(Received for publication, June 12, 1995; and in revised form, September 5, 1995)
From the
The abundant, cytoplasmic 90-kDa heat-shock protein associates
transiently with the Rous sarcoma virus oncogenic protein tyrosine
kinase, pp60, directs its cellular
trafficking and negatively regulates its kinase activity. Here we
report that the serine/threonine phosphatase inhibitor, okadaic acid,
destabilized the heat-shock protein
90-pp60
chaperone complex in
v-src-transfected cells. Concomitant with complex
destabilization by okadaic acid, phosphoserine was doubled and
phosphothreonine was increased 20-fold in the heat-shock protein 90.
Although phosphorylation of the total pool of immunoprecipitable
pp60
was unchanged, okadaic acid
slightly increased phosphoserine and phosphothreonine levels
specifically in pp60
bound to
heat-shock protein 90. The low level of tyrosine phosphorylation in the
pp60
complexed with heat-shock
protein 90 was further decreased by okadaic acid. Interestingly,
okadaic acid-stabilized hyperphosphorylation of the heat-shock protein
90-pp60
complex lowered the level
of pp60
in cell membranes, the
functional location for pp60
. We
suggest that serine/threonine phosphorylation of heat-shock protein 90
and/or pp60
functions as a
regulatory molecular trigger to release pp60
from the chaperone complex at the inner surface of cell
membranes.
Neoplastic transformation of avian or mammalian cells by the
Rous sarcoma virus (RSV) ()results directly from the
protein-tyrosine kinase activity of a single phosphoprotein,
pp60
, that is encoded by the
v-src oncogene(1) . The phosphorylation cascade
initiated by pp60
kinase activity
at or near the plasma membrane is considered to be critical to the cell
transformation process (reviewed in (2, 3, 4) ).
Although it has been
established that nascent pp60protein is synthesized on cytosolic free
polyribosomes(5) , the mature kinase localizes predominantly to
the cytoplasmic surface of the plasma membrane where it is anchored via
a myristic acid residue(6) . Additionally, some
pp60
is associated with perinuclear
membranous structures(7, 8) . During its intracellular
trafficking, v-Src oncoprotein is transiently associated with a
constitutive cytosolic 90-kDa heat-shock or stress protein (hsp90) and
a second host cell 50-kDa phosphoprotein of unknown
function(9, 10, 11) . Complexation with hsp90
sharply diminishes the intrinsic kinase activity of
pp60
, and both autophosphorylation
of the v-Src protein at tyrosine 416 and phosphorylation of cellular
substrate proteins on tyrosine are minimalized until after
pp60
is attached to the plasma
membrane(12) . Therefore, the formation of the
hsp90-pp60
complex tightly
regulates the kinase activity of pp60
and plays an obligatory biochemical function in cell
transformation by this viral oncogene(13) .
The aim of the
present study was to further characterize the transient interaction of
pp60 with hsp90. Specifically, we
were interested in the role of phosphorylation/dephosphorylation of
either hsp90 or pp60
in the
molecular interactions of these two proteins. Toward this end, we
attempted to manipulate the phosphate status of both proteins by
treating v-src-transfected cells with okadaic acid (OA), a
potent inhibitor of the serine/threonine protein phosphatases PP1 and
PP2A (see (14) for review). OA has been shown to stabilize the
hyperphosphorylation of many cellular proteins, notably retinoblastoma
protein and p53(15) , and to reverse the neoplastic phenotypic
characteristics of v-src-transformed 3T3 cells(16) .
Figure 1:
Coimmunoprecipitation of hsp90 with
pp60. TSv-src3T3 cells were lysed with TNESV buffer
supplemented with protease inhibitors, and
pp60
was immunoadsorbed from
lysates (1 mg of total protein) with anti-src mAb (UBI). hsp90
was immunoprecipitated with SPA-830 anti-hsp90 mAb (StressGen),
anti-hsp86 polyclonal antibody (NeoMarkers), or GA beads, with the
appropriate secondary antibodies coupled to protein A-Sepharose beads.
Proteins were resolved by reducing SDS-PAGE and transferred to
nitrocellulose membranes. A, coprecipitated hsp90 was
determined by Western immunoblotting using either anti-hsp90 or
anti-hsp86 antibodies followed by secondary sheep anti-mouse or donkey
anti-rabbit IgG antibodies coupled to horseradish peroxidase and
visualized by chemiluminescence reagents (Renaissance, Dupont). Equal
amounts of protein were precipitated. Lane 1, anti-src
immunoprecipitation; lane 2, hsp90 standard, 1 µg; lane 3, SPA-830 anti-hsp90 immunoprecipitation; lane
4, anti-src immunoprecipitation; lane 5, 25 µg of
cell lysate. B, coprecipitated pp60
was determined by probing Western blots with the 327
anti-src mAb followed by sheep anti-mouse IgG coupled to
horseradish peroxidase with chemiluminescence detection. Lane
6, SPA-830 anti-hsp90 immunoprecipitation; lane 7,
anti-hsp86 immunoprecipitation; lane 8, GA bead precipitation.
Figure 2:
Effects of
OA treatment on the hsp90-pp60 heteroprotein complex. Cells were treated with OA
(10-100 nM) for 2 h; control cells were treated with an
equivalent concentration of Me
SO (0.2%). Cells were lysed
in TNESV buffer with added protease inhibitors, and after protein
assay, UBI anti-src mAb was used to immunoprecipitate
pp60
(lanes 1-4),
and GA beads were used to precipitate hsp90 (lanes 5-8). Lane 9 shows 1 µg of hsp90 standard. Aliquots of lysates
(25 µg of protein) from control and OA-treated cells are included
for comparison (lanes 10-13) and show that both hsp90
and pp60
levels were not decreased
by OA within 2 h. Following SDS-PAGE separation of proteins, Western
immunoblots for hsp90 and pp60
were
probed on nitrocellulose membranes using the SPA-830 anti-hsp90 mAb and
the 327 anti-src mAb, followed by appropriate horseradish
peroxidase-coupled secondary antibodies, and detection was by
chemiluminescence.
Figure 3:
OA alters the phosphorylation of both
hsp90 and pp60. Control and
OA-treated cells were exposed to
[
P]orthophosphate for 2 h in phosphate-free
medium containing 10% dialyzed calf serum prior to lysis with TNESV
buffer containing protease inhibitors; hsp90 and
pp60
were immunoprecipitated using
appropriate antibodies as described under ``Materials and
Methods.'' Following resolution of proteins by reducing SDS-PAGE,
the bands were transferred to PVDF membranes, and phosphoproteins were
visualized by autoradiography. OA (100 nM) was added to the
cells 30 min prior to [
P]orthophosphate. Shown
are cell lysates (10 µg of total protein, lanes 1 and 2), anti-v-src immunoprecipitations (lanes 3 and 4), anti-hsp90 immunoprecipitations (lanes 5 and 6), and GA bead precipitations (lanes 7 and 8) from 1.5 mg of cell lysates. Control samples are indicated
by(-) and samples from cells treated with OA by (+).
Molecular weight markers are shown on the left side of the lysate lanes.
Figure 4:
Phosphoamino acid analysis of
pp60 and hsp90 following OA
treatment. hsp90 and pp60
bands
were cut from the PVDF membranes and treated as described under
``Materials and Methods.'' The locations of unlabeled marker
phosphoamino acids on the thin-layer plates were determined by
ninhydrin staining and are indicated by the dotted lines.
Radioactivity was measured by direct autoradiography and by scanning
the plates with a PhosphorImager analyzer (Molecular Dynamics) capable
of integrating radioactive areas and correcting for background values
(see also Table 1). Shown are phosphoamino acids from control
pp60
immunoprecipitated using UBI
anti-src mAb (A), pp60
coprecipitated with hsp90 from control cells using the
SPA-830 anti-hsp90 mAb (B), and pp60
coprecipitated from OA-treated cells with hsp90 using the
SPA-830 anti-hsp90 mAb (C). Note that most of the
radioactivity resides on phosphoserine in hsp90-associated
pp60
, while the general pool of
pp60
is predominantly labeled on
phosphotyrosine. D, hsp90 immunoprecipitated from control
cells with the SPA-830 mAb; E, hsp90 immunoprecipitated from
OA-treated cells with the SPA-830 mAb. It should be noted that the
control hsp90 sample (D) includes two bands cut from the PVDF
membrane, while the OA-treated hsp90 sample (E) represents
only one band. This was done to permit comparison of hsp90 samples with
similar radioactivity. The relative positions of the phosphoamino acids
from the acid hydrolysates are indicated by PS (phosphoserine), PT (phosphothreonine), and PY (phosphotyrosine), and they are in the same positions in each panel.
In contrast to the rather modest changes in the
phosphorylation of amino acids in pp60,
serine phosphorylation in hsp90 was doubled by OA treatment, and the
phosphothreonine content was increased approximately 20-fold (Fig. 4, compare panel D from two hsp90 bands and panel E from one hsp90 band). Expressed as a percentage of the
total phosphoamino acid content per sample, phosphothreonine in hsp90
was increased 9-fold by OA (Table 1). Although a trace of
phosphotyrosine was found in the hsp90 band (0.7% of total phosphoamino
acids), this was not appreciably altered by OA (0.5%) and may have been
due to a contaminating, unresolved protein.
Figure 5:
Membrane and cytosol hsp90 and
pp60 levels after OA treatment.
After treatment of cells with OA (100 nM) for 2 or 16 h, cell
sonicates were prepared and then separated into cytosolic and membrane
fractions by ultracentrifugation. Following SDS-PAGE, these fractions
were analyzed by Western immunoblotting for both hsp90 and
pp60
. Lanes 1 and 4, control fractions; lanes 2 and 5, OA
treatment for 2 h; lanes 3 and 6, OA treatment for 16
h. Different amounts of total protein from the cell fractions were
used: hsp90 from cytosol, 50 µg (lanes 1-3); hsp90
from membranes, 100 µg (lanes 4-6);
pp60
from cytosol, 100 µg (lanes 1-3); and pp60
from membranes, 10 µg (lanes 4-6).
The steady-state level and functional activity of
pp60 are determined in part by its
interaction with the cellular chaperone phosphoprotein hsp90. Any
compromise of the integrity of the
pp60
-hsp90 complex appears to lead to
destabilization and loss of the v-Src
protein(2, 3, 5, 13, 19) .
Because pp60
coupled to hsp90 is
underphosphorylated on tyrosine (5, 12) and has
greatly diminished kinase activity(5) , a
biochemical/physiologic mechanism must exist for the dissociation of
pp60
from hsp90 at the appropriate time to
permit the normal function of its kinase activity. The data presented
in this paper suggest that phosphorylation of either hsp90 or
pp60
(less likely) might be such a
mechanism. This concept is reinforced by our observations that
treatment of the pp60
-hsp90 complex with
various phosphatases did not dissociate the proteins.
Brief exposure
of cells to the serine/threonine phosphatase inhibitor OA resulted in a
concentration-dependent dissociation of the
pp60-hsp90 complex without initially
affecting the steady-state constituent level of either protein.
However, within 16 h of OA exposure, the steady-state level of
membrane-associated, but not the cytoplasmic, v-Src protein was
markedly diminished. These data are consistent with destabilization of
pp60
following disruption or prevention of
its association with hsp90(13, 19) .
Interestingly,
OA did not affect the phosphorylation status of the total pool of
pp60, but the phosphatase inhibitor did
moderately increase serine and threonine phosphorylation of the
pp60
that coprecipitated with hsp90, while
decreasing dramatically the amount of v-Src protein that coprecipitated
with hsp90. The predominant amino acids that are phosphorylated in
pp60
are the tyrosine 416
autophosphorylation site (2, 3, 4, 25) and the serine 17
residue, known to be phosphorylated by cAMP-dependent protein
kinase(28) . In addition, v-Src protein is also phosphorylated
on serine-12 and serine-48 by protein kinase
C(29, 30) . All of these phosphoserine residues are
within the amino-terminal modulatory domain and close to the
membrane-binding domain of pp60
. It should
be pointed out, however, that Resh and Erikson (31) have shown
amino-terminal phosphorylation sites in pp60
to be very labile, presumably because of their susceptibility to
phosphoprotein phosphatases. Although the molecular consequences of
phosphorylation of pp60
at amino-terminal
serine residues is not well understood, Jove and Hanafusa (2) have suggested that transient phosphorylation within its
amino-terminal modulatory domain might regulate
pp60
kinase activity. Interestingly,
Gottesman et al.(32) have shown that cAMP-dependent
phosphorylation of pp60
at serine 17 in
RSV-transformed cells stimulates the tyrosine kinase activity of
pp60
in vitro and enhances the
tumorigenicity of the transformed cells in nude mice.
On the other
hand, deletion of serine 17, as well as mutation of serine 12, appears
to have little influence on the transforming capability of
pp60(33) . Analysis of several
pp60
amino-terminal deletion mutants for
their ability to stably associate with cellular hsp90 and pp50 suggests
that these particular phosphoserine residues are not necessary for the
association of pp60
with either hsp90 or
pp50, although the mutations may negatively influence the stability of
the complex(24) . The majority of pp60
mutant proteins containing amino-terminal deletions, however, are
more stably associated with hsp90 than is the wild-type v-Src
protein(3, 24) .
Although phosphoserine is present
in both the total pool of pp60 and in the
fraction of pp60
associated with hsp90,
phosphothreonine appears only in the hsp90-bound v-Src protein (see Fig. 4and Table 1). These data suggest that threonine
phosphorylation in pp60
may be necessary for
pp60
to associate with hsp90. OA treatment
appears to only moderately increase the level of v-Src protein
phosphothreonine residues (150%). Very little is known about the role
of threonine phosphorylation in the regulation of
pp60
activity (2, 3) , and
the significance of this finding remains to be determined.
Both
and
hsp90 isoforms are constitutively phosphorylated by
casein kinase II exclusively on serine residues and within a highly
charged region of the protein between amino acids 222 and
290(26, 34) . A characterization of avian hsp90
purified in the presence of the serine phosphatase inhibitor, fluoride,
revealed 6 mol of phosphate/mol of hsp90 dimer; these investigators
also suggested that the phosphorylation state of hsp90 could affect its
affinities for other proteins to which it binds(35) . Recently,
double-stranded DNA-activated protein kinase has been reported to
phosphorylate two amino-terminal threonine residues unique to the hsp90
isoform(36) . Since our data demonstrate that both
and
hsp90 isoforms are hyperphosphorylated after OA (see Fig. 3), it is unlikely that this particular kinase is
responsible for the increased phosphorylation of hsp90 we have
observed. Additionally, since both casein kinase II (37) and
the DNA-activated protein kinase (38) are localized within the
nucleus, it is unlikely that they could be responsible for our
findings, unless the nuclear membrane was damaged. It is more likely
that a plasma membrane or cytosolic serine/threonine kinase is
responsible for hsp90 hyperphosphorylation following OA treatment. We
are presently investigating a number of purified kinases for their
ability to phosphorylate hsp90 on threonine and to dissociate preformed
hsp90-pp60
complexes. From a functional
physiologic standpoint, it would be logical for the particular kinase
responsible for hsp90 threonine phosphorylation to be localized at the
inner surface of the plasma membrane, where it could promote the
release of pp60
to be anchored to the
membrane.
The dramatic, 20-fold increase in hsp90 phosphothreonine
content following brief OA exposure, compared with the more modest
increase in hsp90 phosphoserine, suggests a very robust, specific, and
tightly controlled phosphorylation/dephosphorylation cycle for this
amino acid in hsp90. Such a rapid and controlled cycle presumably has
essential biological significance. We propose that the level of hsp90
threonine phosphorylation regulates the hsp90-pp60 complex stability. This model is similar to one advanced for the
regulation of the interaction of retinoblastoma protein with either the
adenovirus E1A protein or the cellular E2F-1 transcription factor
whereby hyperphosphorylation of retinoblastoma protein by
cyclin-dependent serine/threonine kinases causes E1A or E2F proteins to
release from the complex within the nucleus, allowing for
transactivation of genes involved in cell
proliferation(39, 40) .
It will be intriguing to
determine whether hsp90 hyperphosphorylation on threonine residues
caused by OA treatment disrupts other hsp90-protein complexes, such as
those formed with the glucocorticoid receptor or the serine/threonine
kinase c-Raf-1(41, 42) . Perhaps not surprisingly,
hsp90 association is required for stability and proper intracellular
targeting of c-Raf-1 protein(43) . Preliminary data in our
laboratory also suggest that c-Raf-1 protein is, in fact, rapidly
depleted from cells following OA treatment, conceivably as a result of
OA-induced c-Raf-1-hsp90 complex instability. ()
In
summary, we have shown that brief OA treatment of
v-src-infected 3T3 cells results in modest increases in the
phosphorylation of serine and threonine in pp60 bound to hsp90 and serine residues in hsp90. At the same time, OA
stabilizes the hyperphosphorylation of hsp90 threonine residues.
Subsequent to these phosphorylation changes, the
hsp90-pp60
complex is destabilized, and
eventually pp60
is lost from cell membranes.
We propose that a phosphorylation/dephosphorylation cycle, likely
involving hsp90 threonine residues, functions as a molecular trigger
that initiates the assembly and disassembly of the
hsp90-pp60
heteroprotein complex, thereby
directing the cellular trafficking of pp60
and ultimately regulating cell transformation.