CHIP did not influence the ATPase cycle of Hsp90 in the absence of co-chaperones or in the pres-ence of the Hsp90 cochaperones Aha1 or p23.. D Single-turnover ATPase rates of Hsc70 and Hs
Trang 1preferentially ubiquitinating Hsp70-bound substrates
Marta Stankiewicz1, Rainer Nikolay1,*, Vladimir Rybin2and Matthias P Mayer1
1 Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ–ZMBH Alliance, Heidelberg, Germany
2 European Molecular Biology Laboratory, Heidelberg, Germany
Introduction
CHIP consists of an N-terminal TPR (tetratricopeptide
repeat) domain, which binds to the C-terminal EEVD
motif that is present in all cytosolic Hsp70 and Hsp90 chaperones, a central helical domain, which is essential
Keywords
chaperones; Hsp70; Hsp90; protein triage;
ubiquitination
Correspondence
M P Mayer, Zentrum fu¨r Molekulare
Biologie der Universita¨t Heidelberg (ZMBH),
DKFZ–ZMBH Alliance, Im Neuenheimer Feld
282, 69120 Heidelberg, Germany
Fax: +49 6221 545894
Tel: +49 6221 546829
E-mail: M.Mayer@zmbh.uni-heidelberg.de
*Present address
Biochemisches Institut der Universita¨t
Zu¨rich, Winterthurerstrasse 190, 8057
Zu¨rich, Switzerland
(Received 3 May 2010, revised 8 June
2010, accepted 14 June 2010)
doi:10.1111/j.1742-4658.2010.07737.x
The E3 ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is believed to be a central player in the cellular triage decision, as it links the molecular chaperones Hsp70⁄ Hsc70 and Hsp90 to the ubiquitin proteaso-mal degradation pathway To better understand the decision process, we determined the affinity of CHIP for Hsp70 and Hsp90 using isothermal titration calorimetry We analyzed the influence of CHIP on the ATPase cycles of both chaperones in the presence of co-chaperones and a substrate, and determined the ubiquitination efficacy of CHIP in the presence of the chaperones We found that CHIP has a sixfold higher affinity for Hsp90 compared with Hsc70 CHIP had no influence on ADP dissociation or ATP association, but reduced the Hsp70 cochaperone Hdj1-stimulated sin-gle-turnover ATPase rates of Hsc70 and Hsp70 CHIP did not influence the ATPase cycle of Hsp90 in the absence of co-chaperones or in the pres-ence of the Hsp90 cochaperones Aha1 or p23 Polyubiquitination of heat-denatured luciferase and the native substrate p53 was much more efficient
in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicat-ing that CHIP preferentially ubiquitinates Hsp70-bound substrates
Structured digital abstract
l MINT-7904367 : CHIP (uniprotkb: Q9UNE7 ) and HSP 90-beta (uniprotkb: P08238 ) physically interact ( MI:0915 ) by molecular sieving ( MI:0071 )
l MINT-7904785 : HSP 90-beta (uniprotkb: P08238 ) and p23 (uniprotkb: Q15185 ) bind ( MI:0407 ) by molecular sieving ( MI:0071 )
l MINT-7904047 : CHIP (uniprotkb: Q9UNE7 ), HSP 90-beta (uniprotkb: P08238 ) and p23 (uni-protkb: Q15185 ) physically interact ( MI:0915 ) by molecular sieving ( MI:0071 )
l MINT-7903424 : Alpha-lactalbumin (uniprotkb: P00711 ), HSP70 (uniprotkb: P08107 ) and CHIP (uniprotkb: Q9UNE7 ) physically interact ( MI:0915 ) by molecular sieving ( MI:0071 )
l MINT-7903354 : CHIP (uniprotkb: Q9UNE7 ) and HSC70 (uniprotkb: P11142 ) bind ( MI:0407 )
by isothermal titration calorimetry ( MI:0065 )
l MINT-7903373 : CHIP (uniprotkb: Q9UNE7 ) and HSP90-beta (uniprotkb: P08238 ) bind ( MI:0407 ) by isothermal titration calorimetry ( MI:0065 )
Abbreviations
CHIP, C-terminus of Hsc70-interacting protein; Hsp70, 70 kDa heat shock protein; Hsp90, 90 kDa heat shock protein; Hsc70, 70 kDa heat shock cognate; ITC, isothermal titration calorimetry; MABA-ADP ⁄ MABA-ATP, N 8 -(4-N¢-methylanthraniloylaminobutyl)-8-aminoadenosine 5¢-di ⁄ triphosphate; RCMLA, reduced carboxymethylated a-lactalbumin; TPR, tetratricopeptide repeat.
Trang 2for dimerization, and a C-terminal U-box domain,
which is responsible for interaction with E2
ubiquitin-conjugating enzymes [1,2] This interaction with
molec-ular chaperones suggests that CHIP is involved in the
triage decision [3–5] Hsp70 chaperones are essential
components of the cellular quality control network,
interacting with virtually all misfolded proteins,
pre-venting their aggregation and assisting their refolding
into the native state [6–8] Hsp90 chaperones have also
been shown to bind to misfolded proteins [9], but their
main essential function in all eukaryotic cells is
believed to be interaction with a large number of
regu-latory proteins, called client proteins, including
recep-tors, protein kinases and transcription factors [10–12]
The question therefore arises as to whether CHIP
pref-erentially interacts with Hsp70 to mark misfolded
proteins, which cannot be refolded to the native
state, for degradation, or whether it assumes a more
regulatory role by interacting mainly with Hsp90 to
ubiquitinate signaling proteins
It has been shown in vitro and in cell culture that
sev-eral Hsp90 clients are ubiquitinated in a
CHIP-depen-dent manner, and that this ubiquitination depends on
CHIP’s U-box domain [13–17] In some cases, CHIP
appears to directly bind its substrates and ubiquitinate
them in a chaperone-independent manner [18–20] Such
specific substrate recognition is a typical feature of E3
ubiquitin ligases; however, the majority of CHIP’s
sub-strates described so far are bona fide Hsp70 and Hsp90
clients, and their degradation depends on the presence
of the chaperones [13,21–23] Among the CHIP
substrates are many regulatory proteins that are
ubiqui-tinated and degraded even in the presence of an Hsp90
inhibitor such as geldanamycin [21]
For most if not all functions, Hsp90 cooperates with
Hsp70 in a chaperone cycle, which was first proposed
for steroid hormone receptors and involves a number
of co-chaperones [24,25] Steroid hormone receptors
first interact with Hsp40 and Hsp70 The dimeric
pro-tein Hop (Hsp70-Hsp90 organizing Propro-tein), which has
separate TPR domains for binding to Hsp70 (TPR1)
and Hsp90 (TPR2a), assembles the early client
com-plex with Hsp70 and Hsp90 Hop and Hsp70 are then
replaced by p23 and a TPR domain-containing
pept-idyl-prolyl-cis⁄ trans-isomerase (e.g the 51 and 52 kDa
FK506-binding proteins FKBP51 or FKBP52) The
mature complex decays with a half life of
approxi-mately 5 min, and the hormone receptor re-enters the
cycle by binding to Hsp40 and Hsp70 As CHIP can
interact with Hsp70 and Hsp90, it is not clear whether
ubiquitination of the chaperone substrate occurs while
the substrate is bound to Hsp70 or Hsp90 Another
intriguing question is how the triage decision is made
As CHIP is a TPR-containing co-chaperone, it com-petes with numerous other TPR-containing co-chaper-ones for binding to Hsp70 and Hsp90 [26]
Here we provide new insights into the triage decision
by assessing the physical interaction of CHIP with Hsp70⁄ Hsc70 and Hsp90, and analyzing the functional consequences for the chaperone substrates
Results CHIP–chaperone interaction and competition with Hop
To determine how the triage decision is made, we first addressed the question of protein affinities and cellular concentrations CHIP directs proteins to the degrada-tion pathway, and Hop is an essential co-chaperone for protein folding Because they both interact with the same C-terminal EEVD motif of Hsc70 and Hsp90, we determined the affinities of CHIP for Hsc70 and Hsp90 using isothermal titration calorimetry (ITC) (Fig 1A,B) We also investigated the interaction
of CHIP with heat shock-induced Hsp70, to determine whether CHIP has a preference for this homolog to enhance quality control processes during heat shock CHIP’s affinity for Hsp90 (KD= 0.38 ± 0.04 lm) was approximately six times higher than that for Hsc70 (KD= 2.3 ± 0.3 lm), and two and a half times higher than that for Hsp70 (KD= 0.95 ± 0.01 lm) The affinities of CHIP for Hsc70 and Hsp70 were in the same range as the value measured for the Hop– Hsc70 interaction (KD= 1.5 ± 0.2 lm) using surface plasmon resonance spectroscopy [27] In contrast, for the interaction of Hop with Hsp90, a KD value
of 0.1 ± 0.02 lm was determined by surface plasmon resonance spectroscopy, which is only one quarter of the value measured here for the CHIP–Hsp90 interac-tion These results indicate that Hop and CHIP com-pete efficiently with each other for binding to Hsc70⁄ Hsp70 when the C-termini of Hsc70 ⁄ Hsp70 are limiting when the concentration of Hsc70/Hsp70 is lower than the combined concentration of CHIP and Hop, but Hop appears to be at an advantage com-pared to CHIP when binding to Hsp90
The cellular concentrations of Hsc70⁄ Hsp70, Hsp90, CHIP and Hop where determined by quantitative Wes-tern blot using HEK293, a commonly used epithelial cell line, and Jurkat cells, which are a model for acute T-cell leukemia (Fig 1C and Table 1) The values determined for Hsc70⁄ Hsp70 (0.9 and 0.4% of total protein for HEK293 and Jurkat cells, respectively) and Hsp90 (0.6 and 0.8% of total protein for HEK293 and Jurkat cells, respectively) were somewhat lower than
Trang 3the values of 1–2% reported for other cell lines [27a].
For Hop, we determined a relative amount of 0.2% in
both cell lines In contrast, the relative amount of
CHIP varied significantly, being 0.07% in HEK293
and only 0.01% in Jurkat cells As the total protein
concentration was 154 mgÆmL)1 in HEK293 cells [28]
and 127 mgÆmL)1 in Jurkat cells [29], the
concentra-tions of Hsc70⁄ Hsp70 are 20 and 7 lm, those of
Hsp90 are 11 and 12 lm, those of Hop are 5 and
4 lm, and those of CHIP are 3.1 and 0.4 lm in
HEK293 and Jurkat cells, respectively Given these
concentrations of Hsc70, Hsp90, CHIP and Hop and
the KD values, approximately 8.5 and 1.4% of Hsc70
and 4.6 and 0.7% of Hsp90 molecules have CHIP
bound to their C-termini at any given moment in
HEK293 and Jurkat cells, respectively As there are a large number of TPR domain proteins in higher eukaryotic cells, many of which bind to Hsp90 with a similar affinity as CHIP and Hop do [30,31], the amount of Hsp90 occupied by CHIP is probably much lower than estimated above Fewer TPR proteins have been shown to bind Hsc70⁄ Hsp70 [26] Therefore, based on our affinity determination and quantitative Western blots, the amount of Hsc70 occupied by CHIP is estimated to be 1–9% (in Jurkat and HEK293 cells)
Influence of CHIP on substrate binding of Hsp70 RING and U-box E3 ligases do not transfer ubiquitin themselves but generally bring substrates and E2 ubiquitin-conjugating enzymes in close proximity by binding to both proteins It has been shown that CHIP has the ability to bind substrates [18–20] If CHIP also contacts substrates when bound to Hsp70, it might increase the stability of the Hsp70–substrate complex, thereby allowing more time for ubiquitin transfer by the E2 enzyme
We therefore assessed whether CHIP affects the equilibrium dissociation constant (KD) or the dissocia-tion rate constant (koff) of the Hsp70–substrate complex by analyzing the formation of complexes of Hsp70 with reduced carboxymethylated a-lactalbumin
Fig 1 Interaction of CHIP with Hsc70 and
Hsp90 and in vivo concentrations of the
chaperones and co-chaperones (A,B)
Deter-mination of the interaction parameters of
the CHIP–Hsc70 (A) and CHIP–Hsp90 (B)
complexes using isothermal titration
calorim-etry (C) Quantitative immunoblot for
deter-mination of the in vivo concentrations of
Hsp70 ⁄ Hsc70, Hsp90, CHIP and Hop in
HEK293 and Jurkat cells Various amounts
of purified protein (15–400 ng, left panels)
and cleared protein extracts (10–100 lg) of
HEK293 (middle panels) and Jurkat cells
(right panels), as indicated, were separated
by SDS ⁄ PAGE and analyzed by
immunoblot-ting with specific antisera The upper bands
detected in vivo for CHIP and Hop most
likely represent phosphorylated variants of
the proteins [72,73].
Table 1 Relative amounts of CHIP, Hop, Hsp70 and Hsp90 in
HEK and Jurkat cells The relative amounts of chaperone and
co-chaperones were determined by quantitative immunoblotting as
shown in Fig 1C using purified proteins as standards.
Percentage of
Trang 4(RCMLA), a model chaperone substrate [32], in the
presence and absence of CHIP using gel filtration and
3H-RCMLA (Fig 2A,B) When Hsp70 was
pre-incu-bated with CHIP, the amount of RCMLA bound to
Hsp70 decreased by approximately 40% (Fig 2B)
This was not observed with the CHIP-K30A variant,
which does not bind to the C-terminal EEVD motif of
Hsp70 and Hsp90 [21], suggesting that CHIP does not
compete for Hsp70’s substrate binding pocket but
affects the chaperone–substrate interaction in an
indi-rect way This result clearly indicates that CHIP does
not prolong the half life of the high-affinity Hsp70–
chaperone complex
To investigate substrate release, we chased the
pre-formed Hsp70–3H-RCMLA complexes with unlabeled
RCMLA in the absence and presence of CHIP As
shown in Fig 2C, CHIP did not influence substrate
release by Hsp70, suggesting that the decrease in
detectable Hsp70–RCMLA complex is due to a
decreased association rate CHIP bound to the
C-ter-minus of Hsp70 possibly creates a steric hindrance to
substrate binding We did not observe any direct
inter-action of CHIP with the chaperone substrate RCMLA,
suggesting that substrate binding by CHIP may be a
specific interaction limited to certain proteins In
con-clusion, CHIP did not increase the half life of the
Hsp70–RCMLA complex by directly stabilizing the
chaperone–substrate interaction, but instead decreased
the amount of Hsp70-bound RCMLA by 40%
Influence of CHIP on the ATPase cycle of Hsp70
As nucleotide exchange by Hsp70 is rate-limiting for
substrate release under physiological ATP
concentra-tions, CHIP could also affect the half-life of the
Hsp70–substrate complex by altering the ATPase cycle
of Hsp70 proteins It has been reported that CHIP
decreases the ATPase rate stimulated by the J-domain
co-chaperones Hdj1 and Hdj2 but not the basal
ATPase rate under steady-state conditions [14,33] In
the absence of a J-domain co-chaperone, c-phosphate
cleavage is rate-limiting in the ATPase cycle of Hsp70
proteins [34,35] In the presence of a J-domain protein,
nucleotide exchange becomes rate-limiting [36,37] The
CHIP-induced reduction of the Hdj1⁄ Hdj2-stimulated
ATPase rate of Hsc70 could therefore be caused by a
reduced nucleotide exchange, which in turn would
increase the dwell time of the substrate on the Hsp70
chaperone To address this point, we analyzed the
influence of CHIP on ADP dissociation from and
ATP association with Hsc70 and Hsp70 using the
fluorescent nucleotide analogs N8
-(4-N¢-methylanthra-niloylaminobutyl)-8-aminoadenosine 5¢-di ⁄ triphosphate
(MABA-ADP⁄ MABA-ATP) [38] and stopped-flow instrumentation To measure the basal ADP dissocia-tion rate, Hsc70 or Hsp70 were pre-incubated with MABA-ADP in the absence or presence of a 20-fold excess of CHIP, and subsequently mixed with an
Fig 2 CHIP reduces the affinity of Hsp70 for a model substrate without affecting the dissociation rate (A) Size-exclusion chroma-tography of 3 H-RCMLA (reduced carboxymethylated a-lactalbumin) after pre-incubation in the absence or presence of Hsp70 and CHIP
as indicated (B) Quantification for the size-exclusion chromatogra-phy experiments shown in (A) 3 H-RCMLA was pre-incubated with the indicated proteins The amount of radioactivity in elution volume 9–11.5 mL, in which the RCMLA–Hsp70 complex elutes, is shown relative to the radioactivity in elution volume 12–15.5 mL, in which free RCMLA elutes (C) Dissociation of the RCMLA–Hsp70 com-plex.3H-RCMLA was pre-incubated with Hsp70 before addition of CHIP where indicated At time point 0, a fivefold excess of unla-beled RCMLA was added The complex was analyzed by size-exclu-sion chromatography at various time points The dissociation rate constants (koff) were determined by fitting a single exponential decay function to the data The inset shows the dissociation rate constants in the absence and presence of CHIP (mean ± SEM of two independent determinations).
Trang 5excess of ATP As shown in Fig 3A, CHIP had no
influence on the basal ADP dissociation rates of Hsc70
and Hsp70 These results did not explain the
CHIP-mediated decrease in the Hdj1⁄ Hdj2-stimulated steady-state ATPase rate
In vivo nucleotide exchange factors such as Bag-1 accelerate ADP dissociation by Hsc70 and Hsp70 [37,39] It has been reported that CHIP and Bag-1 interact with each other [40] We therefore determined whether CHIP could influence Bag-1-stimulated nucle-otide exchange To address this question, we pre-incu-bated Hsc70⁄ Hsp70 with MABA-ADP in the absence and presence of a large excess of CHIP, and subse-quently rapidly mixed the reaction mixture with Bag-1 and an excess of ATP As expected, Bag-1 stimulated the ADP dissociation rate by approximately 20-fold at stoichiometic concentrations Even a large excess of CHIP only slightly decreased the Bag-1-stimulated ADP dissociation rate of Hsc70 and Hsp70 (Fig 3A)
No effect of CHIP on the Bag-1-stimulated ADP dis-sociation rate was observed when CHIP was added together with Bag-1 instead of pre-incubated with the Hsp70 protein (data not shown) Therefore, the reported interaction of CHIP and Bag-1 has no strik-ing effect on the nucleotide release function of Bag-1
To analyze the second step of nucleotide exchange, ATP association, we pre-incubated Hsc70 or Hsp70 in the absence and presence of Bag-1 and a 20-fold excess
of CHIP, and subsequently mixed the reaction mixture with MABA-ATP As shown in Fig 3B,C, CHIP did not slow down ATP association significantly in the absence or presence of Bag-1 Instead we observed a slight increase in ATP association rate for Hsc70 in the presence of CHIP
As neither ADP dissociation nor ATP association are negatively affected by CHIP, the reduction in the ATPase activity must be due to an effect on c-phos-phate cleavage To verify this hypothesis, we performed single-turnover ATPase experiments The basal ATPase rate of Hsp70 proteins is very low but can be stimulated
by a J-domain co-chaperone at high concentrations (> 10-fold) As shown in Fig 3D, high concentrations
of CHIP had no effect on the basal single-turnover ATPase rate but decreased the Hdj1-stimulated ATPase
Fig 3 CHIP affects Hdj1-stimulated c-phosphate cleavage by Hsc70 ⁄ Hsp70 but not nucleotide exchange (A) MABA-ADP dissoci-ation rates of Hsc70 and Hsp70 in the absence and presence of CHIP and Bag-1 (B) Fluorescence traces of MABA-ATP association with Hsc70 in the absence and presence of CHIP and Bag-1 (C) Association rates for MABA-ATP in the absence and presence of Bag-1 and CHIP The columns show the rates for the fast phase (k1) and the slow phase (k2) of a fit of a two-phase exponential equation to the traces in (B) and additional data (D) Single-turnover ATPase rates of Hsc70 and Hsp70 in the absence and presence of Hdj1 and CHIP as indicated.
Trang 6rate, consistent with previous steady-state ATPase data
[14,33] Taken together, we found no evidence that
CHIP influences the chaperone cycle of Hsp70 proteins
to prolong the life-time of the substrate–Hsp70–CHIP
complex and thereby to increase the possibility of
recruitment of the E2 ubiquitin-conjugating enzyme and
ubiquitination In contrast, CHIP decreased the
Hdj1-triggered c-phosphate cleavage, thereby
decelerat-ing transition from the low-affinity to the high-affinity
substrate-binding state
Influence of CHIP on the ATPase activity and
co-chaperone binding of Hsp90
We next determined whether CHIP influences the
ATPase cycle of Hsp90 in order to increase the
possibil-ity of ubiquitination of an Hsp90-bound client protein
We performed steady-state ATPase assays of Hsp90 in
the absence and presence of CHIP, and in the absence
and presence of Aha1 and p23, two co-chaperones that
are known to influence the ATPase activity of Hsp90
We obtained a value of 1.2 ± 0.1· 10)3s)1 for the
basal ATPase activity of Hsp90 This rate was
stimu-lated fivefold by a threefold excess of Aha1 over Hsp90,
and inhibited to 50% of the basal rate by a tenfold
excess of p23, consistent with published data [41,42] As
shown in Fig 4, CHIP did not significantly affect the
basal ATPase rate of Hsp90, and also had no influence
on the Aha1-stimulated or p23-inhibited rate, even at a
tenfold excess over Hsp90
A previous study suggested that p23 competes with
CHIP for binding to Hsp90 [13] As CHIP did not
reduce the inhibitory effect of p23 on the ATPase
activity of Hsp90, we determined whether this is due
to the inability of CHIP to bind to Hsp90 in the
pres-ence of p23 We therefore incubated Hsp90 with CHIP
and p23 and used gel filtration to analyze the
com-plexes formed As evident from Figs 5A,B and S1,
CHIP forms a stable complex with Hsp90 and p23 and
does not prevent p23 binding to Hsp90 In contrast,
Hop reduced binding of p23 to Hsp90, consistent with
previous observations [43] Aha1 also reduced binding
of p23 to Hsp90, and CHIP could not reverse this
effect of Aha1 Taken together, CHIP did not reduce
the ATP hydrolysis rates of Hsp90 As ATP hydrolysis
leads to substrate release [44], CHIP should not
increase the half-life of Hsp90–client complexes
Unfolded proteins are more efficiently
ubiquitinated in the presence of the Hsp70 system
As CHIP interacts with both Hsp70 and Hsp90 and
the interaction is mutually exclusive, we wished to
directly compare the two chaperone systems in terms
of their influence on the efficiency of CHIP-mediated ubiquitination of a substrate It has already been shown, that both systems are able to support ubiqui-tination in vitro, but quantitative time-resolved ubiq-uitination experiments are necessary to compare their relative efficiency We pre-incubated the chaperone substrate firefly luciferase in the presence of various concentrations of Hsc70 plus Hdj1 or Hsp90 at
43C, and subsequently shifted the temperature to
30C before adding CHIP, the E2 enzyme UbcH5c, the E1 ubiquitin-activating enzyme and ubiquitin In the presence of Hsc70 and Hdj1, ubiquitination was very efficient even at low chaperone:luciferase ratios
Fig 4 CHIP has no influence on the ATPase rate of Hsp90 (A) Steady-state ATPase rate of Hsp90 in the absence or presence of the indicated concentrations of CHIP (B) Steady-state ATPase rate
of Hsp90 in the absence or presence of the indicated concentra-tions of p23 and CHIP (C) Steady-state ATPase rate of Hsp90 in the absence or presence of the indicated concentrations of Aha1 and CHIP.
Trang 7(1 : 1), but not in the absence of Hdj1 (Fig 6A,B, left
panel) In contrast, ubiquitination in the presence of
Hsp90 was not very efficient and required high
con-centrations of chaperone This indicates that many
more substrate molecules can be successfully
ubiquiti-nated per one Hsc70⁄ CHIP complex than per one
Hsp90⁄ CHIP complex Time-resolved experiments
also showed that CHIP-mediated polyubiquitination
was faster in the presence of the Hsc70⁄ Hdj1 system than in the presence of Hsp90 (Fig 6B, right panel) Interestingly, when we used the lysine-free variant of ubiquitin (Ubi-K0) to prevent polyubiquitination, we also detected multiple bands of luciferase in the pres-ence of Hsc70 and Hsp90, indicating that both chap-erones allow attachment of ubiquitin to several lysines of luciferase These data suggest that, several lysines of the substrate are modified even in the pres-ence of wild-type ubiquitin As multiple bands of ubiquitinated luciferase are already visible at the
20 min time point in the case of wild-type ubiquitin but appear later in the case of the K0 ubiquitin vari-ant, polyubiquitination may occur in the presence of Hsp70 with a certain processivity, or alternatively the lysines in ubiquitin (presumably Lys48) are better substrates for ubiquitination than lysines in the sub-strate
In addition, the co-chaperones of Hsp70 regulate the reaction in a dynamic manner Hdj1 strongly enhanced ubiquitination, as mentioned above (Fig 6A), but Bag-1 reduced the ubiquitination efficacy (Fig 6C, left panels) In contrast, neither p23 nor Aha1 had an impact on the basal Hsp90-dependent ubiquitination (Fig 6C, right panels) The presence of Hop reduced the ubiquitination of luciferase for both Hsc70 and Hsp90; however, the effect was observed only after shorter time periods, and polyubiquitinated species accumulate after longer time periods, despite increas-ing Hop concentrations (Fig 6D) Both systems gener-ate substrgener-ates with multiple ubiquitingener-ated sites, as shown for the reaction using a lysine-free ubiquitin mutant (Fig 6B) However, unfolded proteins are more efficiently ubiquitinated in the presence of the Hsp70 system
Ubiquitination of a native chaperone substrate protein
Hsp70 and Hsp90 not only interact with misfolded proteins but also with native or near-native proteins
To investigate CHIP-mediated ubiquitination of a native protein substrate, we chose the tumor suppres-sor p53, which has been shown to interact with Hsp70 and Hsp90 [45,46] At 25C, p53 was efficiently mono-ubiquitinated by CHIP in the absence of chaper-ones (Fig 7A) Neither Hsc70⁄ Hdj1 nor Hsp90 enhanced this ubiquitination reaction At 37C, Hsc70⁄ Hdj1 but not Hsp90 stimulated CHIP-mediated polyubiquitination of p53 Aha1 and p23 had only minor effects on CHIP-mediated ubiquitination in the presence of Hsp90 (Fig 7B) Taken together, as in the case of luciferase, ubiquitination of the native
Fig 5 Influence of CHIP on p23 binding to Hsp90 Hsp90 and p23
were incubated in the absence or presence of CHIP, Hop and Aha1
as indicated, and subsequently separated by size-exclusion
chroma-tography on a Superose TM 12 10 ⁄ 300 column (GE Healthcare,
Frei-burg, Germany), and analyzed by SDS ⁄ PAGE and Coomassie Blue
staining (A) Representative SDS gels (B) Quantification of the gels
shown in (A), Fig S 1 and additional data: the bands representing
p23 were quantified in all lanes The bar graph shows the amount
of p23 co-eluting with Hsp90 (lanes 4–6) relative to the total
amount of p23 (sum of all lanes).
Trang 8chaperone substrate p53 was more efficient in the
pres-ence of Hsc70 and Hdj1 than in the prespres-ence of Hsp90
and its co-chaperones Aha1 or p23
Discussion
Our study shows that CHIP cooperates with Hsc70
and Hsp90 in a rather passive manner As CHIP has
no effect on substrate dissociation, ADP dissociation
or ATP association, it does not increase the half-life of
an Hsp70–substrate complex to provide more time for recruitment of the E2 ubiquitin-conjugating enzyme Similarly, CHIP had no influence on the ATPase cycle
of Hsp90 to prolong the bound state, the ATP-bound state has a high affinity for substrates We con-clude that CHIP to sample available Hsc70–substrate and Hsp90–substrate complexes in a stochastic process and thereby occasionally effects ubiquitination Sub-strates that are efficiently folded or refolded and there-fore spend a relatively short time in complex with Hsc70 or Hsp90 have only a small chance of being ubiquitinated In contrast, substrates that cannot be folded efficiently and consequently cycle on and off the chaperones continuously, or remain bound to chaper-one for a prolonged time interval, will eventually be ubiquitinated by CHIP and targeted for degradation Such substrates may be misfolded proteins such as heat-denatured luciferase, which we have used in our study, or de novo folding substrates as the cystic
Fig 6 CHIP-mediated ubiquitination of a denatured substrate is more efficient in the presence of Hsc70 and Hdj1 than in the pres-ence of Hsp90 (A–D) Immunoblots of SDS ⁄ PAGE -separated ubiq-uitination reactions using a luciferase-specific antiserum (A) Time course of ubiquitination of heat-denatured firefly luciferase in the presence of Hsc70 and in the presence and absence of Hdj1 Heat-denatured firefly luciferase was ubiquitinated in the presence of
50 n M E1, 1 l M UbcH5c, 1 l M CHIP (except lane 1), 100 l M ubiqu-itin (except lane 2) and 5 l M Hsc70, in the absence (lanes 3–8) and presence (lanes 9–14) of 5 l M Hdj1 for 1–20 min as indicated (B) Comparison of CHIP-dependent poly- and multi-ubiquitination effi-ciency in the presence of Hsc70 ⁄ Hdj1 and Hsp90 Left panel, ubiq-uitination of firefly luciferase at various concentrations of Hsc70 (0.2–6 l M ) with 5 l M Hdj1 and various concentrations of Hsp90 (0.2–6 l M ) as indicated Luciferase was heat-denatured in the pres-ence of the chaperones, and the ubiquitination mix consisting of
50 n M E1, 1 l M UbcH5c, 1 l M CHIP and 100 l M ubiquitin was added Right panel, CHIP-dependent ubiquitination of heat-dena-tured luciferase in the presence of 5 l M Hsc70 plus 5 l M Hdj1 (lanes 9–16) or 5 l M Hsp90 (lanes 17–24) with wild-type ubiquitin (lanes 9–12 and 17–20) or the lysine-free ubiquitin variant Ubi-K0, in which all lysines were replaced by arginines (lanes 13–16 and 21–24) for 10–80 min as indicated (C) Ubiquitination of firefly lucif-erase in the presence of various chaperones and co-chaperones Lanes 1–12: ubiquitination of luciferase in the presence of 5 l M Hsc70 plus 5 l M Hdj1 and the absence (lanes 1–6) or presence (lanes 7–12) of 5 l M Bag-1 for 5–120 min as indicated Lanes 13 to 25: ubiquitination of luciferase in the presence of 5 l M Hsp90 and the absence of co-chaperones (lanes 13–17) or the presence of
5 l M Aha1 (lanes 18–21) or 5 l M p23 (lanes 22–25) for 5–40 min as indicated Lane 13 shows the ubiquitination of luciferase in the absence of CHIP but the presence of Hsp90 (D) CHIP-dependent ubiquitination of heat-denatured firefly luciferase in the presence of
5 l M Hsc70 plus 5 l M Hdj1 (lanes 1–5) or 5 l M Hsp90 (lanes 6–16) and increasing concentrations of Hop (0–23 l M ) for 20 and 120 min
as indicated.
Trang 9fibrosis transmembrane regulator CFTR, a
slow-fold-ing variant (CFTRDF508) of which is known to be
efficiently degraded and has been shown to be
ubiquiti-nated in a CHIP-dependent way [14] Such a
mecha-nism is also consistent with the phenotype of
CHIP) ⁄ )-knockout mice, which accumulate aggregated
proteins [47] The small amount of proteins that are
ubiquitinated erroneously is the price to be paid for
efficient quality control Such a mechanism is
reminis-cent of the quality control in the endoplasmic
reticu-lum, where newly synthesized glycoproteins are folded
in the calnexin⁄ calreticulin cycle [48] Misfolded
glyco-proteins are bound in turn by the chaperones calnexin
or calreticulin and the folding sensor
UDP-glucose-glycoprotein-glucosyltransferase Proteins that fold
properly exit this cycle Glycoproteins that remain in the cycle for an extended period of time have a high probability that their N-linked glycans will be trimmed
by a-1,2-mannosidase I, marking the protein for degra-dation by the ER-associated degradegra-dation pathway The affinity of dimeric CHIP for dimeric Hsp90 (0.38 lm) was approximately sixfold higher than its affinity for monomeric Hsc70 (2.3 lm) This result sug-gests binding of the dimeric CHIP to both C-termini
of the dimeric Hsp90, in agreement with a recent amide hydrogen exchange study analyzing the interac-tion of Hsc70 and Hsp90 with CHIP [49] A KD of 2.4 lm was found previously for the interaction of CHIP with a peptide comprising the ten C-terminal residues of Hsp90 [1] This value most likely represents the KDfor the initial binding of one TPR domain to a single EEVD motif of the Hsp90 dimer in a two-step sequential binding mechanism KD values in the high nanomolar range have also ben obtained for the inter-action of other TPR proteins with Hsp90 [27,30] Therefore, TPR domain proteins compete efficiently with CHIP for binding to Hsp90, and only a small amount of Hsp90 is bound to CHIP at equilibrium This contrasts with the situation for Hsc70⁄ Hsp70, whose C-termini interact with only the TPR domain proteins Hop and CHIP [26] As the concentration of Hsc70 is greater than the concentrations of Hop and CHIP together, changes in the CHIP concentration change the concentration of the Hsc70–CHIP complex, making the system very sensitive to CHIP concentra-tions Despite the lower affinity of CHIP for Hsc70 compared to Hsp90, CHIP is more frequently in com-plex with Hsc70 in the cell than with Hsp90
We further demonstrate that ubiquitination of heat-denatured luciferase is much more efficient in the pres-ence of Hsc70 and Hdj1 than in the prespres-ence of Hsp90 This observation suggests that misfolded proteins at least are targeted to the ubiquitin⁄ proteasomal path-way through the Hsp70 system rather than through the Hsp90 system Such a mechanism might also be true for bona fide Hsp90 clients once an Hsp90-specific inhibitor is added This has been indicated by data for the glucocorticoid receptor, which was found to co-localize with Hsp70 and CHIP after addition of gel-danamycin but not with Hsp90 and FKBP52 [50] However, if CHIP is over-expressed ectopically or as a consequence of a pathological process, even Hsp90-bound clients may be ubiquitinated and degraded [13– 17] As all Hsp90 clients, which are degraded upon CHIP over-expression, are also substrates of Hsc70, the overproduced CHIP may act on the Hsc70–client complex rather than the Hsp90–client complex Our data with p53 support this hypothesis Therefore, it
Fig 7 Chip-mediated ubiquitination of the native chaperone
sub-strate p53 (A) Temperature dependence of p53 ubiquitination p53
was ubiquitinated in the absence and presence of Hsc70⁄ Hdj1,
Hsp90 or both at 25 C (left panel) or 37 C (right panel)
Immuno-blot of SDS ⁄ PAGE-separated ubiquitination reactions using a
p53-specific antiserum (B) The Hsp90 co-chaperones Aha1 and
p23 have no influence on CHIP-mediated ubiquitination of p53 p53
was ubiquitinated in the presence of Hsp70 ⁄ Hdj1, Hsp90 and Aha1
and p23 as indicated.
Trang 10remains unclear whether CHIP can selectively
ubiquiti-nate folded Hsp90-associated clients, thereby
perform-ing regulatory functions in the cell If such a function
of CHIP exists, it seems to be of minor importance, as
the stability of several bona fide Hsp90 substrates is
not affected in CHIP) ⁄ ) mouse embryonic fibroblasts
[51] The CHIP) ⁄ ) mice show phenotypes related to
abnormal protein aggregation [47] rather than
break-down of signaling pathways (compare with FKBP52
knockout mice [52,53]) All these facts speak in favor
of CHIP being an E3 ubiquitin ligase with low
sub-strate specificity that is responsible for the clearance of
hopeless cases of protein folding However, the role of
CHIP in direct substrate binding and its E4 ligase
function [54,55] remain puzzling
The native chaperone substrate p53 was only
mono-ubiquitinated by CHIP at 25C, and the chaperones
did not enhance this ubiquitination nor stimulate
poly-ubiquitination at this temperature At 37C,
Hsc70⁄ Hdj1 but not Hsp90 stimulated CHIP-mediated
polyubiquitination NMR experiments with p53 core
domain showed that the core domain starts unfolding
at 37C and binds concomitantly to Hsp90 [56] The
Hsc70⁄ Hdj1-stimulated polyubiquitination may
there-fore be due to recognition of unfolded regions within
p53 by the chaperone Therefore, ubiquitination of p53
appears to be very similar to ubiquitination of the
denatured firefly luciferase
Our study also clarified the previously observed
effect of CHIP on the Hdj1⁄ Hdj2-stimulated
steady-state ATPase rate of Hsc70 [14,33] We show here that
nucleotide exchange of Hsc70 is not affected by CHIP
In contrast, CHIP decreased the Hdj1-stimulated
c-phosphate cleavage, as demonstrated by
single-turn-over ATPase experiments CHIP slows down the
transition of Hsc70 from a low-affinity state with high
substrate association and dissociation rates to a
high-affinity state with low substrate dissociation rates
CHIP therefore counteracts the targeting function of
the J-domain protein The molecular basis for this
observation could be a reduced association rate for
substrates It was shown previously that Hsp70
pro-teins require two signals for highly efficient hydrolysis
of ATP: one signal provided by the J-domain and a
second signal provided by the substrate [57–62] High
concentrations of some J-domain proteins can provide
both signals by interaction with the substrate binding
pocket as well as the ATPase domain [59,63–65] As
CHIP reduces the affinity for substrates without
affect-ing the dissociation rate, substrate association is
conse-quently reduced This in turn reduces the substrate
signal for ATP hydrolysis It may be advantageous if
substrates do not associate with Hsc70 when CHIP is
already bound Such a mechanism would prevent ubiq-uitination of a substrate that has not had the opportu-nity to refold
In summary, our results suggest the model shown in Fig 8 Proteins in an intermediate folding state after
de novosynthesis at the ribosome or proteins misfolded under stressful conditions are bound by Hsp70s in an Hdj-dependent manner and folded⁄ refolded to the native state Proteins that do not fold or that are diffi-cult to fold are released and rebound by Hsp70s sev-eral times (black symbols and arrows in Fig 8) In a stochastic process, CHIP associates with Hsp70–sub-strate complexes and recruits the E2 conjugating enzyme for ubiquitination of the substrate As Hsp70s are approximately 10–40 times more abundant than CHIP, only approximately 1–10% of the Hsp70– substrate complexes will be bound by CHIP with possible ubiquitination of the substrate Efficiently folding substrates (gray symbols and arrows in Fig 8) have only a small chance of being ubiquitinated Hsc70–CHIP complexes are less likely to bind
misfold-ed proteins The likelihood of at least one round of refolding is thereby increased This model of the triage decision allows sufficient time for refolding attempts
by the chaperones, keeping the amount of erroneously degraded chaperone substrates low Any increase in CHIP concentration due to physiological or patho-physiological processes will increase the clearance rate for damaged proteins, at an increased cost of degrad-ing proteins that are still useful
Experimental procedures Protein expression and purification
Human CHIP was produced in Escherichia coli and puri-fied by a combination of cation- and anion-exchange chromatography as described previously [2] Human Hdj1 and human Bag-1 were purified as described previously [37] Human Hop was purified from over-producing E coli strains as described previously [66,67] All proteins were quantified as described previously [68] using the Bio-Rad reagent (Bio-Rad Laboratories, Mu¨nchen, Germany) Human wild-type Hsp90b, Hsc70, Hsp70 and Aha1 were expressed with an Ulp1 cleavable N-terminal His6–Smt3 tag in E coli for 5 h at 30C (20 C for Aha1) and purified
as described previously [69] with some modifications The cells were lysed in a French press in 25 mm HEPES⁄ KOH
further purified on a Resource-Q column (GE Healthcare, Freiburg, Germany) Hsp90 and Aha1 were further purified
on a Superdex 200 Hiload 16⁄ 60 column (GE Healthcare)