(Received for publication, December 5, 1996, and in revised form, January 30, 1997)
From the Department of Biochemistry and Molecular
Biology, Oklahoma State University, Stillwater, Oklahoma 74078 and
§ Harvard-Massachusetts Institute of Technology Division of
Health Sciences and Technology,
Cambridge, Massachusetts 02139
The heme-regulated eukaryotic initiation factor
2 (eIF-2
) kinase (HRI) interacts with hsp90 in situ
in rabbit reticulocyte lysate (RRL). In this report, we have examined
the role of hsp90 in the maturation of newly synthesized HRI in both
hemin-supplemented and heme-deficient RRL. Analysis of translating
polyribosomes indicated that hsp90 interacts with nascent HRI
cotranslationally. Coimmunoadsorption of HRI with hsp90 by the 8D3
anti-hsp90 antibody indicated that this interaction persisted after
release of newly synthesized HRI from ribosomes. Incubation of HRI in
heme-deficient lysate resulted in the transformation of a portion of
the HRI polypeptides into an active heme-regulatable eIF-2
kinase
that exhibited slower electrophoretic mobility. Transformation of HRI was dependent on autophosphorylation, and transformed HRI was resistant
to aggregation induced by treatment of RRL with
N-ethylmaleimide. Transformed HRI did not coimmunoadsorb
with hsp90, and regulation of the activity of transformed HRI by hemin
was not hsp90-dependent. The hsp90 binding drug
geldanamycin disrupted the interaction of hsp90 with HRI and inhibited
the maturation of HRI into a form that was competent to undergo
autophosphorylation. Additionally geldanamycin inhibited the
transformation of HRI into a stable heme-regulatable kinase. These
results indicate that hsp90 plays an obligatory role in HRI acquiring
and maintaining a conformation that is competent to become transformed
into an aggregation-resistant activable kinase.
In rabbit reticulocyte lysate (RRL),1
initiation of protein synthesis is regulated by heme-regulated
inhibitor (HRI), a protein kinase that specifically phosphorylates the
38-kDa -subunit of eukaryotic initiation factor-2, eIF-2 (reviewed
in Refs. 1-5). In the eIF-2·GTP·Met-tRNAf ternary
complex, GTP is hydrolyzed during the last step of the initiation of
translation. Subsequent exchange of GDP for GTP to recycle eIF-2 is
catalyzed by eIF-2B. Phosphorylation of the
-subunit of eIF-2 by
activated HRI results in the binding and sequestration of eIF-2B in a
poorly dissociable complex (reviewed in Refs. 6-8). This
unavailability of eIF-2B results in the accumulation of eIF-2 in
complexes with GDP, thus inhibiting the formation of the ternary
complex and arresting initiation of protein synthesis.
In RRL, HRI is activated by a number of conditions as follows: heme deficiency, heat shock, sulfhydryl reagents, oxidants, glucose deficiency, and ethanol (reviewed in Ref. 7). Activation of HRI in response to heme deficiency is thought to proceed through a number of stages. HRI is initially present as an inactive pro-inhibitor. During heme deficiency, HRI is thought to be activated to a form that is completely reversible by hemin. Heme-reversible HRI is subsequently converted into an intermediate form whose activity is partially inhibited by hemin. Finally, after prolonged incubation in the absence of heme, HRI progresses to a heme-irreversible form (reviewed in Refs. 3 and 7). The relationship between the molecular forms of HRI activated in response to heme deficiency and forms of active HRI generated in hemin-supplemented lysate is not well understood.
As HRI is activated, it becomes progressively more phosphorylated (9). While this process in known to involve autophosphorylation, the possibility that HRI activation may involve phosphorylation of HRI by other protein kinases has also been proposed (10). Activation of HRI may also involve sulfhydryl oxidation or sulfhydryl/disulfide bond rearrangements (3, 11, 12). Covalent modification of some critical HRI sulfhydryl groups by N-ethylmaleimide causes hyperphosphorylation and irreversible activation of HRI (13).
In addition to these posttranslational modifications, activation of HRI correlates with dissociation from specific chaperones (14-17). In RRL, HRI occurs in heteromeric complexes containing the heat shock proteins hsp90, hsc70, and their associated cohorts FKBP52 and p232 (14, 18). The interaction of the hsp90 chaperone machinery with HRI appears to be dynamic rather than static2 (18). The association of hsp90 with HRI is enhanced in the presence of hemin (18) and requires the presence of Mg2+ and ATP.2 These observations have led to hypotheses proposing that interaction of hsp90 with HRI in the presence of hemin inhibits HRI activation and that the dissociation of hsp90 from HRI causes HRI activation (15). However, our recent results do not support these hypotheses.2 Rather, the interaction of hsp90 with HRI appears to stabilize HRI and protect it from denaturation. This positive role for hsp90 is more consistent with the known function of hsp90 as a molecular chaperone of certain signal transduction proteins (reviewed in Ref. 19).
In this study, we have used the hsp90-specific inhibitor, geldanamycin, to investigate the role of hsp90 in the regulation of HRI in RRL. Our findings indicate that (i) hsp90 plays an obligatory role as a molecular chaperone in the maturation3 of newly synthesized HRI into a conformation that is competent to autophosphorylate and transform3 into a heme-regulatable kinase; and (ii) continuous chaperone support is required to maintain HRI in this competent conformation prior to its transformation.
The NcoI-EcoRI
fragments from the pSP64 plasmids containing the coding sequence of
wild type (20) and the K199R mutant (21) of HRI were cloned into the
BglII site of pSP64T (22). A short synthetic double-stranded
DNA sequence with NcoI overhangs (5 C ATG CAT CAC CAT CAC
CAT CAC CA 3
and 3
GTA GTG GTA GTG GTA GTG GTG TAC 5
) coding for
Met-His7 residues was then inserted into the HRI
NcoI site. The reading frame was confirmed by DNA sequencing. Different plasmids used in the study are wild type HRI in
pSP64T with no His-tag, HRI; wild type HRI with His-tag in pSP64T,
His7-HRI; and K199R mutant with His tag in pSP64T, His7-K199R HRI.
Coupled transcription/translation of HRI and His7-HRI were initiated in nuclease-treated rabbit reticulocyte lysate (TnT RRL, Promega) at 30 °C in the absence of [35S]Met for 15 min. Preliminary experiments indicated that HRI synthesis began in TnT lysates between 10 and 15 min of incubation. At 15 min, a pulse of 460 µCi/ml [35S]Met was given. After 4 min of radiolabeling, 1 volume of TnT protein synthesis mix containing [35S]Met-labeled HRI ([35S]His7-HRI) was mixed with 4 volumes of normal heme-deficient or hemin-supplemented (10 µM hemin) protein synthesis mixes (11) containing non-nuclease-treated RRL and the protein synthesis initiation inhibitors edeine (10 µM) and/or aurintricarboxylic acid (60 µM). [35S]His7-HRI was then incubated for 60 min at 30 °C. HRI synthesis was found to be completed after 8-12 min of chase with no further incorporation of [35S]Met.
Assay of the Kinase Activity of [35S]His7-HRI Adsorbed to Ni-NTA ResinNi2+-nitrilotriacetic acid coupled to agarose
(Ni-NTA resin, Qiagen) was equilibrated with adsorption buffer (50 mM Tris-HCl (pH 7.5), 20 mM sodium molybdate,
and 10 mM imidazole). RRL mixes containing
[35S]His7-HRI were adjusted to 20 mM sodium molybdate, incubated for 3 min on ice, and
centrifuged at 10,000 rpm for 5 min before adsorption to Ni-NTA resin.
[35S]His7-HRI from 25 µl of RRL reaction
mixes was bound to the resin (10 µl) for 1 h on ice, followed by
3 washes with 500 µl of buffer containing 50 mM Tris-HCl
(pH 7.5), 20 mM sodium molybdate, and 50 mM
imidazole (wash buffer). Assays for the kinase activity of
[35S]His7-HRI bound to Ni-NTA resin were
performed for 4 min at 30 °C as described (14). Samples were
analyzed by 10% SDS-PAGE, followed by transfer to PVDF membrane and
autoradiography as described previously (23). Autophosphorylation of
HRI was assayed by the incorporation of 32Pi
into HRI during eIF-2 kinase assays incubated with
[
-32P]ATP. 32P-Labeled HRI and eIF-2
were detected by quantitatively quenching [35S] emission
with two intervening layers of previously developed x-ray film.
The same membranes used for kinase assays were used for Western blot analysis. The interaction of hsp90 with His7-HRI was detected using anti-hsp90 84/86 peptide polyclonal antiserum (provided by Dr. Stephen Ullrich, NCI) and standard protocols (18). Expression of wild type HRI with no His-tag under similar conditions was used as control for nonspecific binding to Ni-NTA resin in all the studies.
ImmunoadsorptionPreparation of goat anti-mouse IgM cross-linked to agarose, binding of 8D3 anti-hsp90 antibody or nonimmune control to goat anti-mouse-agarose, and coimmunoadsorption of HRI with hsp90 were carried out as described previously (18). Briefly 5 µl of RRL mixes containing [35S]HRI were adjusted to 20 mM sodium molybdate. After 90 min of binding on ice, immunopellets were washed 3 times with 500 µl of TBS (50 mM Tris-HCl (pH 7.5), and 150 mM NaCl), and the immunopellets were eluted with SDS sample buffer. Proteins present in immunopellets and supernatants were separated on 10% SDS-PAGE and transferred to PVDF membrane. [35S]HRI was detected by autoradiography.
Cotranslational Association of hsp90 with HRITnT RRL lysates programmed with either luciferase, HRI, or no template were labeled with [35S]Met as described above. After 18.5 min of synthesis, the protein synthesis mix was diluted with 2 volumes of ice-cold buffer containing 20 mM Tris-HCl (pH 7.5), 25 mM KCl, and either 2.5 mM magnesium acetate or 10 mM EDTA. Diluted translations were layered on top of 15-40% sucrose gradients containing buffers and salts as above and centrifuged for 4.5 h at 40,000 rpm in an AH650 rotor. The supernatant was removed, and the ribosomal pellets were dissolved in SDS sample buffer. Proteins present in ribosomal pellets were separated on 10% SDS-PAGE and transferred to PVDF membrane. Hsp90 was detected by Western blotting as described above.
HPLC Analysis of the Molecular Size of HRIDe novo synthesis and maturation of [35S]HRI in heme-deficient and hemin-supplemented RRL by pulse-chase were performed as described above. At the end of 45 min maturation, RRL was treated with 1 mM NEM for 15 min followed by analysis of 40 µl of protein synthesis mix on a Pharmacia HR 10/30 Superdex 200 column pre-equilibrated at 4 °C in HPLC buffer containing 25 mM HEPES·NaOH, 25 mM sodium glycerophosphate, 2 mM EDTA, 0.5% Tween, 10% (w/v) glycerol, and 100 mM KCl (pH 7.4). Fractions (0.2 ml) were collected and analyzed by 10% SDS-PAGE, autoradiography, and densitometry as described (23).
Because the activation
of HRI in RRL appears to be a multistage process, the endogenous HRI
present in RRL may represent a heterogeneous population of molecules.
Thus, pulse-chase [35S]Met labeling of synchronized
translations of an N-terminal His7-tagged HRI,
His7-HRI were used to study the maturation and activation of a homogeneous population of HRI molecules in RRL. Following initial
synthesis and radiolabeling, [35S]His7-HRI
was subsequently incubated in hemin-supplemented or heme-deficient RRL,
adsorbed to Ni-NTA resin, and separated by SDS-PAGE (Fig.
1). HRI that lacked the His7-tag was
similarly analyzed to provide a measure of nonspecific binding of
endogenous HRI and [35S]HRI to the resin (Fig. 1,
A-B, lanes 1 and 8).
In hemin-supplemented RRL, [35S]His7-HRI
occurred as an apparently homogeneous population of molecules,
represented by the detection of a single 35S-labeled
polypeptide (Fig. 1A). In contrast, incubation of
[35S]His7-HRI in heme-deficient RRL produced
a heterogeneous population of [35S]His7-HRI
molecules, 50% that had a slower electrophoretic mobility than that
observed for [35S]His7-HRI in
heme-supplemented RRL (Fig. 1B). The proportion of HRI
undergoing the electrophoretic shift was proportional to hemin
concentration (Fig. 1C). No further increase in the amount of the slow mobility form of [35S]His7-HRI
was produced following incubations in the heme-deficient RRL for up to
2 h (not shown). The presence of the His7-tag had no
effect on the electrophoretic mobility of HRI (not shown). The slow
moving [35S]His7-HRI comigrated with active,
autophosphorylated HRI purified from rabbit reticulocyte lysate and
labeled by incubation with [-32P]ATP in
vitro (Fig. 1B, lane 9). Thus, HRI was
posttranslationally modified in heme-deficient lysate, resulting in
reduced electrophoretic mobility, and this mobility shift may have been
related to events occurring during HRI activation.
To determine whether the shift in HRI mobility correlated with
acquisition of activity, kinase assays were carried out on [35S]His7-HRI adsorbed to Ni-NTA resin (Fig.
2). The eIF-2 kinase activity adsorbed to the Ni-NTA
resin was specific to the adsorption of the His7-tagged
HRI, as little eIF-2
kinase activity was recovered from control
adsorption of reactions containing HRI lacking the His7-tag
(Fig. 2, lanes 1-4). In hemin-supplemented and
heme-deficient lysates, no eIF-2
kinase activity (Fig. 2, lane
5 and 6, lower panel) and little of the slow mobility
form of [35S]His7-HRI (Fig. 2, lane
5 and 6, upper panel) were present after a short 8-min
incubation of pulse-labeled [35S]His7-HRI.
After a 60 min incubation of [35S]His7-HRI in
heme-deficient RRL, both the mobility shift (Fig. 2, lane 8, upper panel) and enhanced eIF-2
kinase activity (Fig. 2,
lane 8, lower panel) were observed. In hemin-supplemented
RRL, [35S]His7-HRI remained inactive even
after 60 min of incubation, as the eIF-2
kinase activity detected
was equal to the background activity (Fig. 2, lanes 5 and
7, lower panel). These results indicate that the mobility
shift of HRI in heme-deficient RRL correlated with acquisition of
eIF-2
kinase activity.
To test the hypothesis that acquisition of reduced electrophoretic
mobility and eIF-2 kinase activity required the
autophosphorylation of HRI, we studied the electrophoretic
mobility of the inactive K199R mutant of HRI, which does not
undergo autophosphorylation (21).
[35S]His7-HRI and
[35S]His7-K199R HRI were incubated in
hemin-supplemented and heme-deficient RRL for 60 min, adsorbed to
Ni-NTA resin, and assayed for kinase activity (Fig. 3).
In contrast to wild type [35S]His7-HRI (Fig.
3A, lane 4), incubation of
[35S]His7-K199R HRI in heme-deficient RRL did
not produce kinase molecules with reduced electrophoretic mobility
(Fig. 3A, lane 6). The
[35S]His7-K199R HRI was inactive in
autophosphorylation (Fig. 3B, lane 6, upper panel) and did
not increase eIF-2
phosphorylation above background levels
representing nonspecifically bound activity (Fig. 3B, lane 6 versus lane 2, lower panel). These findings indicated that
intramolecular autophosphorylation of HRI was required for its
activation.
Association of hsp90 with HRI
Physical interactions of HRI
with hsp90 are well documented (14, 15, 18). To determine if hsp90
associates with HRI prior to its release from the ribosomes,
polyribosomes were isolated from translation mixtures programmed with
either HRI, luciferase, or no template. Consistent with previous
results (24), hsp90 was not detected in the polysomal pellets
programmed with luciferase (Fig. 4B, lane 2).
Also, hsp90 was not detected in the no-template control translation
(Fig. 4A, lane 2). In contrast, hsp90 was specifically
detected in the polyribosomal pellets isolated from translations
programmed with HRI template (Fig. 4, A-B, lane 4). This
association of hsp90 with the ribosomal pellet was not observed when
peptide chains were released from the ribosomes by treatment with EDTA
(Fig. 4, A-B, lane 3). However, EDTA did not destabilize the interaction of hsp90 with HRI in RRL (not shown). These results demonstrated that hsp90 associated with nascent HRI
cotranslationally.
We next examined whether the association of hsp90 with HRI was
maintained after release of newly synthesized HRI from the ribosomes.
Coimmunoadsorption of HRI by anti-hsp90 antibodies following an 8-min
chase of translations demonstrated that the association of
[35S]HRI with hsp90 was maintained even after HRI's
release from ribosomes (Fig. 5A, lanes 3 and
4). This interaction persisted even after 60 min of
incubation in heme-supplemented RRL (Fig. 5A, lane 7). In
contrast, incubation in heme-deficient RRL resulted in the recovery of
less HRI as an hsp90·HRI complex. While both the fast and slow
mobility forms of [35S]HRI were produced in
heme-deficient RRL after 60 min incubation (Fig. 5A, SUP, lane
8), only the fast mobility form of [35S]HRI was
coimmunoadsorbed by anti-hsp90 antibodies (Fig. 5A, PEL, lane
8). Thus, HRI did not associate with hsp90 following HRI
autophosphorylation and transformation.
To confirm this conclusion, we examined the interaction of hsp90 with the [35S]K199R HRI mutant (Fig. 5B). Similar to wild type [35S]HRI, hsp90 association with newly synthesized [35S]K199R HRI mutant was maintained posttranslationally after an 8-min chase in heme-deficient and hemin-supplemented RRL (Fig. 5B, lanes 3 and 4). However, in contrast to active HRI, the [35S]K199R HRI mutant coimmunoadsorbed with hsp90 to the same extent in heme-deficient or hemin-supplemented RRL at the end of the 60-min incubation (Fig. 5B, lanes 5 and 6). Thus, transformation of HRI into an hsp90-free form required autophosphorylation.
Effects of Geldanamycin on the Interaction of hsp90 with HRITo determine whether hsp90 played a direct role in the
maturation and transformation of HRI, we utilized the hsp90 inhibitor, geldanamycin (25). At the concentrations used in this study, the drug
vehicle Me2SO had no effect on the following: (i) the association of hsp90 with HRI; (ii) protein synthesis; (iii)
interaction of chaperones with substrate proteins (e.g.
thermally denatured luciferase (26)); or (iv) eIF-2 kinase activity.
Hsp90 coadsorbed with His7-HRI on Ni-NTA resins, and this
coadsorption was enhanced in hemin-supplemented RRL relative to heme-deficient RRL (Fig. 6A, lane 3 and
4). In the presence of geldanamycin, the amount of hsp90
recovered during adsorption of hsp90·His7-HRI complexes
was equal to the amount of hsp90 that nonspecifically bound to the
resin (Fig. 6A, lanes 1 and 2 versus lanes 5 and
6). Additionally, geldanamycin inhibited the coadsorption of
hsp90·His7-HRI complexes from either heme-deficient or
hemin-supplemented RRL even after an initial 45-min incubation of HRI
in the absence of the drug (Fig. 6A, lanes 7 and
8). These results indicated that geldanamycin disrupted the
association of hsp90 with both newly synthesized and matured
His7-HRI in heme-deficient and hemin-supplemented RRL.
Effects of Geldanamycin on the Maturation of HRI
To determine
whether the interaction of hsp90 with HRI is essential for HRI
maturation, we examined the effects of geldanamycin on the
transformation of newly synthesized HRI into an active kinase. When
newly synthesized [35S]His7-HRI was incubated
in heme-deficient lysate, the drug vehicle Me2SO did not
inhibit HRI transformation into the slow mobility form (Fig. 6B,
lane 4, upper panel) and did not inhibit HRI acquisition of
eIF-2 kinase activity (Fig. 6B, lane 4, lower panel). In
contrast, when newly synthesized
[35S]His7-HRI was incubated in
geldanamycin-treated heme-deficient RRL, geldanamycin specifically
inhibited HRI transformation into the slow mobility form (Fig.
6B, lane 6, upper panel) and inhibited HRI acquisition of
eIF-2
kinase activity (Fig. 6B, lane 6, lower panel). The
pharmacologically inactive benzoquinoid ansamycin, geldampicin (25),
had no effect on [35S]His7-HRI maturation,
activation, nor the interaction of HRI with hsp90, indicating that the
effects of geldanamycin were specific (not shown). Geldanamycin had no
direct inhibitory effect on [35S]His7-HRI
activity when added directly to the kinase assays (not shown).
Geldanamycin did not reverse the electrophoretic mobility shift (Fig.
6B, lane 8, upper panel) or eIF-2
kinase activity (Fig.
6B, lane 8, lower panel) once such was acquired in
heme-deficient lysate lacking geldanamycin, indicating that the
geldanamycin effects did not reflect the reversal of HRI activation nor
the suppression of active HRI by geldanamycin. These results
demonstrated that geldanamycin-sensitive hsp90 activity is required for
newly synthesized HRI to become an active kinase.
The
experiments described above demonstrated an essential positive role for
geldanamycin-inhibitable hsp90 function in de novo folding
and activation of HRI in heme-deficient RRL. However, interactions
between hsp90 and HRI persist in hemin-supplemented RRL (Fig. 5 and
Ref. 18). This persistence suggested that in hemin-supplemented RRL,
hsp90 maintained or provided mature HRI molecules with the competence
to activate. To test this hypothesis, [35S]His7-HRI was subjected to three
successive incubations in RRL lacking or containing hemin and in which
hsp90 function was or was not inhibited by geldanamycin.
[35S]His7-HRI was initially incubated in
hemin-supplemented RRL lacking geldanamycin for 50 min to mature the
HRI (incubation 1). Subsequently, hsp90 function was or was not
inhibited by the addition of geldanamycin, and the RRL reactions were
incubated an additional 10 min (incubation 2). The purpose of
incubation 2 was to disrupt hsp90's post-maturational maintenance of
HRI during this time. After incubation 2, aliquots of these reactions
were mixed with fresh RRL (1:2, v/v) containing or lacking hemin, and
these reactions were incubated an additional 60 min to assay HRI's
competence to transform and activate (incubation 3). These
activating/transforming RRLs (lacking or containing hemin) used for
incubation 3 contained the same concentration of geldanamycin or
Me2SO as were used for incubation 2. Subsequent to these
successive incubations, we examined the electrophoretic mobility (Fig.
7, top panel), autophosphorylation (Fig. 7,
middle panel), and eIF-2 kinase activity (Fig. 7,
bottom panel) of [35S]His7-HRI
recovered in Ni-NTA resin adsorption pellets. Background kinase
activity that was nonspecifically bound to the Ni-NTA resin was
determined in assays of activity recovered from untreated RRLs that
were programmed with HRI lacking the His7-tag (Fig. 7,
NS, lanes 1-4). Additionally, these experiments
were designed to confirm that hsp90 was neither necessary to maintain
the activity of transformed HRI nor to inhibit HRI activity in the
presence of hemin.
In the absence of geldanamycin, [35S]His7-HRI became activated in response to heme deficiency (Fig. 7, lane 5 versus lane 6). However, in the presence of geldanamycin, [35S]His7-HRI was not activated in response to heme deficiency (Fig. 7, lane 9 and 10 versus lane 5 and 6), despite initial de novo maturation of [35S]His7-HRI by hsp90 in the absence of geldanamycin. Geldanamycin did not inhibit previously activated [35S]His7-HRI (Fig. 7, lane 8 versus lane 12) nor did geldanamycin activate the matured [35S]His7-HRI (Fig. 7, lane 5 versus lane 9) in hemin-supplemented RRL. Thus, geldanamycin-inhibitable hsp90 function was required for mature HRI to be competent to activate, even after a prior 50 min of maturation in hemin-supplemented RRL. Furthermore, geldanamycin did not inibit heme-mediated inactivation of previously activated [35S]His7-HRI (Fig. 7, lanes 7 and 8 versus lanes 11 and 12).
The Effect of N-Ethylmaleimide (NEM)The results from the
above experiment indicated that geldanamycin-inhibitable hsp90 function
was required for HRI to be competent to activate. To characterize
mature but inactive molecules of HRI, NEM was used to unmask the kinase
potential of HRI molecules independently of the presence of hemin (1,
3). NEM activation of HRI was examined in assays of HRI
autophosphorylation (Fig. 8, upper panel) and
eIF-2 phosphorylation (Fig. 8, lower panel) using equal
amounts of [35S]His7-HRI adsorbed to Ni-NTA
resin. [35S]His7-HRI was not activated when
hemin-supplemented lysate was treated with NEM 8 min after the arrest
of HRI synthesis (Fig. 8, lane 9). Similarly, in
hemin-deficient RRL, NEM treatment of immature newly synthesized HRI
(treatment 8 min after arrest of HRI synthesis) induced little
autophosphorylation (Fig. 8, lane 10 versus lane 8, upper
panel) and eIF-2
kinase activity (Fig. 8, lane 10 versus
lane 8, lower panel) over activity recovered in a nonspecific
fashion (Fig. 8, lane 10 versus lane 8). In addition, the
slow mobility form of [35S]His7-HRI
characteristic of HRI folding into an active kinase was not observed
(not shown). Thus, HRI needed to be folded to be activated by NEM.
Following a 45-min maturation in hemin-supplemented lysate, a portion
of the mature [35S]His7-HRI molecules were
activated by NEM in the presence of hemin, as indicated by increased
levels of autophosphorylation (Fig. 8, lane 5, upper panel)
and eIF-2 phosphorylation (Fig. 8, lane 5, lower panel)
over those observed in non-NEM-treated reactions (Fig. 8, lane
3) and over levels nonspecifically bound to the Ni-NTA resin (Fig.
8, lanes 1 and 7). These results indicated that
at the end of 45 min incubation in hemin-supplemented RRL, [35S]His7-HRI was matured to a form that was
competent to be activated. However, only a small portion of
[35S]His7-HRI was activated upon NEM
treatment, since NEM inactivates the chaperone machinery of
RRL4 in addition to activating HRI by
derivatization of sensitive sulfhydryl groups (1, 3). As further
controls for NEM activation, HRI was also matured in heme-deficient RRL
and treated with NEM. NEM hyperactivated HRI previously activated in
heme-deficient lysate (Fig. 8, lane 6 versus lane 4).
NEM inactivates the ability of the RRL chaperone
machinery to renature thermally denatured firefly
luciferase.4 To determine if prolonged interaction of hsp90
with the fast mobility form of HRI (Fig. 5) plays a role in stabilizing
the structure of HRI, we examined the effect of NEM treatment on the stability of [35S]HRI synthesized in pulse-chase
translations in RRL. HPLC gel exclusion chromatography of
[35S]HRI indicated that HRI was present as multiple
molecular weight species (Fig. 9). The slow mobility
form of HRI generated in heme-deficient RRL was present as a more
discrete peak of material (Fig. 9C). When NEM treatment was
given after a 45-min incubation in heme-deficient and
hemin-supplemented RRL, the fast mobility form of HRI from both
heme-deficient and hemin-supplemented RRL aggregated and was observed
to elute in the void volume (Mr >1.3 × 106) (Fig. 9, A-B). Only the slow mobility form
that was present in NEM-treated heme-deficient RRL eluted normally
(Fig. 9C) and was active in phosphorylating eIF-2 (not
shown). Thus, in RRL, only HRI that had been autophosphorylated and
transformed (fast mobility form) was resistant to denaturation and
aggregation following inactivation of chaperone machinery by NEM. This
was the population of HRI that did not physically nor functionally
associate with hsp90.
Hsp90's support of HRI function was studied using a homogeneous population of [35S]His7-HRI synthesized in synchronized pulse-chased translations. This population was distinguished from endogenous HRI via the recombinant addition of an His7-tag. Using this approach, and the hsp90 inhibitor geldanamycin, we have delineated two essential roles for HRI as follows: (i) hsp90 is required to fold nascent HRI into an active/activable kinase; and (ii) hsp90 is required for maintenance and stabilization of kinase structure prior to HRI activation. These essential and positive roles of hsp90 are clearly incompatible with other models suggesting that hsp90 functions to suppress HRI activity (15).
Based on our results, we present a model for the positive role of hsp90
in maturation of HRI and its transformation into a stable, active
heme-regulated kinase (Fig. 10). According to the model, hsp90 binds to nascent HRI during synthesis of HRI on
polyribosomes (Fig. 4). This cotranslational interaction of hsp90 with
HRI implies that hsp90 facilitates the de novo folding of
HRI. However, the role of hsp90 in folding of nascent polypeptides
appears to be substrate-specific, since hsp90 does not appear to be
associated with nascent luciferase (Fig. 4, and Refs. 23 and 24), and the hsp90 inhibitor geldanamycin has no effect on the folding of newly
synthesized luciferase.5
After the release of HRI from ribosomes (Fig. 10, early HRI folding intermediates), hsp90 continues to interact with HRI (Fig. 5). However, these early HRI folding intermediates are not active (Fig. 2) and cannot be activated by NEM treatment of either hemin-supplemented or heme-deficient RRL (Fig. 8). Thus, at this stage of maturation, HRI is not folded into a conformation that is competent to undergo autophosphorylation and activation. Hsp90 function in subsequent maturation is inferred from the physical association at this point and is consistent with the inhibition by geldanamycin of subsequent maturation of these molecules.
Upon further incubation, early HRI folding intermediates mature into a competent form (Fig. 10, mature competent HRI), as indicated by the acquisition by HRI molecules of the ability to be unmasked by NEM (Fig. 8, lane 5 versus lane 3). However, physical and functional interactions between HRI and hsp90 persist at this time since HRI previously matured in geldanamycin-free hemin-supplemented RRL fails to activate upon subsequent incubations in RRL containing geldanamycin (Fig. 7). This observation suggests that matured competent HRI becomes misfolded in the absence of hsp90 support (Fig. 10, matured misfolded HRI). In the model, mature misfolded HRI is presented as being off the de novo folding pathway. However, it is equally possible that "mature misfolded HRI" might be part of normal HRI folding intermediates. Regardless, in addition to its role in de novo folding, the sustained interaction of hsp90 with HRI in the presence of hemin (Fig. 5) plays an important role in maintaining HRI in a competent conformation prior to its activation, autophosphorylation, and transformation.
In the absence of functional chaperone machinery, mature misfolded HRI aggregates. NEM inactivates the capacity of RRL chaperone machinery to renature and maintain the activity of firefly luciferase.4 NEM treatment causes the aggregation of mature-but-inactive [35S]HRI (Fig. 9). Thus, besides the general requirement for hsp90 to fold HRI into a competent conformation, hsp90 also functions to protect competent HRI from denaturation while HRI awaits stimuli that induce its activation.
Competent HRI is activated in response to heme deficiency. Initial events leading to the activation of HRI likely occur while HRI is still bound to hsp90 or may involve reiterative cycles of binding and release of HRI by the hsp90 chaperone machinery. We have recently observed that active hsp90-bound HRI can be detected.2 We thus postulate an active but unstable transition state during HRI transformation and a potential role for hsp90 during this step of the activation process (Fig. 10, unstable active HRI).
Phosphorylation events play an obligatory role in HRI activation in response to heme deficiency. The proportion of the HRI molecules that are transformed into the stable slow mobility form of HRI is dependent on hemin concentration (Fig. 1C). In the absence of the ability to catalyze autophosphorylation, the reduced electrophoretic mobility characteristic of activation (Fig. 2) is not observed with the inactive K199R HRI mutant (Fig. 3). This result indicates that autophosphorylation is a prerequisite for activation in response to heme deficiency since active endogenous HRI is present. However, the number and sequence of subsequent phosphorylation events that reduce the electrophoretic mobility of HRI and lead to its complete activation and stabilization is not clear from the present data. Phosphorylation of HRI by other kinases in RRL has also been proposed (e.g. casein kinase II (10)).
Phosphorylation of HRI stabilizes the kinase structure such that it no longer needs hsp90 to support its function. This conclusion is supported by the several observations: (i) the slow mobility form of HRI does not associate with hsp90 (Fig. 5); (ii) the inactive K199R mutant, which can not autophosphorylate, remains bound to hsp90 in heme-deficient RRL (Fig. 6); (iii) geldanamycin has no inhibitory effect on previously matured and activated HRI activity in heme-deficient lysate (Figs. 6 and 7); (iv) the transformed slow mobility form of HRI is stable and resistant to aggregation in NEM-treated RRL (Fig. 10C).
Although hsp90 functions to protect mature HRI from denaturation and to maintain its competence to activate in response to heme deficiency, subsequent hemin regulation of transformed HRI is hsp90-independent. The activity of transformed HRI, which is not associated with hsp90, is still responsive to hemin (Fig. 7). Additionally, while geldanamycin blocks the interaction of hsp90 with HRI, it does not inhibit the ability of hemin to repress the activity of transformed HRI (Fig. 7). Furthermore, hemin-induced repression of the activity of transformed HRI is not accompanied by reassociation of hsp90 with HRI (not shown). In addition, the ratio of the slow to fast mobility forms of [35S]HRI matured and transformed in heme-deficient RRL did not change upon subsequent return of the [35S]HRI to hemin-supplemented RRL (Fig. 7). Thus, although the activation of HRI kinase activity by hemin is reversible, the transformation of HRI, as judged by its electrophoretic mobility shift on SDS-PAGE, is not. We have not yet investigated whether transformed HRI that has been repressed by hemin can reactivate in response to heme deficiency.
The model presented for HRI maturation and activation may represent a heuristic model for other hsp90-dependent kinases as well. Hsp90 plays an essential positive role in the function of the viral protein tyrosine kinase p60src (27) and a cellular member of this family, p56lck (28). The nature of this role is to support kinase folding (28). This role appears to be manifested, in part, during the biogenesis of these kinases (28-30). However, hsp90 occurs in complexes with numerous other kinases, and these complexes do not appear to be specific to nascent molecules (28, 31-36). Thus, in addition to its role in the kinase biogenesis, hsp90 may support the maintenance folding of kinases subsequent to their de novo folding. Consistent with these observations, hsp90 mediates the potential of both newly synthesized and matured molecules of HRI.
However, the dependence of kinase molecules on hsp90 machinery may be conditional. The interaction of Cdk4 with hsp90 and the 50-kDa hsp90 cohort cdc37 appears to be specific for that population lacking the essential cyclin subunit (33). Similarly, the interaction of hsp90 with raf appears to occur at specific time points in the cell cycle (37). Additionally, although p56lck requires hsp90 to maintain kinase function in RRL, hsp90-bound p56lck appears to represent a small portion of the total p56lck population occurring in T cells (28). Thus, we have previously postulated that in addition to facilitating de novo folding, hsp90 may provide reiterative support of kinase structure prior to specific stabilizing events (28). This model is consistent with a model postulating reiterative hsp90-mediated support of the progesterone receptor to maintain hormone binding competence until such binding stabilizes the structure of the receptor (38). For HRI, transformation to an hsp90-independent state involves autophosphorylation induced by heme deficiency. Thus, hsp90's previously documented roles in kinase biogenesis and in maintenance of protein structure can be integrated in light of the model presented here for the pathway of HRI maturation and activation. This model for HRI maturation, transformation, and activation postulates overlapping pathways of kinase folding and regulation.
We thank Dr. Stephen Ullrich (NCI) for providing 84/86 anti-hsp90 antibody, the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute for providing geldanamycin, and Dr. Kenneth Rinehart (University of Illinois, Urbana) for providing geldampicin.