(Received for publication, April 25, 1995; and in revised form, August 11, 1995)
From the
Rat PC12 cells respond to extracellular peptide growth factors in at least two distinct ways. When treated with nerve growth factor (NGF) PC12 cells exit the cell cycle and differentiate to a neuronal phenotype, whereas when treated with epidermal growth factor, they proliferate. We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatases (PTPs) in the differentiation process. PC12 cells express substantial amounts of both SH-PTP1 and 2. SH-PTP1, but not SH-PTP2, becomes tyrosine phosphorylated following NGF, but not epidermal growth factor treatment. The enzymatic activity of SH-PTP1 toward an exogenous substrate following NGF treatment is increased 2-fold. We found that SH-PTP1 binds to the NGF receptor TrkA in vitro and that anti-TrkA immunoprecipitates have PTP activity. These results show that SH-PTP1 is differentially phosphorylated and activated by NGF in PC12 cells and suggest that this activation may play a role in NGF-induced differentiation.
Diverse extracellular growth factors use one of a few common
strategies to transmit their messages to the cell interior. Among the
best understood of these strategies is that utilized by ligands that
bind to receptor protein tyrosine kinases (RPTKs). ()The
general scheme by which these signals are transduced includes receptor
dimerization and autophosphorylation, followed by the recruitment of an
array of Src homology 2 (SH2)-containing molecules(1) . These
recruited SH2-containing proteins include enzymes such as kinases,
phosphatases, GTPase-activating proteins, and phospholipases, and
adaptor proteins, which bind to activators of the Ras family of
GTPases, thus linking the signaling complex to downstream
effectors(2) .
One of the SH2-containing proteins recruited
by several different RPTKs is protein tyrosine phosphatase (PTP)
SH-PTP2(3, 4, 5, 6) . In the case of
the platelet-derived growth factor (PDGF)- receptor SH-PTP2 binds
to phosphorylated tyrosine 1009, and itself becomes tyrosine
phosphorylated by the receptor(7, 8) .
Tyrosine-phosphorylated SH-PTP2 then serves as a docking site for the
SH2-containing adaptor protein Grb2(8, 9) . In this
way, one function of SH-PTP2 binding to the PDGF receptor appears to be
as a platform for the assembly of additional adaptor proteins required
to generate an active signaling complex. The role of the phosphatase
activity itself is not well established at present.
Although structurally similar to SH-PTP2, the second known SH2-containing PTP (SH-PTP1) (10, 11, 12) appears to have a different role in signal transduction. Disruption of SH-PTP1 function gives rise to the motheaten and viable motheaten mouse strains, which have severe immunologic defects as well as multiple other hematopoietic abnormalities(13, 14, 15) . These mice exhibit a large increase in the number of myeloid precursor cells as a result of colony-stimulating factor 1-independent proliferation of macrophages and increased sensitivity of colony-forming units to erythropoietin. In addition, there is also an increase in the numbers of erythroid precursors, consistent with a defect in interleukin-3 signaling. Biochemical evidence shows that SH-PTP1 is coupled to c-Kit, interleukin-3, and the insulin receptors but not to the colony-stimulating factor-1 receptor(16, 17, 18, 19) . SH-PTP1 dephosphorylates a variety of RPTKs when coexpressed in 293 cells (5) and has been shown to down-regulate interleukin-3-induced tyrosine phosphorylation and mitogenesis(16) . Thus, unlike SH-PTP2, this enzyme appears to act as a negative regulator of signal transduction.
In PC12 cells, engagement of the nerve growth factor (NGF) receptor TrkA results in differentiation of the cells to a neuronal phenotype, whereas engagement of the epidermal growth factor (EGF) receptor causes proliferation(20, 21) . One possible reason for these differences could be that these receptors recruit and/or activate distinct populations of SH2 proteins. The role of SH2-containing PTPs in PC12 cell differentiation and proliferation has not previously been reported. We therefore examined the effect of NGF and EGF treatment on the receptor binding, phosphorylation, and activity of both known SH-PTPs in PC12 cells. We report here that both SH-PTP1 and 2 are present in these cells and that the NGF receptor tyrosine phosphorylates and activates only SH-PTP1.
Proteins were separated in 8% SDS-polyacrylamide gels (SDS-PAGE). After separation, the proteins were transferred to a nitrocellulose membrane that was then stained with Coomassie Blue and destained for 10-30 min to visualize proteins. Stained blots were washed several times with Tris-buffered saline, 0.05% Tween 20 buffer prior to blocking in 5% defatted milk (Carnation) in Tris-buffered saline, 0.05% Tween 20. Immunoblots were performed by enhanced chemiluminescence (Amersham Corp.) according to the manufacturer's protocol.
Figure 1:
SH-PTP1 mRNA
and protein are present in PC12 cells. A, 10 µg of total
RNA isolated from Rat1 fibroblasts, PC12 cells, and rat lymphocytes
were separated on a denaturing agarose gel. The resulting Northern blot
was hybridized as described under ``Materials and Methods''
with a P-labeled human SH-PTP1 probe. B, 20
µg of cell lysate from Rat1 fibroblasts, PC12 cells, and rat
lymphocytes were separated on an 8% SDS-PAGE gel. After transfer to a
nitrocellulose membrane, an immunoblot was performed with either
polyclonal anti-SH-PTP1 or monoclonal anti-SH-PTP2
antibodies.
Figure 2:
SH-PTP1 is tyrosine phosphorylated in
response to NGF. A, lysates (10 µg) and TrkA
immunoprecipitates (from 400 µg of lysate) from control and
NGF-treated 6-24 cells were separated on 8% SDS-PAGE gels, transferred
to nitrocellulose membranes, and probed with anti-phosphotyrosine (-PY), anti-SH-PTP1, and anti-SH-PTP2. B,
SH-PTP1 and 2 from control and NGF-treated 6-24 cells were
immunoprecipitated from 50 µg of cell lysate and probed with
anti-SH-PTP1, 2, or anti-phosphotyrosine antibody. C, SH-PTP1
from control, NGF-, and EGF-treated PC12 cells were immunoprecipitated
from 100 µg of cell lysate and probed with anti-SH-PTP1 or
anti-phosphotyrosine antibody.
Figure 3: Time and concentration dependence of SH-PTP1 tyrosine phosphorylation. A, SH-PTP1 was immunoprecipitated from 6-24 cells treated with 50 ng/ml NGF for the indicated times. B, SH-PTP1 was immunoprecipitated from 6-24 cells treated with varying amounts of NGF for 5 min. Anti-phosphotyrosine and anti-SH-PTP1 immunoblots are shown.
Figure 4: NGF treatment activates SH-PTP1. 6-24 (top panel) or PC12 (bottom panel) cells were left untreated (open symbols) or treated with either 100 ng/ml NGF (filled symbols) for 5 min, lysed, and 300 µg of protein was immunoprecipitated with polyclonal anti-SH-PTP1 (squares) and 2 (diamonds) antibodies. The washed immunoprecipitates were assayed for PTP activity as described under ``Materials and Methods.'' Activity values from 6-24 cells are derived from triplicate experiments; those from PC12 cells represent the average from two experiments.
Figure 5: NGF-dependent binding of TrkA to SH-PTP1 in vitro. GST-SH-PTP1 on glutathione-agarose beads was incubated with lysates (660 µg of protein) from control and NGF-treated 6-24 cells for 1 h at 4 °C. The beads were washed five times with Nonidet P-40 lysis buffer and then boiled in SDS-PAGE sample buffer and chromatographed on an 8% SDS-PAGE gel. An immunoblot was performed with antibodies against TrkA.
Figure 6: TrkA coimmunoprecipitates with NGF-stimulated PTP activity. Lysates from control (open rectangles) and NGF-treated (filled rectangles) 6-24 cells were immunoprecipitated with control serum (lane 1) or anti-TrkA (lanes 2-4). In lane 3, an excess of blocking peptide was added to the TrkA antibody prior to the immunoprecipitation; in lane 4, 1 mM sodium vanadate was added. The washed immunoprecipitates were assayed for PTP activity for 10 min using Raytide as substrate as described under ``Materials and Methods.'' The data represent the average values obtained from two experiments.
Upon exposure to NGF, PC12 (rat adrenal pheochromocytoma)
cells differentiate into sympathetic nerve cells producing long
neurites (20) . We became interested in the possible role of
PTPs in this signaling process. Like many other receptor PTKs,
engagement of the TrkA by ligand leads to autophosphorylation of a
number of specific residues in the carboxyl terminus of the receptor
which serve as docking sites for SH2-containing
proteins(28, 29, 30) . There is ample
precedent for involvement of SH-PTPs in signaling through many receptor
PTKs. For example, SH-PTP2 binds to the activated PDGF- receptor
at tyrosine 1009(7, 8) . The bound PTP is
phosphorylated by the receptor and serves as a docking site for the
adaptor Grb2(8, 9) . SH-PTP2 appears to be required
for PDGF signaling, as microinjection of neutralizing antisera or
catalytically inactive forms of the enzyme inhibits PDGF-mediated
mitogenesis in NIH-3T3 cells(31, 32) . Similar results
have been obtained regarding insulin-mediated signaling(33) .
The positive role of SH-PTP2 in generating growth signals has also been
confirmed in developmental studies in both Drosophila and Xenopus(34, 35) . In these organisms,
disruption of SH-PTP2 function results in marked developmental
abnormalities consistent with loss of function of RPTK-regulated
pathways. Thus, SH-PTP2 appears to be a required element in some
receptor PTK signaling pathways. Our findings in PC12 cells indicate
that although SH-PTP2 is abundantly expressed, it does not associate
with the NGF receptor, and it is neither tyrosine phosphorylated nor
activated in response to NGF. Thus, it is unlikely that this PTP is
involved in the regulation of differentiation in PC12 cells by NGF.
Although SH-PTP1 is usually thought of as a hematopoietic cell PTP (indeed, one of its many pseudonyms is HCP), its tissue distribution includes a variety of epithelial cell types in addition to cells of hematopoietic lineage(10, 11, 12) . Here, we report that SH-PTP1 is also abundant in PC12 cells. Although we could not demonstrate binding of SH-PTP1 to TrkA in vivo, we show that this PTP is tyrosine phosphorylated and activated in NGF-treated PC12 cells and that it binds to TrkA in vitro in a NGF-dependent manner. It is interesting to note that Aparicio et al.(36) described the activation of three PTPs by NGF in PC12 cells, one of which has a molecular mass of about 60 kDa. Our results suggest that this PTP may be SH-PTP1.
Our findings are similar to those reported by Uchida et al.(18) , who showed that SH-PTP1 is a direct substrate of the insulin receptor kinase. As in our studies with TrkA, in vitro binding of SH-PTP1 to the insulin receptor was shown to be ligand-dependent, and insulin stimulated the tyrosine phosphorylation and activity of SH-PTP1. These authors were not, however, able to detect direct binding of SH-PTP1 to the insulin receptor by coimmunoprecipitation. In our studies, it is possible that the Trk antibody partially displaced the Trk-bound PTP during immunoprecipitation. The Trk antibody used for these experiments was raised against the 14 carboxyl-terminal amino acids of the TrkA receptor, where at least one of the phosphorylated tyrosines that is slated to transmit signals is located(28, 29, 30) . Whether partially displaced by TrkA antibody or not, it is possible that the amount of SH-PTP1 bound to immunoprecipitated TrkA is too low to be detected by immunoblot using the polyclonal anti-SH-PTP1 antiserum. The activity assay used to detect the presence of PTPs in TrkA immunoprecipitates (Fig. 6) is much more sensitive than immunoblotting techniques and thus is not inconsistent with the coimmunoprecipitation results shown in Fig. 2A and Fig. 3A. A more detailed characterization of the PTP activity present in TrkA immunoprecipitates may resolve this issue.
As none of the known TrkA autophosphorylation sites matches the consensus for SH-PTP1 binding, the interaction between this PTP and TrkA may be mediated through an unconventional binding motif. Indeed, Uchida et al.(18) found that the binding of SH-PTP1 to the insulin receptor is mediated not through SH2-phosphotyrosine interactions but rather through an element in the carboxyl terminus of SH-PTP1. Alternatively, the NGF-induced tyrosine phosphorylation and activation of SH-PTP1 may be indirectly mediated by TrkA. In this scenario, NGF receptor activation leads to tyrosine phosphorylation of SH-PTP1 through an intermediate rather than by direct binding. For example, in PC12 cells the Src tyrosine kinase is activated by NGF and is required for differentiation(37) . Matozaki et al.(38) have shown that Src can phosphorylate SH-PTP1 in vitro and that cells transformed with v-src have high levels of tyrosine-phosphorylated SH-PTP1, whereas nontransformed cells do not. Their results suggest that SH-PTP1 may be a direct target for the Src kinase. In murine T cells, similar findings have been noted regarding the Src-like protein kinase Lck and SH-PTP1(27) .
Although
it has been suggested that the selection of differentiation versus proliferation pathways in PC12 cells is a result of quantitative
differences among the PTK receptors(39) , there must also be
important qualitative differences, since native PC12 cells have
approximately equal numbers of NGF and EGF receptors yet respond quite
differently to each agent. Furthermore, certain proteins appear to be
tyrosine phosphorylated in response to differentiation agents but not
proliferative agents. To date, the most convincing example of such a
protein is SNT, an 80-kDa p13-binding
protein(40) , which is heavily tyrosine phosphorylated in
response to NGF, but not EGF. To our knowledge, SH-PTP1 represents the
second example of a protein of this type. Although we do not yet know
if the tyrosine phosphorylation and activation of SH-PTP1 by NGF are
required for PC12 differentiation, our results suggest that this enzyme
plays a role in modulating NGF-induced signaling events.