(Received for publication, November 22, 1995; and in revised form, December 18, 1995)
From the
A membrane-associated form of Raf-1 in v-Ras transformed NIH 3T3
cells can be inactivated by protein phosphatases regulated by GTP.
Herein, a distinct protein-tyrosine phosphatase (PTPase) in membrane
preparations from v-Ras transformed NIH 3T3 cells was found to be
activated by guanyl-5`-yl imidodiphosphate (GMPPNP) and was identified
as an effector for pertussis toxin (PTx)-sensitive G-protein
subunits. PTPase activation was blocked by prior treatment of cells
with PTx. PTPase activation by GTP, but not GMPPNP, was transient. A
GMPPNP-stimulated PTPase (PTPase-G) co-purified with G
subunits during Superose 6 and Mono Q chromatography. PTPase-G
activity in Superose 6 fractions from GDP-treated membranes was
reconstituted by activated G
, but not
G
, subunits. PTPase-G may contribute to
GMPPNP-stimulated inactivation of Raf-1 in v-Ras cell membranes because
Raf-1 inactivation was PTx-sensitive and PTPase-G inactivated exogenous
Raf-1.
Raf-1 is a protein serine/threonine kinase that functions
downstream of Ras in the MAP ()kinase cascade,
phosphorylating and activating mitogen-activated protein kinase kinase
(MKK or MEK) (reviewed in (1) ). The mechanism of Raf-1
activation and inactivation is incompletely understood. Raf-1
activation occurs at the plasma membrane following binding to Ras-GTP (2, 3) . Activation of Raf-1 in vitro by a
mixture of membranes from v-Ras transformed and v-Src transformed
fibroblasts requires ATP/Mg, compatible with a requirement for
phosphorylation of Raf-1 for enzymatic activation(4) . Raf-1
becomes phosphorylated on tyrosine and serine residues and activated
upon co-expression with Ras and Src in Sf9 cells (reviewed in (4) ) or NIH 3T3 cells(5) . Both tyrosine and serine
phosphorylation of Raf-1 appear to be required for Raf-1 activation
because Raf-1 isolated from Sf9 cells also expressing Ras alone (
)or Ras and Src
(6) can be
inactivated in vitro by treatment with either
protein-serine/threonine or -tyrosine phosphatases.
Membranes from mammalian cells transformed with oncogenic Ras contain a portion of cellular Raf-1 in a constitutively active form(7) . This membrane-associated form of Raf-1 can be inactivated by protein phosphatases regulated directly or indirectly by GTP(6) . We performed experiments to characterize the protein phosphatase responsible for GTP-stimulated inactivation of Raf-1 and to define the mechanism of its regulation.
Incubation of membranes from v-Ras cells with an antibody
that binds the Ras effector domain did not prevent GMPPNP-stimulated
inactivation of membrane-associated Raf-1(6) , suggesting
involvement of G-proteins other than Ras in Raf-1 inactivation (data
not shown). To test for involvement of PTx-sensitive heterotrimeric
G-proteins, we compared the abilities of guanine nucleotides to
stimulate inactivation of endogenous Raf-1 in membranes from untreated
v-Ras transformed cells in comparison with membranes from cells that
were treated overnight with PTx (Table 1). GTP or GMPPNP, but not
GDP, stimulated inactivation of Raf-1 in membranes prepared from
untreated cells, and inactivation was blocked by inclusion of
microcystin-LR and vanadate to inhibit protein-serine/threonine and
-tyrosine phosphatases. Raf-1 in membranes prepared from PTx-treated
cells was not susceptible to guanine nucleotide-stimulated inactivation (Table 1). PTx specifically catalyzes ADP-ribosylation of
subunits of heterotrimeric G-proteins of the G
family
(except G
) (reviewed in (12) ). Thus, these results
strongly suggested that GTP-stimulated inactivation of Raf-1 in the
mixture of membranes from v-Ras cells was mediated by activation,
directly or indirectly, of protein phosphatases by heterotrimeric
G-proteins of the G
family.
Schally and co-workers (13) previously demonstrated that addition of somatostatin to membranes of pancreatic cancer cells promoted dephosphorylation of epidermal growth factor receptors autophosphorylated on tyrosine, implying activation of a PTPase by G-protein-coupled receptors. Stork and coworkers (14) extended this observation by demonstrating that GMPPNP addition to membranes could stimulate a PTPase activity, assayed using p-nitrophenyl phosphate as substrate, and that activation of PTPase activity by somatostatin was PTx-sensitive. Since Raf-1 deactivation was PTx-sensitive (Table 1) and a purified PTPase can inactivate Raf-1 in vitro(6) , we hypothesized that a G-protein-activated PTPase might be responsible for guanine nucleotide-stimulated inactivation of Raf-1.
To test this
hypothesis, we determined whether loading of membranes isolated from
v-Ras cells with GTP or GMPPNP stimulated PTPase activity, using as
substrate [[P]Tyr]RCM-lysozyme (Fig. 1). GTP significantly increased the rate of
P
release in comparison to GDP (Fig. 1A). When membranes
were reassayed after 30 min, the increase in PTPase activity due to GTP
was absent (Fig. 1B,
-
).
Transient activation of PTPase activity by GTP is consistent with the
transient activation of a G-protein
subunit due to timed GTP
hydrolysis(12) . Reloading these same membranes with GTP
partially recovered enhanced activity (Fig. 1B,
- - -
), proving that the decrease in
activity was not due simply to protein lability. GMPPNP, which is not
hydrolyzed by G
subunits, caused a persistent elevation
of PTPase activity (Fig. 1B). Activation also occurred
when 100 µM concentrations of guanine nucleotide were used
(data not shown). Neither GTP nor GMPPNP caused an increase in PTPase
activity in membranes from PTx-treated cells (data not shown).
Together, these data demonstrate activation of a
[[
P]Tyr]RCM-lysozyme phosphatase by a
PTx-sensitive G-protein.
Figure 1:
Activation of a
[[P]Tyr]RCM-lysozyme phosphatase in
isolated membranes by GTP and GMPPNP. Membranes from v-Ras transformed
cells were incubated with GTP (
), GMPPNP (
), or GDP
(
), spiked with Mg
in excess of chelators,
recovered by centrifugation, and portions (0.5 µl) were assayed for
PTPase activity at the times indicated (see ``Experimental
Procedures''). A, initial PTPase assays. B,
assays of samples left for 30 min on ice with periodic mixing. Dashed line in B, membranes were repelleted and reincubated
with GTP before assay. Representative of 4
experiments.
In parallel experiments, GMPPNP or GTP
stimulated PTPase activity in membranes isolated from parental NIH 3T3
cells with results similar to those in Fig. 1(data not shown).
Membranes from parental cells, however, contained 5-fold less
GMPPNP-stimulated PTPase activity than membranes from v-Ras cells (data
not shown). Membranes from cells transformed with a different oncogenic
Ras, Ras, also showed significantly increased levels of
GMPPNP-stimulated PTPase activity relative to membranes from parental
cells. Thus, the greater specific activity of GMPPNP-stimulated PTPase
is a consequence of Ras transformation.
The GMPPNP-stimulated PTPase
was characterized by gel permeation chromatography in buffer containing
0.01% (v/v) Triton X-100 following solubilization of guanine
nucleotide-treated membranes with 1% (v/v) detergent. Solubilization per se increased total PTPase activity 4-5-fold, but a
2-3-fold stimulation of activity in GMPPNP-treated versus GDP-treated membranes was preserved (data not shown). The
principal peak of PTPase activity observed with GDP-treated membranes
from v-Ras cells eluted between the positions of the standards bovine
-globulin (150 kDa) and BSA (67 kDa) with an apparent mass of
70 kDa (Fig. 2A). Treatment of membranes with
GMPPNP reproducibly caused the appearance of a peak of PTPase activity
that eluted earlier, at
100 kDa (Fig. 2A). We
refer herein to this GMPPNP-stimulated PTPase as PTPase-G. An
additional species of GMPPNP-stimulated PTPase of higher M
(
150,000) was occasionally observed as a
smaller peak or leading shoulder (data not shown; see also Fig. 3B). The latter may correspond to a labile PTPase.
Figure 2:
Characterization of PTPase-G by Superose 6
and Mono Q chromatographies. Membranes from v-Ras transformed NIH 3T3
cells were incubated with GMPPNP () or GDP (
), solubilized
with Triton X-100, and fractionated for analyses (see
``Experimental Procedures''). A and B,
portions (2 µl) of fractions were assayed for
[[
P] Tyr]RCM-lysozyme phosphatase
activity. B, Raf-1 was treated with portions (2 µl) of
fractions from GMPPNP-treated membranes and assayed (
) by MEK
phosphorylation. Arrows a and b and bar c,
markers for elution of bovine
-globulin, BSA, and soybean trypsin
inhibitor, respectively. C, Immunoblot with
anti-G
antibody C-20 of proteins from
GMPPNP-treated membranes in indicated fractions from Superose 6
chromatography. D, fractions 33 to 39 inclusive were pooled,
subjected to Mono Q chromatography, and assayed for PTPase activity. E, immunoblot with anti-G
antibody of
proteins from GMPPNP-treated membranes in indicated pairs of pooled
fractions from Mono Q chromatography (D). Data (A-E) are representative of 3
experiments.
Figure 3:
Regulation of PTPase-G by
G. Membranes from v-Ras cells were treated with
GMPPNP or GDP and fractionated as in Fig. 2A. A, PTPase activity in portions of fractions from
GMPPNP-treated membranes after treatment to substitute GMPPNP (
)
or GDP (
) for bound GMPPNP (see ``Experimental
Procedures'');
, GDP-treated membranes. B, PTPase
activity in portions of fractions from GDP-treated membranes incubated
with GMPPNP/Mg brain G
(
), GDP/Mg brain
G
(
), or buffer C (
- -
-
) (see ``Experimental Procedures''). C,
PTPase activity in portions of fractions from GDP-treated membranes
incubated with 25 ng of activated (see ``Experimental
Procedures'') recombinant G
(
),
G
(
), G
(
), or
buffer/GMPPNP/Mg (
). Data for G
superimposed
upon the control profile and were not plotted. Data are Representative
of 2 (C) or 3 (A and B)
experiments.
We tested whether PTPase-G could deactivate active Raf-1, using
FLAG-Raf-1 purified from Sf9 cells co-expressing Ras and
Src. A single Gaussian-shaped peak of activity causing
deactivation of Raf-1 was detected and corresponded to PTPase-G (Fig. 2B). The deactivation of Raf-1 occurred in the
presence of 2.4 µM microcystin-LR and was abolished by 0.1
mM vanadate, consistent with the action of a PTPase (data not
shown). Raf-1 deactivating activity was increased 5-7-fold by
GMPPNP in comparison with GDP (data not shown). Thus, activation of
PTPase-G can explain, at least in part, the ability of GMPPNP to cause
inactivation of endogenous Raf-1 in membranes from v-Ras
cells(6) .
Our membrane preparations contain membranes
derived from the endoplasmic reticulum in addition to plasma membranes
and thus contain PTP1B(15) . PTP1B was detected by
immunoblotting (data not shown), and its elution corresponded to the
major peak of [[P]Tyr]RCM-lysozyme
phosphatase activity migrating with an apparent mass of
70 kDa
(centered at fraction 42) and not to the peak of GMPPNP-stimulated
PTPase (centered at fraction 39). The peak of
[[
P]Tyr]RCM-lysozyme phosphatase
containing 50-kDa PTP1B (Fig. 2B) did not coincide with
deactivation of exogenous Raf-1, suggesting that PTP1B is not PTPase-G.
The COOH-truncated, 37-kDa form of PTP1B deactivated Raf-1(6) ,
but this may be due to its reactivity in vitro at high
concentrations or the absence of regulatory sequences(15) .
Using an antibody that recognizes G, we
detected G
subunit(s) in fractions containing
GMPPNP-stimulated PTPase (Fig. 2C). Antibodies C-10 and
K-20 to G
and G
also
detected a protein of 41 kDa in these fractions (data not shown). No
G
subunits were detected in these fractions by
immunoblotting with antibody T-20 that recognizes G
(data not shown). The profile of the elution of immunoreactive
41-kDa protein correlated well with the profile of GMPPNP-stimulated
PTPase activity (Fig. 2, compare A and C).
G
subunits were absent from corresponding fractions in
profiles from GDP-treated membranes, indicating that co-elution of
G
and PTPase in these fractions required G-protein
activation (data not shown). These results are consistent with
activation of a PTPase by binding of G
subunits and not
G
subunits.
Superose 6 fractions encompassing
the leading edge of the PTPase-G peak were pooled to reduce
cross-contamination with the peak of PTP1B and subjected to ion
exchange chromatography on Mono Q. PTPase-G was resolved into multiple
forms that eluted in the middle of the gradient (Fig. 2D). The reasons for this heterogeneity are
unknown. Each of these forms co-eluted with 41-kDa proteins recognized
by antibodies to G (Fig. 2E) and
also by antibodies to G
and G
(data not shown). PTP1B eluted in fractions 17-19 and did
not co-elute with a GMPPNP-stimulated peak of PTPase activity or with
G
subunits. Recovery of PTPase-G activity after Mono Q
chromatography was low,
20-30%, which may be indicative of
disruption of a PTPase-G complex.
Reversibility of activation was
demonstrated by substitution of GDP for bound GMPPNP in PTPase-G in
fractions from Superose 6 chromatography (Fig. 3A).
Portions of column fractions from GMPPNP-treated membranes were
incubated with GDP or GMPPNP in the presence of Mg chelators to promote nucleotide dissociation (9) prior to
readdition of Mg
and assay. Replacement of GMPPNP
with GMPPNP preserved the PTPase-G activity. Replacement of GMPPNP with
GDP abolished the peak of PTPase-G, resulting in a profile after
reassay nearly identical with the profile obtained by assay of
fractions from GDP-treated membranes. Since G
was
not detected in the fractions containing PTPase activity, reversal of
activation by GDP strongly supports regulation by G
subunits.
To definitively test this hypothesis, we examined
the ability of purified G-protein subunits to activate PTPase in
fractions from GDP-treated membranes (Fig. 3, B and C). Preparations of brain G and
G
subunits were utilized to provide a range of
subunits principally derived from members of the G
family(10) . Addition of brain G
-GMPPNP,
but not brain G
-GDP, increased activity of PTPase that
eluted in the leading shoulder of the constitutive
70-kDa peak of
PTPase activity (Fig. 3B). Brain
G
-GMPPNP also activated a species of M
150,000 that may correspond to the occasionally observed
species of GMPPNP-stimulated PTPase activity noted above. Addition of a
mixture of brain G
subunits in the presence of GDP
or GMPPNP did not alter PTPase activity (data not shown). Importantly,
addition of activated recombinant G
,
G
, and G
also stimulated PTPase
activity (Fig. 3C). G
appeared to
cause a greater activation than G
and G
suggesting specificity in the interaction with the effector
PTPase, but detailed titration studies will be required to determine
the relative potency and efficacy of various PTx-sensitive G
subunits.
G-protein-coupled receptors may initiate both
positive signals for MAP kinase activation via G subunits and negative signals via G
subunits,
depending on context and the specific G-protein coupled. For example,
epitope-tagged MAP kinase expressed in Cos-7 cells is transiently
activated by isoproterenol via endogenously expressed
-adrenergic
receptors(16) . The stimulatory signal is provided by
G
and is Ras-dependent. An inhibitory signal is
provided by G
-mediated elevation of cAMP
concentration; elevation of cAMP in cells prior to stimulation blocks
MAP kinase activation. In pheromone signaling in Saccharomyces
cerevisiae, G
stimulates an adaptive pathway that
antagonizes G
-mediated activation of the MAP
kinase-related enzyme FUS3(17) . The signaling mechanisms for
MAP kinase activation by mammalian G-coupled receptors are
complex(18) ; it appears that specific G
subunits may promote, inhibit, or possibly be indifferent to
activation of MAP kinase by G
. Complexity is also
indicated by reports that overexpression of GTPase-impaired
G
caused transformation and activation of MAP kinase
in Rat-1 but not NIH 3T3 cells(19) , and that overexpression of
GTPase-impaired G
transformed NIH 3T3
cells(20) .
Thus, a balance of positive and negative signals
determines the extent of MAP kinase activation by G-protein-coupled
receptors. PTPase-G may deliver a negative signal from receptors that
couple to responsible subtypes of G to modulate the
timing or extent of activation of MAP kinase. While our experiments
reveal a negative modulation by PTPase-G of membrane-associated Raf-1
that has been already activated by v-Ras, we cannot exclude the
possibility that PTPase-G may also act positively in other contexts or
temporal sequences.
Identification of unambiguous effectors for
G, as well as G
, subunits has been
elusive (reviewed in (21) ). Our findings strongly implicate
PTPases as effectors for activated PTx-sensitive G
protein(s), fulfilling each of the classical biochemical criteria
utilized to establish adenylate cyclase as an effector for
G
. Our findings also show that reconstitution with
G
subunits can serve as an assay for purification
and identification of PTPase-G. Elucidation of the pathways regulated
by G
-regulated PTPases should provide insight into the
mechanisms of mitogenic signaling and cell cycle control.