(Received for publication, November 7, 1995)
From the
Treatment of SKBr3 cells with benzoquinone ansamycins, such as
geldanamycin (GA), depletes p185, the receptor
tyrosine kinase encoded by the erbB2 gene. In the same cells,
a biologically active benzoquinone photoaffinity label specifically
binds a protein of about 100 kDa, and the ability of various GA
derivatives to reduce the intracellular level of p185
correlates with their ability to compete with the
photoaffinity label for binding to this protein. In this report, we
present evidence that the
100-kDa ansamycin-binding protein is
GRP94. Membrane-associated p185
exists in a
stable complex with GRP94. GA binding to GRP94 disrupts this complex,
leading to degradation of pre-existing p185
protein, and resulting in an altered subcellular
distribution of newly synthesized p185
.
Herbimycin A (HA) ()and geldanamycin (GA) are
benzoquinone ansamycins with potent antiproliferative activity that
specifically bind to the heat shock protein hsp90(1) , with
which several tyrosine kinases, as well as other intracellular signal
transduction molecules, are
complexed(2, 3, 4, 5, 6, 7, 8, 9) .
Geldanamycin dissociates certain multi-molecular complexes containing
HSP90(1, 10) , leading to target protein (i.e. v-Src, c-Raf-1) instability(1, 9) .
The erbB2 gene (also known as her-2/neu) encodes a
185-kDa receptor-like protein (p185) with
tyrosine kinase activity. This protein is overexpressed in many breast,
ovarian, and prostate carcinomas and is associated with poor prognosis.
Miller et al.(11) have reported that
p185
is rapidly depleted in human breast
cancer cells (SKBr3) following exposure to HA or GA. Although the
p185
protein level is markedly reduced,
p185
mRNA and protein synthesis are only
slightly affected, and it appears that one of the primary effects of
these drugs is to significantly reduce the half-life of
p185
(11, 12) . However,
unlike v-Src and c-Raf-1 (see above), p185
cannot be demonstrated to form a complex with
HSP90(12) . Intriguingly, when they used a biologically active,
I-labeled photoaffinity derivative of GA (CP202509),
Miller et al.(13) demonstrated specific binding to a
100-kDa protein in SKBr3 cells, but not to p185
itself. The ability of various GA derivatives to reduce the
intracellular level of p185
correlated with
their ability to specifically compete with the
I-photoaffinity label for binding to this protein.
In
this report, we present evidence that this ansamycin-binding protein is
the glucose-regulated protein GRP94, an endoplasmic/sarcoplasmic
reticulum protein with homology to the molecular chaperone HSP90 (14, 15, 16) . An abundant cellular
glycoprotein, GRP94 is induced in response to glucose deprivation,
hypoxia, calcium ionophores, glycosylation inhibitors, and low pH, and
its expression is up-regulated in pathological states during which
these conditions naturally occur, such as ischemia and tumor growth
(reviewed in (17) ). While the function of GRP94 has not been
well defined, it has been implicated in Ca regulation(18) , protein folding, and antigen
presentation(19, 20) . In addition, we demonstrate
here the existence of a stable heterocomplex between p185
and GRP94. Finally, we show that GA rapidly dissociates
GRP94 from p185
and that this occurs prior to
degradation of p185
protein.
To verify the specificity of the immunoprecipitation, lysates of unlabeled SKBr3 cells were prepared in parallel with the above samples. Immunoprecipitation and immunoblotting with both antibodies revealed that neither cross-reacted with the other's target (native or denatured), demonstrating that both antibodies, whether used for immunoprecipitation or immunoblotting, are specific for their cognate antigens.
Figure 1:
p185 and GRP94
exist as a complex in SKBr3 cells. SKBr3 cells were incubated 18 h in
the presence (+) or absence(-) of GA (3 µM).
Prior to SDS-PAGE analysis, proteins contained in membrane (M)
or cytosol (C) fractions were (panel B) or were not (panels A and D) immunoprecipitated with rat
monoclonal anti-human GRP94. In panel C, proteins in total
lysate (L) were (+) or were not(-) subjected to
immunoprecipitation with monoclonal anti-human
p185
. The proteins were separated by
electrophoresis and immunoblotted with monoclonal antibodies to
p185
(panels A and B) or to
GRP94 (panels C and D) as described under
``Materials and Methods.''
In order
to determine whether disappearance of the
p185GRP94 complex following overnight exposure to
GA was the result of loss of p185
, or the result of
complex dissociation prior to loss of p185
, we
performed the same experiment following a brief exposure of SKBr3 cells
to GA. As seen in Fig. 2, after a 75-min exposure of SKBr3 cells
to GA, total p185
, as measured by Western blotting of
cell lysates, was only minimally reduced (see Fig. 2A).
However, co-precipitation of a p185
GRP94 complex
was dramatically reduced (see Fig. 2B).
Figure 2:
The native complex between
p185and GRP94 is dissociated by GA prior to
p185
depletion. SKBr3 cells were incubated for
75 min in the presence (+) or absence(-) of GA (3
µM). Prior to SDS-PAGE analysis, proteins contained in
membrane (M) or cytosol (C) fractions were (panel
B) or were not (panel A) immunoprecipitated with rat
anti-human GRP94. Immunoblotting of p185
was
performed using a murine anti-human p185
antibody.
Figure 3:
GA binds to cellular GRP94. SKBr3 cells
were labeled with I-CP202509 and lysed as described under
``Materials and Methods.'' Lysates were subjected to
immunoprecipitation with monoclonal antibodies to GRP94 or HSP90 as
described. The positions of protein molecular size standards are
indicated (in kDa). Lane 1, lysate of cells incubated with
I-CP202509 but not exposed to UV light. Lane 2,
lysate of cells incubated with
I-CP202509 followed by
photolysis. Lane 3, lysate in lane 2 after clearing
by immunoprecipitation with anti-GRP94. Lane 4, anti-GRP94
immunoprecipitate from lysate in lane 2. Lane 5,
lysate of cells incubated with
I-CP202509 and 5
µM CP127374 followed by photolysis. Lane 6,
anti-GRP94 immunoprecipitate from lysate in lane 5. Lane
7, lysate in lane 2 after clearing with anti-HSP90. Lane 8, anti-HSP90 immunoprecipitate of lysate in lane
2.
Figure 4:
GA
alters the subcellular distribution of newly synthesized
p185. A, cells, grown on coverslips,
were treated with GA (2 uM for 22 h), and then
p185
immunofluorescence was detected as
described under ``Materials and Methods.'' Results are
compared to untreated cells. B, cells treated as in A were pulse-labeled with
[
S]methionine/cysteine for the final 6 h.
p185
was immunoprecipitated from total cell
lysate and subjected to overnight digestion with endoglycosidase H as
described under ``Materials and Methods.'' Lanes
1-3 represent p185
immunoprecipitations from control cells, and lanes
4-6 represent drug-treated cells. Lanes 1 and 4, no endoglycosidase H; lanes 2 and 5, 10
milliunits of endoglycosidase H; lanes 3 and 6, 20
milliunits of endoglycosidase H.
After 22 h in
the presence of GA, however, immunofluorescence analysis revealed that
p185 was no longer localized to the plasma membrane,
but instead appeared restricted to cytoplasmic inclusions, as
visualized by a punctate pattern of perinuclear fluorescence (Fig. 4A). Since GA depletes pre-existing
p185
protein within several hours(11) , and
reduces the half-life of newly synthesized protein from greater than 9
h to approximately 2 h(12) , the immunofluorescent signal
observed in Fig. 4A most likely represents
p185
newly synthesized in the presence of GA.
Immunoprecipitation and PAGE analysis of newly synthesized
p185
(using a 6-h pulse with
[
S]methionine) from cells treated with GA for 24
h revealed that the ratio of distribution of the protein between
membrane and cytosol remained the same as in untreated cells (83% of
total signal in membrane fraction; data not shown). However, drug
treatment rendered all of the newly synthesized material sensitive to
endoglycosidase H, while p185
synthesized in the
absence of GA was only slightly sensitive to endoglycosdiase H (Fig. 4B). Endoglycosidase H sensitivity is
characteristic of incomplete glycosylation and is consistent with the
lower apparent molecular weight of p185
synthesized in
the presence of GA (Fig. 4B, compare lanes 1 and 4). Since resistance to endoglycosidase H is acquired
in the trans-Golgi (21) , these data are further consistent
with the trapping of p185
synthesized in the presence
of GA in the endoplasmic reticulum or cis-Golgi. Thus, although
overnight exposure to GA does not change the fraction of protein that
is membrane associated, it causes dramatic subcellular redistribution
of newly synthesized p185
.
In this report, we demonstrate that the p185 receptor tyrosine kinase forms a stable complex with the
glucose-regulated chaperone protein GRP94. The benzoquinone ansamycin
GA destabilizes this complex within 75 min, prior to significant loss
of p185
, which occurs over the next several hours.
Exposure of SKBr3 cells to GA and other benzoquinoid ansamycins has
been shown previously to result in rapid loss of
p185
(11) . This effect appears to be mediated
primarily at the level of protein stability, since p185
mRNA level and rate of synthesis remain essentially unaltered,
while the half-life of the protein is reduced from 9.5 to 2
h(11, 12) . Effects of benzoquinone ansamycins on
other receptor tyrosine kinases have been noted. Thus, Murakami et
al.(22, 23) reported destabilizing effects of HA
on the epidermal growth factor receptor, while Sepp-Lorenzino et
al.(24) described similar effects of HA on both the
insulin receptor and the insulin-like growth factor receptor.
Sepp-Lorenzino et al.(24) suggest that the protein
instability caused by HA is mediated by the 20 S proteasome in a
ubiquitin-dependent manner.
Previously, using an iodinated
photoaffinity analog of GA, Miller et al.(13) demonstrated that this compound did not label
p185, but instead bound an unknown protein of
approximately 100 kDa. Furthermore, the ability of various GA analogs
to deplete cellular p185
correlated with their ability
to interact with p100(13) . The data presented here identify
p100 as GRP94, which can be co-precipitated in a complex with
p185
. By binding to GRP94, GA either induces rapid
dissociation of this complex or interferes with the dynamic equilibrium
of complex association-dissociation. This occurs prior to significant
loss of p185
, suggesting a role for GRP94 in
maintaining p185
stability. Whether ansamycin
disruption of GRP94
p185
complexes leads to
ubiquitination of p185
and its subsequent proteolysis
by the 20 S proteasome is currently under investigation.
Other receptors may be regulated in a similar fashion. For example, the type I tumor necrosis factor receptor, which has no tyrosine kinase activity, has recently been shown to form a native complex, whose function is as yet undetermined, with a protein showing strong homology to members of the HSP90 family(25) .
Our current data
further suggest that failure of newly synthesized p185 to associate with GRP94 prevents the translocation of the newly
synthesized protein to the plasma membrane, instead trapping it in an
intracellular vesicular compartment consistent with the endoplasmic
reticulum/cis-Golgi. The data in Fig. 4demonstrate that,
following overnight exposure to GA, p185
is only
detectable in this vesicular compartment and not on the plasma
membrane. Perhaps an association with GRP94 is required for both the
proper intracellular trafficking and stability of a family of receptor
proteins.
GA has previously been shown to bind to the heat shock
protein HSP90, with which GRP94 shares 50% homology(26) . The
drug causes dissociation of heterocomplexes composed of this protein
and various signal transduction proteins, including v-Src, c-Raf-1, and
the progesterone receptor(1, 9, 10) . In the
case of c-Raf-1 and v-Src, heterocomplex dissociation results in
protein instability and altered subcellular
localization(1, 9, 27) . These findings are
thus quite similar to those reported here, except p185 apparently associates with GRP94 and not HSP90. It is not clear
why the GA photoaffinity label only recognizes GRP94 and not HSP90 in
either intact cells or cell lysates. We have documented that SKBr3
cells contain normal amounts of HSP90, and that this HSP90 efficiently
binds to solid phase-immobilized GA in a competable manner (1) . (
)In addition, the photoaffinity label binds
effectively to purified HSP90 in a photolysis-dependent manner in
vitro.
Conversely, GRP94 binds to solid
phase-immobilized GA, corroborating the photoaffinity label results,
but GRP94 binding is less efficient than that of HSP90.
A
possible explanation for these apparent discrepancies is that GA and
its photoaffinity label derivative, although both effective in
depleting p185
(13) , possess different
affinities for GRP94 and HSP90. At the same time, substitution with a
bulky group at the 17-position of GA (necessary for solid phase
immobilization of GA; see (1) ) may decrease the drug's
binding affinity to GRP94 without affecting binding to HSP90. In fact,
certain 17-substituted GA derivatives are poor depletors of
p185
(28) ,
presumably because of
their failure to interact with GRP94.
The current results, together
with previous reports of GA effects on HSP90(1, 10) ,
identify the molecular site of action of the benzoquinone ansamycins as
the chaperonins, specifically HSP90 and GRP94. Binding of these
inhibitors to the chaperonins appears to destabilize the complex of an
array of signaling proteins, such as p185, v-Src, or
c-Raf-1, with the chaperone protein, thereby directing the signaling
protein to a degradative pathway. Further studies should better define
the role of these chaperones in regulating signal transduction as well
as the potential of the benzoquinone ansamycins in the pharmacologic
manipulation of this process.