (24) containing four different
concentrations of trypsin as specified in the figure, followed by a
6-min digestion on ice. Reaction was terminated by adding boiling SDS
sample buffer to the samples. Proteolytic peptide fragments were
separated by 12% SDS-PAGE, followed by autoradiography analysis.
FL denotes full-length p50cdc37. C,
(His7)-[35S]HRI was pulse-labeled in TnT RRL.
Aliquots (10 µl) of TnT RRL containing
(His7)-[35S]HRI were then chased by
incubation in hemin-supplemented RRL containing previously synthesized
[35S]p50cdc37 (PEL: lanes 1 and
2) or [35S]p50cdc37/N8aa (PEL:
lanes 3 and 4) for 8 min at 30 °C as described under
"Experimental Procedures."
(His7)-[35S]HRI was immunoadsorbed from the
reaction mixtures with anti-(His5) antibody (lanes
2 and 3) or equivalent amount of nonimmune mouse IgG
(lanes 1 and 4). Samples were analyzed by
SDS-PAGE, followed by transfer to polyvinylidene difluoride membrane.
Immunoadsorbed (His7)-[35S]HRI (upper
panel of PEL) and co-adsorbed [35S]p50cdc37
or [35S]p50cdc37/N8aa (lower panel of
PEL) were visualized by autoradiography. A 2-µl aliquot of each
reaction was also taken prior to immunoadsorption to verify that
equivalent amounts of [35S]p50cdc37 or
[35S]p50cdc37/N8aa were synthesized and present
in each reaction mixture (UF panel).
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Fig. 6.
Effect of purified
(His6)-p50cdc37 on HRI transformation
(A) and activity (B) in
heme-deficient RRL. A, [35S]HRI was
pulse-labeled in TnT RRL. TnT RRL containing [35S]HRI (3 µl) was then chased into heme-deficient RRL mixture (22 µl) which
was supplemented with 12 µg of (His6)-p50cdc37
(even-numbered lanes) or an equivalent amount of buffer
(odd-numbered lanes) in which the
(His6)-p50cdc37 was stored as described under
"Experimental Procedures." Aliquots of maturation RRL mixtures were
taken after 0, 10, 20, 30, 45, and 60 min of incubation at 30 °C for
direct analysis of HRI transformation. Samples were separated by
SDS-PAGE, transferred to polyvinylidene difluoride membrane, and
visualized by autoradiography. The amount of transformed
(TR) plus hyperphosphorylated (HP)
[35S]HRI was quantified by scanning densitometry and
expressed as optical density × mm2 (numbers
above lanes in panel). B, for analysis of HRI activity,
aliquots (50 µl) of the RRL reaction mixtures containing
(His7)-[35S]HRI ((His7)-HRI:
lanes 3 and 4) or [35S]HRI
(NS: lanes 1 and 2) matured in the presence of 25 µg of (His6)-p50cdc37 (lanes 1 and
3), or an equivalent volume of storage buffer (lanes
2 and 4) were taken after 60 min of incubation at
30 °C and adsorbed to Ni2+-NTA-agarose as described
under "Experimental Procedures." After washing away unbound
materials, the eIF2 kinase activity of the adsorbed HRI was assayed
as described under "Experimental Procedures." Samples were
separated by SDS-PAGE, transferred to polyvinylidene difluoride
membrane, and [35S]HRI (B, lower panel) and
[32P]eIF2
(B, upper panel) were detected by
autoradiography. The amount of [32P]eIF2
was
quantified by scanning densitometry and expressed as optical
density × mm2 (numbers above the eIF2
panel).
Densitometry indicated that equivalent amounts of total
[35S]HRI were specifically immunoadsorbed (lanes
3 and 4). HP, hyperphosphorylated form of
HRI; TR, transformed form of HRI; MC, mature
competent form of HRI.
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Fig. 7.
The effect of geldanamycin on the ability of
GST-p50cdc37 to enhance HRI transformation and activation in
heme-deficient RRL. (His7)-[35S]HRI
(lanes 2, 4, 6, and 8) and [35S]HRI
lacking the (His7)-tag (lanes 1, 3, 5, and
7) were pulse-labeled in TnT RRL and then chased and matured
at 30 °C for 1 h in heme-deficient RRL containing
GST-p50cdc37 (lanes 1-4) or GST-p50cdc37
purification buffer (lanes 5-8) in the presence of 10 µg/ml geldanamycin (lanes 3, 4, 7, and 8) or
Me2SO (lanes 1, 2, 5, and 6) as
described under "Experimental Procedures." RRL maturation mixtures
were adjusted to 20 mM sodium molybdate and immunoadsorbed
with GaG-agarose containing bound anti-(His5) monoclonal
antibody. After washing away unbound material, the eIF2 kinase
activity of adsorbed HRI was assayed as described under "Experimental
Procedures." Samples were separated by SDS-PAGE, followed by transfer
to polyvinylidene difluoride membrane.
(His7)-[35S]HRI that was specifically
immunoadsorbed (lower panel) and [32P]eIF2
(upper panel) were detected by autoradiography. The amount
of [32P]eIF2
was quantified by scanning densitometry
and expressed as optical density × mm2 (numbers
above the eIF2
panel). Densitometry indicated that equivalent
amounts of total (His7)-[35S]HRI were
specifically immunoadsorbed (even lanes). HP,
hyperphosphorylated form of HRI; TR, transformed form of
HRI; MC, mature competent form of HRI.
kinase activity of HRI
was also assessed (Fig. 6B). After 60 min of incubation, the
eIF2
kinase activity of HRI matured in p50cdc37-supplemented
heme-deficient RRL was nearly 3-fold higher relative to HRI matured in
control RRL supplemented with buffer alone. Thus, the physical
association of p50cdc37 with untransformed intermediates of HRI
appeared to reflect a positive role for this co-chaperone in HRI
maturation/activation pathway, as supplementation of RRL with
p50cdc37 promoted the acquisition of an active conformation.
kinase activity
of HRI 3-fold over the control, and this enhancement of activity
correlated with the generation of a [35S]HRI species with
a slower electrophoretic mobility (lower panel, denoted
HP). Addition of geldanamycin inhibited the activation of
HRI in both buffer-supplemented control and
p50cdc37-supplemented heme-deficient RRL (Fig. 7, upper
panel). Consistent with this finding, the slow electrophoretic
form of HRI representing transformed HRI molecules was not produced in
geldanamycin-poisoned reactions (Fig. 7, lower panel). These
results indicated that geldanamycin inhibitable Hsp90 function was
essential for the observed stimulatory effects of p50cdc37 on
HRI transformation-activation.
C mutant on the transformation and activation of
HRI in heme-deficient RRL (Fig. 8). The
data presented in Fig. 3 indicate that complexes formed between HRI and
the p50cdc37/
C mutant lacked bound Hsp90. Thus, the
p50cdc37/
C mutant was predicted to retard HRI transformation
and activation when added to heme-deficient RRL. (His7)-HRI
(Fig. 8, even lanes) or non-His-tagged HRI (Fig. 8,
odd lanes) was synthesized and then matured in
heme-deficient RRL in the presence of control M2 anti-FLAG tag-agarose
presaturated with FLAG-peptide, FLAG-tagged-p50cdc37, or
FLAG-tagged p50cdc37/
C. The electrophoretic mobility
of the non-His-tagged [35S]HRI that was specifically
associated with p50cdc37 or p50cdc37/
C indicated
that the HRI was not transformed, and the p50cdc37- and the
p50cdc37/
C-bound non-His-tagged [35S]HRI were
as inactive in phosphorylating eIF2
as the material present in the
control pellet for nonspecifically bound activity (Fig. 8,
lanes 1, 3, and 5).
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Fig. 8.
Effect of removal of the C-terminal region of
p50cdc37 on HRI transformation and activation in heme-deficient
RRL. (His7)-[35S]HRI (lanes 2, 4, and 6) and [35S]HRI lacking the
(His7)-tag (lanes 1, 3, and 5) were
pulse-labeled in TnT RRL and then chased and matured at 30 °C for
1 h in normal heme-deficient RRL containing M2-agarose that had
been pre-saturated with FLAG peptide (Eastman Kodak Co.) (lanes
1 and 2), FLAG-tagged p50cdc37 (lanes
3 and 4), or FLAG-tagged p50cdc37/ C
(lanes 5 and 6) as described under
"Experimental Procedures." The RRL maturation mixtures were then
immunoadsorbed with GaG-agarose containing bound
anti-(His5) monoclonal antibody. After washing away unbound
material, the eIF2
kinase activity of adsorbed
(His7)-HRI was assayed as described under "Experimental
Procedures." Samples were separated by SDS-PAGE, followed by transfer
to polyvinylidene difluoride membrane.
(His7)-[35S]HRI that was specifically
immunoadsorbed (lower panel) and [32P]eIF2
(upper panel) were detected by autoradiography. The amount
of [32P]eIF2
was quantified by scanning densitometry
and expressed as O.D. × mm2 (numbers above the
eIF2
panel). Densitometry indicated that equivalent amounts of total
(His7)-[35S]HRI were specifically
immunoadsorbed (even lanes).
C,
p50cdc37/
C that was also highly phosphorylated and labeled
by [
-32P]ATP during the kinase assay. "*", an
unknown protein contaminant present in the purified eIF2 preparation,
which became phosphorylated and labeled by [
-32P]ATP
during kinase assay.
C mutant on
the eIF2
kinase activity and electrophoretic properties of the total
(His7)-[35S]HRI population matured in these
RRLs and subsequently adsorbed to anti-His resin were also examined
after adsorption (Fig. 8, lanes 2, 4, and 6). The
eIF2
kinase activity of the
(His7)-[35S]HRI that was matured in
heme-deficient RRL in the presence of wild type p50cdc37 was
almost twice as much as the eIF2
kinase activity present in the
control RRL containing M2 anti-FLAG tag-agarose with bound FLAG-peptide. In contrast, the eIF2
kinase activity of the
(His7)-HRI that was matured in the presence of the
p50cdc37/
C mutant was decreased by 60% relative to the
kinase activity adsorbed from control RRL. A portion of the
(His7)-[35S]HRI that was matured in the
presence of wild type p50cdc37 exhibited a slower
electrophoretic mobility than the
(His7)-[35S]HRI that was matured in control
RRL, indicating it had become hyperphosphorylated (Fig. 8, lower
panel, lane 4 versus 2 (HP)). This shift in
electrophoretic mobility caused the band to appear more diffuse.
However, quantification of the total amount of
(His7)-[35S]HRI molecules exhibiting slower
electrophoretic mobilities indicated that nearly the same amount of
(His7)-[35S]HRI became transformed in RRL
supplemented with wild type p50cdc37 (O.D. × mm2 = 4.14) compared with control RRL (O.D. × mm2 = 4.11). This
observation is consistent with the enhanced eIF2
kinase activity
displayed by the (His7)-[35S]HRI that was
matured in the presence of wild type p50cdc37. In addition, the
fast electrophoretic mobility of the
(His7)-[35S]HRI that was matured in the
presence of p50cdc37/
C indicated that it remained mostly
untransformed, consistent with its suppressed kinase activity (Fig. 8,
lane 6). These results indicate that Cdc37 gene products
which do not interact with Hsp90 may act as dominant negative
inhibitors of p50cdc37 function: a conclusion consistent with
the observation that Hsp90 is required for p50cdc37 to exert
its positive effect on HRI activity upon transformation.
kinase activity of HRI 4-fold compared with the activity
observed in control RRL. In contrast, the addition of an equivalent
amount of recombinant p50cdc37 to heme-deficient RRL subsequent
to HRI transformation caused little further stimulation of HRI's
eIF2
kinase activity (approximately 10%) compared with the control.
Therefore, the stimulatory effects of p50cdc37 were specific to
that HRI population whose transformation had been previously documented
to be functionally dependent upon geldanamycin-inhibitable Hsp90
function.
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Fig. 9.
Effect of recombinant FLAG-p50cdc37
on HRI kinase activity upon addition subsequent to HRI
transformation. (His7)-[35S]HRI was
pulse-labeled in TnT RRL and chased and matured at 30 °C for 1 h in normal heme-deficient RRL as described under "Experimental
Procedures." The effect of FLAG-peptide alone (lane 1),
FLAG-tagged p50cdc37 (lane 2), or FLAG-peptide plus
soluble reduced carboxymethylated bovine serum albumin (lane
3) was evaluated by incubating the RRL maturation mixtures for an
additional 20 min at 30 °C. The RRL mixtures were then
immunoadsorbed with GaG-agarose containing bound
anti-(His5) monoclonal antibody. A 2-µl aliquot of each
maturation mixture was taken prior to immunoprecipitation to detect
different forms of [35S]HRI generated (UF
panel). After washing away the unbound material, the eIF2
kinase activity of adsorbed (His7)-HRI was assayed as
described under "Experimental Procedures." Samples were separated
by SDS-PAGE, followed by transfer to polyvinylidene difluoride
membrane, and autoradiography detection of [32P]eIF2
(PEL panel). The amount of [32P]eIF2
was
quantified by scanning densitometry and expressed as O.D. × mm2 (numbers above the PEL panel).
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Fig. 10.
Endogenous p50cdc37 associates with
HRI in vivo. Cultured human erythroleukemia K562
cells were treated with Me2SO (lane 1) or with 1 µg/ml geldanamycin (lane 2) for 2 h. Endogenous
p50cdc37 was immunoprecipitated as described under
"Experimental Procedures." Immunocomplexes were separated by
SDS-PAGE, transferred to polyvinylidene difluoride membrane, and
analyzed for the presence of p50cdc37 (lower panel)
and associated HRI (upper panel). An equivalent aliquot of
the immunopellet was similarly analyzed for the presence of Hsp90
(middle panel). Proteins that were specifically detected
when membranes were probed with anti-HRI, anti-p50cdc37, or
anti-Hsp90 antibodies are indicated by solid arrowheads. The
detected proteins were of the expected molecular weight. Open
arrowheads denote the position of the heavy chain of the
immunoprecipitating anti-p50cdc37 antibodies. Analysis of
anti-p50cdc37 immunoadsorptions from two additional sets K562
cell extracts gave similar results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C and HRI (Fig. 3) or Raf-1 (21) lack bound
Hsp90. Furthermore, the interaction between p50cdc37/
C and
HRI was enhanced by geldanamycin (Fig. 3), apparently due to lack of
competition from endogenous Hsp90/p50cdc37 in the presence of
geldanamycin. Thus, the N-terminal domain of p50cdc37 binds to
HRI directly rather than associating with HRI via Hsp90. This finding
is consistent with similar conclusions reached previously regarding the
association of p50cdc37 with Raf-1 or Cdk4 (14, 21, 31).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Douglas Melton (Harvard University) for generously providing plasmid DNAs. We also thank Thomas Prince (Oklahoma State University) for technical assistance. Geldanamycin was provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI, Natioinal Institutes of Health. Oligonucleotide synthesis and DNA sequencing were performed by the Oklahoma State University Recombinant DNA/Protein Resource Facility. Polyclonal mouse ascites antibodies against p50cdc37 were prepared by the Oklahoma State University Hybridoma Center for the Agricultural and Biological Sciences. Rabbit antibodies were produced by the Oklahoma State University Lab Animal Resources Unit.
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FOOTNOTES |
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* This work was supported by National Institute of Health Grants DK-53223 (to J.-J. C.), GM51608 (NIGMS) (to R. L. M.), and ES04299 (NIEHS) (to R. L. M.), Oklahoma Center for the Advancement of Science and Technology Grant HN6-018 (to S. D. H.), and by the Oklahoma Agricultural Experiment Station Project 1975.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 246 NRC, Dept. of
Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3035. Tel.: 405-744-6200; Fax: 405-744-7799; E-mail: rmatts@biochem.okstate.edu.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007583200
2 N. Grammatikakis, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
HRI, heme-regulated
eIF2 kinase;
eIF, eukaryotic initiation factor;
eIF2
,
-subunit
of eukaryotic initiation factor 2;
RRL, rabbit reticulocyte lysate;
Hsp90, 90-kDa heat shock protein;
Hsc70, 70-kDa heat shock cognate
protein;
PAGE, polyacrylamide gel electrophoresis;
Ni2+-NTA-agarose, Ni2+-nitrilotriacetic acid
coupled to agarose;
TnT, coupled transcription and translation;
GST, glutathione S-transferase;
GaG-agarose, goat anti-mouse IgG
cross-linked to agarose;
IgM, immunoglobulin M, goat anti-mouse IgM
cross-linked to agarose.
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