Howard Hughes Medical Institute and the Center for Cell Signaling Departments of Internal Medicine and Pharmacology University of Virginia Charlottesville, Virginia 22908
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ABSTRACT |
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INTRODUCTION |
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Somatostatin and selected analogs have antiproliferative effects in vivo and in vitro that, if better understood, could foster development of new cancer therapies (5). Addition of somatostatin inhibits proliferation of several cell lines in culture (6). Removal of somatostatin from cultures of mixed splenic lymphocytes by expression of an antisense oligonucleotide, or from cultures of NIH 3T3 cells expressing sstr2 by addition of antisomatostatin antibody, enhances proliferation (1, 2). These observations and others indicate a direct effect of somatostatin to inhibit cellular proliferation.
Activation of a membrane-associated protein tyrosine phosphatase (PTPase) has been studied as one possible mechanism for the growth inhibition. Schally and co-workers (7) discovered that somatostatin stimulated a PTPase in plasma membranes from MIA PaCa-2 cells (7). The stimulated PTPase dephosphorylated either an endogenous substrate, epidermal growth factor receptors 32P-labeled on tyrosine by autophosphorylation, or an exogenous substrate, [32P]pTyr-histone. Stork and co-workers (8) implicated G proteins in the activation mechanism by demonstrating that PTPase activation by either guanyl-5'-yl imidodiphosphate (GMPPNP) or somatostatin was blocked by pertussis toxin.
More recently, we demonstrated that GMPPNP stimulated a PTPase in
membranes from NIH 3T3 cells transformed with oncogenic Ha-Ras (9).
PTPase activation appeared to be mediated by
Gi/o-subunits for two reasons. G
i/o, but
not Gß
, subunits copurified with a stimulated PTPase
from GMPPNP-treated membranes after solubilization and Superose 6
chromatography. Activated G
i/o-subunits, but not
Gß
-subunits, reconstituted PTPase activity upon
addition to fractions from control membranes. To test whether
somatostatin receptors could couple to these G proteins to stimulate
PTPase activity, we transiently expressed human somatostatin receptor 3
(sstr3) in these cells (10). Somatostatin plus GMPPNP
activated a PTPase in membrane preparations from these transfected
cells, indicating that sstr3 is capable of stimulating this
effector in this background.
The number and identity of PTPases regulated, directly or indirectly, by G proteins is unknown, but recent reports suggest that the Src homology 2 (SH2) domain-containing PTPase SHP-1 (11) may be modulated by somatostatin (2, 12, 13). PTPase activity and a protein band immunoreactive to an anti-SHP-1 antibody were circumstantially present in partially purified preparations of somatostatin receptors from rat acinar membranes (12); this anti-SHP-1 antibody would likely not distinguish SHP-1 from SHP-2. Stable expression of sstr2 in NIH 3T3 cells increases specific cellular content of SHP-1 mRNA and protein (2). SHP-1 activity was also increased 3- to 4-fold in subconfluent sstr2-expressing cells growing in complete medium, assayed by specific immune complex assay (2). More recently, somatostatin analog SMS 201995 (Octreotide) was shown to cause translocation of SHP-1 to the particulate fraction (100,000 Xg) and reciprocal depletion from the supernatant fraction prepared from MCF-7 breast carcinoma cells (13). Octreotide also increased PTPase activity in the particulate fraction, and the majority was recovered with anti-SHP-1 antibody (13). These effects of Octreotide were maximal at 4 h and were specific for SHP-1 and not SHP-2 (13).
Here, we extend our investigations by testing the ability of each of the five cloned somatostatin receptors to promote PTPase activity in membranes from Ras-transformed NIH 3T3 cells. In addition, we tested whether coexpression of a dominant-negative mutant of SHP-2 could interfere with PTPase activation in vitro. Our findings raise some interesting mechanistic possibilities.
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RESULTS |
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We previously chose sstr3 for study (10) because its third
intracellular loop has a small insertion that makes it distinct from
the other cloned somatostatin receptors (3). The third intracellular
loop of G protein-coupled receptors is often important in specific
binding and activation of G proteins (14). Activation of
sstr3 stimulated PTPase activity in vitro (Fig. 1A), consistent with our previous report (10). Treatment
of membranes with somatostatin or somatostatin plus GDP did not
significantly alter PTPase activity in comparison to GDP. GMPPNP alone
usually caused no significant increase in PTPase activity under these
conditions. However, combined treatment with somatostatin plus GMPPNP
reproducibly increased PTPase activity 1.5- to 1.8-fold in comparison
to GDP. No stimulation occurred using membranes from cells transfected
with a control plasmid. Although several factors (transfection
efficiencies < 100%; a background of nonstimulated PTPases;
nonoptimal activation conditions) likely contribute to make the fold
stimulation small, the effect was sufficiently large to allow
qualitative determination of the capacity of sstr1-5 to
stimulate PTPase activity when studied in multiple experiments.
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Transient expression of sstr1 or sstr5 did not
promote in vitro PTPase activation by somatostatin in
multiple experiments in which expression of sstr3 was
studied in parallel as a positive control (Fig. 2). This
inability of sstr1 and sstr5 to support PTPase
activation was not merely due to failure of receptor expression because
membranes from sstr1- and sstr5-expressing
cells contained high-affinity binding sites for the radioligand (Fig. 2
).
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pCMV constructs for expression of active and inactive SHP-2 (15), identical except for absence (wt SHP-2) or presence (C/S SHP-2) of an inactivating Cys to Ser mutation, were obtained from J. Pessin (University of Iowa, Iowa City, IA) (15). As controls, we used pCMV constructs for expression of active (wt) and inactive (C/S) PTP1B (15). The total amount of pCMV DNA transfected per dish from any source was held constant in all coexpression experiments to be described.
We studied effects of phosphatase coexpression on
sstr2 because the earlier reports suggesting a link between
SH2 domain-containing phosphatases used sstr2 (2, 12, 16).
Somatostatin stimulated PTPase activity in membranes prepared from
cells transfected with pCMV-sstr2A (Fig. 3).
Somatostatin also stimulated PTPase activity in membranes cotransfected
with pCMV constructs for PTP1B or C/S PTP1B, although the magnitude of
effect was reduced to a similar extent in each case (Fig. 3
). This
reduction may be due to untoward competition for transcription or
translation factors in the instance where two proteins are being
expressed. Cotransfection with a pCMV construct for wt SHP-2 also
reduced the magnitude of PTPase stimulation, but did not abrogate the
somatostatin response (Fig. 4
).
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DISCUSSION |
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Buscail et al. (16) tested sstr1-5 for ability to stimulate PTPase activity in whole cell homogenates from Chinese hamster ovary cells (CHO), engineered to specifically express a given receptor, and treated in situ with the somatostatin analog RC-160 for 15 min (16). The substrate used was tyrosine-phosphorylated poly(Glu, Tyr). The clearest results were obtained with CHO-sstr2 cells wherein RC-160 stimulated PTPase activity 110% above control. RC-160 stimulated PTPase activity from CHO-sstr1 cells very modestly (30% at 1 µM) but had no effect on PTPase activity in cells expressing sstr3, sstr4, or sstr5 (16). These results confirmed a previous study of sstr1 and sstr2 (17) from the same authors, using similar assays, wherein RC-160 or somatostatin stimulated PTPase activity 2-fold in COS-7 cells transiently expressing sstr2, and only modestly stimulated (1.3-fold) PTPase activity in COS-7 cells expressing sstr1.
Using different methodologies, Florio et al. (18) studied PTPase regulation in CHO-K1 cells stably expressing sstr1 or sstr2. Crude membranes were prepared by differential centrifugation from cells treated in situ with somatostatin for 20120 min, suspended in a HEPES buffer containing 1% NP-40, and assayed for PTPase activity with a radiolabeled c-src peptide substrate. Somatostatin did not affect PTPase activity in CHO-K1-sstr2 cells and stimulated PTPase activity 1- to 6-fold in CHO-K1-sstr1 cells after 2 h.
Our study and those just discussed can not be directly compared because of experimental differences. However, our results confirm an ability of sstr2A to regulate PTPase activity, as reported by Susini and colleagues (16, 17). In addition, we show that sstr3 and sstr4 can couple to PTPase activity. The negative data obtained for sst3 and sstr4 in the Buscail study (16) may be due to the cellular background or a lower sensitivity for detecting activated PTPase. We observed no stimulation of PTPase activity by somatostatin with sstr1, and Buscail et al. (16, 17) observed only very small increases for sstr1 in their studies. Thus, our data and those of Buscail et al. (16, 17) may be substantially in agreement with respect to sstr1. Our data and Buscails data (16, 17) conflict strongly with that of Florio et al. (18) for sstr2. Both Florio et al. (18) and Buscail et al. (16) studied PTPase activation by sstr2 in CHO cell lines. One possibility is that different phosphatase phenomena were being studied; we and Buscail et al. (16, 17) used principally 10- to 15-min stimulations with somatostatin whereas Florio et al. (18) used principally 2-h stimulations.
The laboratories of Susini and Srikant have proposed that the SH2-domain containing PTPase SHP-1, and not SHP-2, is regulated by somatostatin, albeit by different mechanisms. Susini and co-workers have shown that sstr2 expression can up-regulate SHP-1 transcription in NIH 3T3 cells (2) and have furthermore hypothesized that SHP-1 binds to activated somatostatin receptors and is activated in consequence (12). More recently, Srikant and colleagues (13) demonstrated that somatostatin induced translocation of SHP-1 from the cytosolic to the membrane fraction in breast cancer cells and proposed that translocation per se accounts for an observed increase in SHP-1 activity in the particulate fraction.
SHP-1 and SHP-2 are closely related nontransmembrane PTPases of 65,000 and 68,000 Mr (19, 20). The principal features of each are two nonidentical SH2 domains at the NH2 terminus and a catalytic domain located at the COOH terminus. Corresponding SH2 domains and the catalytic domains of SHP-1 and SHP-2 are 5070% similar by alignment (20), but are sufficiently different that their functions could be distinct (20). Strong evidence indicates that SHP-1 and SHP-2 are restrained by intramolecular interactions of their SH2 domains with the COOH-terminal portion of their catalytic domains (21) and, furthermore, that both can be activated in vitro by liganding of their SH2 domains (19, 20, 21). However, the molecular details of their regulation are still poorly understood, both as to the molecular segments involved and as to the individual roles of the SH2 domains.
We used coexpression of catalytically inactive SHP-2 with sstr2 to test whether an SH2 domain-containing phosphatase was involved in our system. We chose to study SHP-2 because expression of SHP-1 is principally confined to hematopoietic cells (19). C/S SHP-2 is catalytically inactive but otherwise capable of binding phosphotyrosine via its SH2-domains and capable of binding substrates. If an SH2 domain-containing phosphatase were involved, we reasoned that C/S SHP-2 would be expressed in greater amounts than endogenous SHP-2 and thus might compete for any pool of SHP-2 in our membrane preparations. There it would behave as a "dominant-negative," blocking PTPase stimulation.
The result that C/S SHP-2 blocks PTPase activation was surprising
to us. SHP-2 is a non-transmembrane phosphatase, generally accepted to
be cytosolic and to be recruited to membrane receptors or docking
proteins such as insulin receptor substrate 1 by
phosphotyrosine:SH2-domain interactions that activate its enzymatic
activity (19). The PTPase that is activated by somatostatin and guanine
nucleotides in our membrane preparations is either membrane-associated
and copurifying, or present as a contaminant from the cytosolic
fraction. Translocation is not involved. Suzuki et al. (22)
performed a careful study of the cellular localization of SHP-2 in rat
brain, utilizing both immunohistochemistry and cellular disruption and
fractionation. SHP-2 was mainly observed in the synaptic plasma
membrane fraction and could be immunoprecipitated in association with a
100-kDa tyrosine-phosphorylated protein. SHP-2 can be detected by
Western blotting in our partially purified membranes (our unpublished
data), but its manner of association and significance are unknown. The
strong evidence supporting one modality of regulation of SHP-2, by
liganding of its SH-2 domains, presents a bias against existence of
another, undiscovered mechanism involving G proteins, but such could
exist. As yet, we have not observed activation in vitro of
purified, bacterially expressed
glutathione-S-transferase-SHP-2 fusion protein by purified,
activated brain Gi/o-subunits. Other component(s) may be
required. Alternatively, C/S SHP-2 may indirectly affect
protein-protein interactions in situ that negate in
vitro activation of another PTPase. Thus, although our results
simply taken are consistent with identification of SHP-2 as a
somatostatin-regulated phosphatase, more work will be required to test
this hypothesis.
Regulation of SHP-2 by Gi/o by a conventional SH2 mechanism
is also possible because Gß oligomers stimulate the
activity in situ of a tyrosine kinase (23), possibly c-Src
(24), that phosphorylates Shc on tyrosine residues. Moreover, SHP-2 can
be found in association with c-Src (25). However, the conditions we
used herein for PTPase activation, omitting ATP and including
millimolar concentrations of GMPPNP, would be expected to preclude
phosphorylation reactions. Thus, phosphorylation is not likely to be
involved in the in vitro activation of PTPase activity by
somatostatin in the experimental system we describe.
A 115-kDa tyrosine phosphorylated protein has been strongly implicated in SHP-2 function. An unidentified protein recognized by anti-pTyr antibodies can be precipitated from insulin-treated cells in association with SHP-2 (26). Daughter of Sevenless, DOS, the likely homolog of this protein in Drosophila, has been identified as a pleckstrin-homology domain containing protein of unknown function, possibly an adapter or docking function (27, 28). pTyr-DOS binds CSW by SH2 domain interactions and serves as substrate for CSW in vitro (27). By epistasis arguments, DOS functions in parallel with CSW and downstream of the tyrosine kinase Sevenless and upstream of, or in parallel with, Ras in the pathway to specify photoreceptor development (27, 28). Interestingly, a portion of DOS is plasma membrane-associated, and this fraction is increased by Sevenless signaling (28).
In summary, evidence from several laboratories demonstrates that somatostatin activates a PTPase(s) that we currently view as unidentified. SH2-domain containing phosphatases are candidates (2, 12, 13), supported here by the demonstration that expression of C/S SHP-2 blocks PTPase activation. Regulation of PTPase activity by somatostatin may occur rapidly, by G protein-dependent pathways (Refs. 2, 8, 10 , and data herein) or by long-term alteration of transcription and expression (2). Somatostatin receptor subtypes 2, 3, and 4 can function to activate PTPase activity in vitro in membranes from v-Ras-transformed NIH 3T3 cells. Elucidation of the PTPases and mechanisms involved is critically important to full understanding of G protein-mediated signaling.
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MATERIALS AND METHODS |
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Cell Culture and Transfection
NIH 3T3 cells stably transformed with Ha-RasG12V
were routinely grown (37 C; 10% CO2 atmosphere) in DMEM
containing 10% (vol/vol) FCS and used throughout. Cells (at 60%
confluency in 100-mm dishes) were transfected using CaPO4
precipitation [1520 µg expression construct(s), 0.125
M CaCl2, 25 mM BES
[N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic
acid (Sigma-Aldrich))], 140 mM NaCl, 0.75 mM
Na2HPO4, pH 6.95] and diluted 1:10 dropwise
onto cells in dishes containing fresh media. Cells were incubated
overnight at 37 C in 5% CO2, washed extensively in PBS to
remove precipitated DNA, and allowed to recover 2448 h in complete
media at 37 C in 10% CO2.
Preparation of Membranes from Transfected Cells
Transfected cells were subjected to serum starvation for
2 h before membrane isolation. Cells were washed in PBS, then
suspended in 5 ml of buffer A [25 mM HEPES, pH 7.6 at 4 C,
10 mM EDTA, 10 mM EGTA, 5 mM
benzamidine, 0.1% (vol/vol) 2-mercaptoethanol, 1 mM fresh
phenylmethylsulfonyl fluoride, 40 µg/ml each of
Na-p-tosyl-L-lysine chloromethyl ketone,
N-tosyl-L-phenylalanine chloromethyl ketone, leupeptin,
pepstatin A, aprotinin, and 1 mg/ml soybean trypsin inhibitor]
containing 8.6% (wt/vol) sucrose. Suspended cells were subjected to 30
passes (4 C) in a motorized Teflon-glass homogenizer. The homogenate
was layered onto buffer A containing 39% (wt/vol) sucrose, and
centrifuged (30 min, 4 C) at 30,000 x g in an SW41 rotor
(Beckman, Palo Alto, CA). Membranes collected at the interface were
washed by dilution to 25 ml with 25 mM Tris, pH 7.9 (4 C),
and centrifugation (30 min, 4 C) at 40,000 x g in a
50.2Ti rotor. Membranes were suspended to 1 mg/ml total protein in
buffer B [buffer A containing 8.6% (wt/vol) sucrose and the above
indicated protease inhibitors], and used immediately.
Stimulation of Membranes with Guanine Nucleotides and
Somatostatin
Membranes were treated with GDP to produce a basal state
by incubation with 1 mM GDP at 22 C and for a further 20
min after adjustment of Mg to 5 mM in excess of total
chelator concentrations. Membranes were recovered for use by
centrifugation at 100,000 x g in an Airfuge (Beckman);
after traces of the supernatant were carefully removed, membranes were
resuspended to 1 mg/ml protein in buffer B, adjusted to 5
mM total Mg+2 in excess of chelators. Portions
(20 µl) were placed on the walls of tubes containing appropriate
amounts of concentrated stocks of guanine nucleotides and/or
somatatostatin. Reactions (37 C) were initiated by tapping the tubes to
mix membranes with stimulators. After 10 min, portions (2 µl) were
removed and assayed in triplicate for PTPase activity.
PTPase Assay
PTPase activity was assayed with
[[32P]pTyr]RCM-lysozyme as substrate as described (10).
Data were expressed as percentage release of total 32P in
the assay.
Ligand Binding and Western Blotting
The binding assay for iodinated somatostatin was modified
from the work of Rens-Domiano et al. (29). Cells were
homogenized in [50 mM Tris, pH 7.4 at 4 C, 1
mM EGTA, 5 mM MgCl2, 10 µg/ml
leupeptin and pepstatin A, 0.5 µg/ml aprotinin, 200 µg/ml
bacitracin]. The particulate fraction was isolated by centrifugation
(45,000 x g, 15 min, 4 C). Crude membranes were
resuspended in homogenization buffer. Membrane protein (200 µg) was
incubated (30 min, 25 C) with
[125I-Tyr11]-somatostatin-14 (50,000 cpm)
with or without nonlabeled competing somatostatin-14 (100
nM). Bound radioligand was determined after filtration and
washing as described (13).
For Western analysis, SHP-2 expression constructs were used to transfect cells as detailed above. Whole-cell lysate (100 µg) dissolved in SDS sample buffer was electrophoresed (10% gel) and transferred to nitrocellulose (0.45 µm). The myc-epitope-tagged SHP-2 protein was detected by enhanced chemiluminescence (Amersham) using anti-myc (1:2,000, Santa Cruz Biochemical, Santa Cruz, CA) as 1 C antibody and horseradish peroxidase-linked anti-mouse (1:5000, Bio-Rad) as 2 C antibody.
Note Added in Proof.
GnRH stimulates PTPase activity upon addition, with GTP--S, to
plasma membranes from ovarian carcinomas (30).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the Howard Hughes Medical Institute and NIH Grant DK-41077 (to T.W.S.).
Received for publication October 14, 1996. Revision received March 19, 1997. Accepted for publication April 17, 1997.
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REFERENCES |
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