(Received for publication, December 23, 1994; and in revised form, July 21, 1995)
From the
Cytosolic Raf-1 exists in a high molecular weight complex with the heat shock protein Hsp90, the purpose of which is unknown. The benzoquinone ansamycin, geldanamycin, specifically binds to Hsp90 and disrupts certain multimolecular complexes containing this protein. Using this drug, we are able to demonstrate rapid dissociation of both Raf-1-Hsp90 and Raf-1-Ras multimolecular complexes, concomitant with a markedly decreased half-life of the Raf-1 protein. Continued disruption of the Raf-1-Hsp90 complex results in apparent loss of Raf-1 protein from the cell, although Raf-1 synthesis is actually increased. Prevention of Raf-1-Hsp90 complex formation interferes with trafficking of newly synthesized Raf-1 from cytosol to plasma membrane. These data indicate that association with Hsp90 is essential for both Raf-1 protein stability and its proper localization in the cell.
Raf-1, a serine/threonine kinase, is part of a highly conserved kinase cascade that mediates signaling by extracellular growth factors and leads to the stimulation of mitogen-activated protein kinases(1, 2) . Raf-1 functions downstream of Ras, which in its active, GTP-bound state binds directly to the amino-terminal regulatory domain of Raf-1 (3) . This interaction is transient and apparently serves to recruit Raf-1 to the cell membrane(4, 5) , a step that is necessary for Raf-1 activation. The requirement for Ras can be bypassed by coupling a plasma membrane targeting signal to Raf-1(6) . Following its recruitment by Ras, Raf-1 associates with cytoskeletal components via an unknown mechanism(7) .
Although activated Raf-1 is plasma membrane-associated, this kinase is primarily cytosolic in location and exists in a native heterocomplex with the heat shock proteins Hsp90 and p50(8) . Hsp90 is an ubiquitously expressed molecular chaperone that has been found in complexes with a variety of proteins including steroid hormone receptors, dioxin receptor, actin, v-src, and other kinases(9, 10, 11, 12, 13) . Raf-1 binds to Hsp90 via its COOH-terminal catalytic domain (8) and remains complexed to Hsp90 and p50 even when bound to Ras at the plasma membrane(14) . It is not clear why native Raf-1 associates with Hsp90, although it has been proposed that this heat shock protein is involved in Raf-1 transport to the cell membrane(15) .
The benzoquinone ansamycin, geldanamycin
(GA), ()has been shown to bind specifically and directly to
Hsp90 and to disrupt the Hsp90-pp60
molecular
complex, leading to destabilization of
pp60
(16) . Although originally described
as tyrosine kinase inhibitors, benzoquinone ansamycins have been shown
to be inactive when added directly to purified tyrosine kinases at
concentrations >1500 times their effective in vivo dose(17, 18) . Additionally, several attempts to
demonstrate direct association of ansamycins with tyrosine kinases in vivo and in vitro have been
unsuccessful(16, 19) . This class of drug is now
thought to exert kinase inhibitory activity indirectly by somehow
destabilizing these proteins(18, 20, 21) .
Consistent with this hypothesis, binding to Hsp90 and destabilization
of the Hsp90-pp60
complex occurs both in
vivo and in vitro at nanomolar concentrations of
ansamycin(16) . These drug levels are very similar to the
concentration of ansamycin previously reported to produce decrements in
cellular lck, v-src, and epidermal growth factor receptor protein level
and activity(18, 20, 21) , leading to the
hypothesis that benzoquinone ansamycins are tyrosine kinase inhibitors
because they disrupt Hsp90-kinase heterocomplexes(16) .
We now report that GA also disrupts the association between Hsp90 and the serine/threonine kinase Raf-1. The purpose of this study was to use GA to analyze the function of the Raf-1-Hsp90 complex.
[S]Methionine-labeled proteins were
immunoprecipitated and electrophoresed as described above. The SDS-PAGE
gel was fixed with a solution of 10% acetic acid and 50% methanol,
washed copiously in water, and enhanced with Enlightning solution
(DuPont NEN) prior to gel drying and autoradiography. Films of either
chemiluminescent or radioactive blots were scanned into a Macintosh
computer using a Foto/Eclipse Gel Analysis system (Fotodyne), and band
intensities were quantified using Collage Analysis software (Fotodyne).
Raf-1 half-life was determined by regression analysis of log
transformed Raf-1-specific band intensities.
Figure 1: GA disrupts multimolecular complexes containing Raf-1 protein. Log phase MCF7 cells were cultured for 4 h with (lanes 2 and 4) or without (lanes 1 and 3) GA (2 µM) before being lysed with TENSV buffer. 1800 µg of total protein were immunoprecipitated with Raf-1 antibody (lanes 1 and 2). As well, 50 µg of total protein from cell lysates were used for Western blotting (lanes 3 and 4).
Although cellular Raf-1 protein was reduced by 55% in
GA-treated cells (compare Raf-1 signal in lanes 3 and 4, Fig. 1), Raf-1-specific immunoprecipitation from 1.8
mg of total protein resulted in apparent antibody saturation, because
drug treatment only minimally reduced the amount of Raf-1 recoverable
by immunoprecipitation (compare Raf-1 signal in lanes 1 and 2, Fig. 1). Because native Raf-1-Hsp90 heterocomplexes
are unstable compared with the pp60-Hsp90
complex(8) , the data shown in Fig. 1do not represent a
stoichiometric coprecipitation. However, this technique yields
qualitative data demonstrating the disruption of existing Raf-1-Hsp90
and Raf-1-Ras complexes. Because Hsp90 is a very abundant protein and
serves as a chaperone for a variety of other proteins, only a minor
fraction of it is associated with Raf-1, although at least all
cytosolic Raf-1 appears to occur in a complex with heat shock
proteins(14) . The amount of GTP-Ras bound to Raf-1 depends on
the activation status of the cell.
Figure 2:
GA increases Raf-1 synthesis while
decreasing Raf-1 half-life. A, CHP100 cells were pretreated
with or without GA (1 µM) and exposed to a 2-h
[S]methionine pulse (100 µCi/ml of
methionine-free medium) before lysing the cells in TENSV buffer. Lanes 1 and 2 show total Raf-1 determined by Western
blotting. Additionally, 200 µg of total protein was
immunoprecipitated with Raf-1 antibody, separated on an 8% SDS-PAGE
gel, and visualized by autoradiography (lanes 3 and 4). Data shown were obtained by immunoprecipitating equal
amounts of protein, but similar results were obtained if equivalent
acid-precipitable radioactivity was immunoprecipitated. B,
determination of Raf-1 half-life in CHP100 and MCF7 cells by
[
S]methionine pulse-chase. Cells were
preincubated with or without GA (2 µM) for 16 h. They were
then pulsed for 2 h with [
S]methionine (100
µCi/ml) and chased with nonradioactive methionine-containing medium
for 3-24 h. The cells were lysed in TENSV buffer, and Raf-1 was
immunoprecipitated and analyzed as above.
In order to test this
hypothesis directly, a [S]methionine pulse-chase
protocol was used to determine Raf-1 half-life in both CHP100 and MCF7
cells exposed to GA for 16 h. GA exposure reduced Raf-1 half-life from
17.5 to 4 h in CHP100 cells and from 11 to 4 h in MCF7 cells (Fig. 2B). The half-maximal GA concentration necessary
to produce these effects was determined to be 25 nM (data not
shown).
Figure 3:
Time course of Raf-1 disappearance from
different cell compartments following GA addition. A, MCF7
cells were grown without GA (lanes 1, 5, and 9) or with GA (2 µM) for 4 h (lanes 2, 6, and 10), 16 h (lanes 3, 7, and 11), or 40 h (lanes 4, 8, and 12).
The cells were lysed, and preparations of cytosol (lanes
1-4) and Nonidet P-40-soluble (lanes 5-8) and
-insoluble (lanes 9-12) membrane fractions were
obtained. 15 µg of total protein/lane were electrophoresed through
10% SDS-PAGE minigels. Western blotting was performed for Raf-1. B, Western blotting for Ras was performed using the fractions
obtained from untreated and 16-h GA-treated samples. Lanes 1 and 2 represent cytosol from untreated and GA-treated
samples, respectively; lanes 3 and 4 represent
Nonidet P-40-soluble membrane preparations from untreated and
GA-treated samples, respectively; and lanes 5 and 6 represent Nonidet P-40-insoluble membrane preparations from
untreated and GA-treated samples, respectively. C, MCF7 cells
were pulsed with [S]methionine after
pretreatment without (lanes 1 and 2) or with (lanes 3 and 4) GA (2 µM) and then
chased for 4 h with nonradioactive media. Cytosol and Nonidet
P-40-soluble membrane preparations were obtained, and equal amounts of
total protein were immunoprecipitated with Raf-1 antibody,
electrophoresed on an 8% SDS-PAGE gel, and visualized by
autoradiography. Cytosolic fractions are represented in lanes 1 and 3; Nonidet P-40-soluble membrane fractions are
represented in lanes 2 and 4. This experiment was
performed twice. Densitometric analysis of the Raf-1 bands in untreated
cells (in the two experiments) revealed 24 and 39% of the radioactivity
in cytosol and 76% and 61% of the radioactivity in membrane. In
GA-treated cells, 65 and 75% of the radioactivity was found in cytosol,
compared with 25 and 35% in the membrane fraction. D, the
experiment described in A was performed four times. Band
densities were determined by densitometric analysis. A graphical
representation of the cumulative results is depicted. Data are
displayed as the fraction of the value obtained in untreated cells
± S.D.
Given the long half-life of Raf-1 in these tumor cells (see Fig. 2B), the data are consistent with several events. First, disruption of preformed cytosolic Raf-1-Hsp90 complexes by GA leads to rapid destabilization of cytosolic Raf-1 protein. Continued exposure of cells to GA also prevented newly synthesized Raf-1 from associating with Hsp90 (data not shown). Second, Raf-1-Hsp90 complex disruption affects the detergent-soluble membrane component of Raf-1 as rapidly as it does the cytosol component (Fig. 3, A and D). Loss of Raf-1 from this fraction occurred too quickly to be due solely to inability to recruit new Raf-1 from the cytosol. Immunoprecipitation of Raf-1 no longer co-precipitated Ras at a time when significant amounts of Raf-1 were still found in the detergent-soluble membrane fraction (compare Fig. 3A, lane 6 with Fig. 1, lane 2), suggesting that Ras association with Raf-1 requires the continued participation of Hsp90. Furthermore, Raf-1 stability, even when the protein is associated with Ras, depends on the presence of Hsp90.
Finally, the kinetics of Raf-1 turnover in the
detergent-insoluble membrane fraction are also affected by GA (Fig. 3, A and D). Whether this means that
anchorage of Raf-1 to cytoskeletal elements requires participation of
Hsp90 remains to be determined, because the harsh conditions necessary
to solubilize cytoskeleton-bound Raf-1 preclude recovery of protein
heterocomplexes. Although a model in which enzymatically active Raf-1
is no longer associated with Hsp90 would be appealing in that such a
model would be strikingly similar to that proposed for association of
Hsp90 with pp60(22) , such a model is not
consistent with our current data.
Together with destabilization,
Raf-1-Hsp90 complex disruption might also affect the ability of
cytosolic Raf-1 to be recruited by Ras. To address this question, we
pulsed MCF7 cells, which had been exposed to GA for 16 h, with
[S]methionine and followed the labeling with a 4
h chase (Fig. 3C). As described above (see Fig. 2A), Raf-1 synthesis in drug-treated cells was
elevated (approximately 3-fold). However, in two separate experiments,
the chase period was sufficient to allow 68% of newly synthesized Raf-1
(61 and 76%, respectively) to appear in the Nonidet P-40-soluble
membrane fraction of untreated cells, whereas 32% of the labeled
protein (24 and 39%, respectively) was recovered from the cytosol. In
contrast, in GA-treated cells only 30% of radiolabeled Raf-1 (25 and
35%, respectively) was recovered from the Nonidet P-40-soluble membrane
fraction, whereas 70% (65 and 75%, respectively) remained associated
with the cytosol. These data are consistent with a model in which
disruption of Raf-1-Hsp90 cytosolic complexes not only destabilizes
Raf-1 but also interferes with its proper intracellular trafficking and
recruitment to the membrane by Ras.
Figure 4: GA depletes Raf-1 from both transformed and untransformed cell lines. Cells were grown in log phase culture for 16 h without (lanes 1, 3, 5, 7, 9, 11, and 13) or with GA (2 µM) (lanes 2, 4, 6, 8, 10, 12, and 14). TENSV lysates were analyzed for Raf-1 content by Western blotting with Raf-1 antibody. The cells used were NIH 3T3 (lanes 1 and 2), CHP100 (lanes 3 and 4), HeLa (lanes 5 and 6), MCF7 (lanes 7 and 8), DU145 (lanes 9 and 10), Raji (lanes 11 and 12), and CEM (lanes 13 and 14).
Taken together, these data point to a role for Hsp90 in Raf-1-mediated signal transduction. Although the half-life of Raf-1 is approximately 10-20 h in the cell lines examined, such prolonged stability requires association of Hsp90 with both cytosolic Raf-1 as well as the Raf-1 found in the cell membrane fraction. Disruption of this complex is followed by a marked reduction in Raf-1 stability, even though synthesis of the protein is actually elevated. However, newly synthesized Raf-1, to which Hsp90 has been prevented from binding, cannot translocate efficiently to the Nonidet P-40-soluble membrane fraction. Additionally, Raf-1, already associated with this fraction at the time of Hsp90 dissociation, rapidly dissociates from Ras. Therefore, Hsp90 association also appears necessary for recruitment of cytosolic Raf-1 to the plasma membrane, as well as for its maintenance there in a Ras-bound state.
Because binding to Ras is part of the mechanism that leads to activation of Raf-1 and because the most highly enzymatically active Raf-1 is found in the Nonidet P-40-insoluble cellular membrane fraction (5) , prevention of Raf-1 binding to Ras and depletion of Raf-1 from this and other cellular compartments are likely to have a negative impact on Raf-1 function.