From the Department of Biochemistry and Molecular
Biology and § Graduate Training Program in Molecular,
Cellular, Biochemical, and Developmental Sciences, Mount Sinai School
of Medicine, Box 1020, New York, New York 10029
Received for publication, October 31, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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Differentiation of neuronal precursor cells in
response to neurotrophic differentiation factors is accompanied by the
activation of membrane-anchored SNT signaling adaptor proteins. Two
classes of differentiation factors, the neurotrophins and fibroblast
growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2 Within the developing vertebrate nervous system, proliferating
progenitor cells differentiate into post-mitotic neurons under the
control of specific secreted polypeptide growth factors. Neurotrophins and fibroblast growth factors
(FGFs),1 which promote
neuronal differentiation, signal through the activation of the Trk and
FGFR classes of receptor tyrosine-specific protein kinases (RTKs),
respectively. Other growth factors and protein hormones, such as
epidermal growth factor and insulin, also signal through RTKs present
on neuronal progenitor cells, but these factors promote proliferative
or anti-apoptotic responses without favoring differentiation.
Elucidating the RTK signaling pathways specific to the neuronal
differentiation response has been the subject of extensive research
over the past decade (1-3).
Following progenitor cell stimulation with differentiation factors, the
earliest marker of neuronal differentiation is tyrosine phosphorylation
of SNTs (suc1-binding neurotrophic targets), which occurs within
15 s of cell stimulation (4). SNT1 (also termed FRS2 Although activation of Ras and Shp2 are critical events in neuronal
differentiation, these proteins are also activated by cell stimulation
with the nondifferentiating growth factors epidermal growth factor and
insulin (14-17). Induction of neuronal differentiation must require
activation of additional signaling pathways or be dependent on the
magnitude or duration of Ras and Shp2 stimulation. A downstream target
of both Ras and Shp2, the ERK, mitogen-activated protein kinase,
undergoes prolonged activation in response to neuronal differentiation
factors but only transient activation in response to other growth
factors (3). It has been proposed that sustained ERK activation is
necessary and sufficient to drive neuronal differentiation, because
only sustained activation allows for substantial ERK nuclear
translocation and potential phosphorylation and activation of key
transcription factors (3).
We have developed a robust methodology to corroborate whether SNT
tyrosine phosphorylation can drive sustained ERK activation and the
neuronal differentiation response, and to analyze SNT effector domains
required for SNT's functional attributes. Using this methodology, we
show that SNT activation drives sustained ERK activity and neuronal
differentiation of PC12 cells. We further identify three effector
functions on SNT1 that act coordinately to mediate downstream responses.
Immunological Reagents--
Mouse monoclonal antibodies used
were 4G10 anti-phosphotyrosine (pY) (Upstate Biotechnology Inc.),
anti-Ras and anti-Shp2 (PTP1D) (Transduction Laboratories), 12CA5
anti-HA tag and 9E10 anti-Myc tag (courtesy of T. Moran), and E10
anti-phospho-ERK (p-ERK1/2) (New England BioLabs). Rabbit polyclonal
antibodies included anti-Grb2 and anti-Sos1 (Santa Cruz Biotechnology)
and anti-ERK (ERK1/2) (New England BioLabs). Conjugated secondary antibodies were from CALTAG Laboratories: Horseradish
peroxidase-conjugated anti-rabbit IgG, mouse IgG1, and mouse IgG2b,
alkaline phosphatase (AP)-conjugated anti-mouse IgG1, FITC-conjugated
anti-mouse IgG1, and biotin-conjugated anti-mouse IgG2b. Texas
Red-conjugated streptavidin was from CALTAG, unconjugated goat
anti-mouse IgG was from Jackson ImmunoResearch Labs, and protein
G-Sepharose was from Amersham Pharmacia Biotech.
Nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate was
obtained from Roche Molecular Biochemicals, and ECL was from Amersham
Pharmacia Biotech.
Cells and Cell Culture Reagents--
Mouse NIH3T3 cells
were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10%
bovine calf serum, rat PC12 cells (variant from M. Chao's laboratory,
kindly provided by R. Krauss) were cultured in DMEM with 10% fetal
bovine serum. Both cells were grown in an atmosphere of 5%
CO2 at 37 °C.
PD98059 was from New England BioLabs. Acidic FGF was purified from
Escherichia coli (18), recombinant rat Cell Lysis, Immunoprecipitation, and Immunoblotting--
Cells
were starved for 2 h in serum-free medium and subsequently treated
with 100 ng/ml FGF plus 5 µg/ml heparin, 5 µg/ml insulin, or 50 ng/ml NGF for times as indicated in the figures. Cells were lysed with
150 mM NaCl, 20 mM Tris, pH 7.6, 50 mM NaF, 1 mM Na3VO4, 5 mM benzamidine, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1% v/v Nonidet P-40, and nuclei were removed by
centrifugation. For immunoprecipitations, antibodies were incubated
with lysates overnight at 4 °C. Immunoprecipitates from polyclonal
antibodies were captured directly with protein G-Sepharose for 1 h, whereas immunoprecipitates from monoclonal antibodies were captured
with protein G-Sepharose preincubated with goat anti-mouse IgG (100 µg/ml gel). Beads were pelleted and washed twice with lysis buffer
before eluting proteins by boiling in sample buffer containing 2% SDS
and 5%
Proteins were electrophoresed through SDS-polyacrylamide gels
(SDS-polyacrylamide gel electrophoresis) (7.5% or 10%
polyacrylamide), electroblotted to polyvinylidene difluoride membranes
(Immobilon-P, Millipore), and probed with primary antibodies as
indicated in the figures, followed by incubation with corresponding
horseradish peroxidase-conjugated secondary antibodies (typically
1:10,000 dilution) and detection by ECL. Apparent molecular weights of proteins were estimated based on migration of prestained molecular weight standards (Amersham Pharmacia Biotech).
Expression of Myc-tagged Proteins in NIH3T3
Cells--
Myc-tagged SNT1 expression vector was derived by inserting
full-length human SNT1 cDNA into the cloning sites of a modified pSR
Myc-tagged SNT1(IRS) in pSR
NIH3T3 cells (8 × 105 per 10-cm dish) were
cotransfected as calcium phosphate precipitates (20) with 5 µg of
SNT1 expression plasmid, 1 µg of pLTRneo, and 25 µg of human
placental carrier DNA and selected with G418 (400 µg/ml, Life
Technologies, Inc.) to establish pools of stably transfected cells.
Expression of Proteins in PC12 Cells--
PC12 cells were
transiently transfected with pSR
For transient cotransfection, 0.5 µg of Myc-tagged pSR
For PC12-stable transfections, each Myc-tagged SNT1(IRS)CX open reading
frame was first shuttled into the expression vector pMIRB (21) (kindly
provided by D. Ornitz) for expression driven by Moloney murine leukemia
virus LTR as a bicistronic mRNA bearing a downstream
IRES-neo-cassette. ~1.5 × 107 PC12 cells were
electroporated (250 V/500 microfarads) in a Gene Pulser (Bio-Rad) in 1 ml of DMEM containing 40 µg of linearized pMIRB vectors, and plated
onto two 10-cm dishes. Three days after transfection, 800 µg/ml G418
was added and maintained for 3 weeks. G418-resistant clones were
picked, expanded, maintained in 200 µg/ml G418, and screened for
similar level of protein expression by both immunoblot and
Myc-immunostaining.
Northern Blot Analysis--
Total cellular RNA was isolated
using the TRIzol reagent (Life Technologies, Inc.). RNA samples (10 µg per lane) were electrophoresed through 1.1% agarose gels
containing 2.2 M formaldehyde in MOPS buffer (20 mM MOPS, pH 7.0, 5 mM sodium acetate, and 1 mM EDTA), and then transferred overnight to HyBond N+ nylon
membrane (Amersham Pharmacia Biotech). Full-length VGF cDNA (kindly
provided by S. Halegoua) was radiolabeled with 32P-dNTPs
(PerkinElmer Life Sciences) by DNA polymerase and random hexamer
primers and hybridized to the blot using ExpressHyb hybridization solution (CLONTECH) at 68 °C for 2 h,
washed to a final stringency of 50 °C, 0.1× SSC, 0.1% SDS, and
autoradiographed. To confirm equivalent loading and transfer of RNA
samples, the blot was reprobed under the same conditions with
32P-labeled rat glyceraldehyde phosphate dehydrogenase cDNA.
Ras Activation Assay--
A GST-RafRBD (Ras-binding domain)
fusion protein, which can bind Ras·GTP (kindly provided by S. Taylor), was expressed in E. coli and purified on
glutathione-agarose at 2 mg of fusion protein per 1 ml of beads, as
previously described (22). PC12 cells were lysed in
Mg2+-containing lysis buffer (25 mM HEPES, pH
7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 10%
glycerol, 10 mM MgCl2, 1 mM
Na3VO4, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin, and 1% Nonidet P-40). 250 µg of clarified lysates was
incubated with 15 µl of GST-RafRBD-agarose for 1 h at 4 °C,
resolved on 12% SDS-polyacrylamide gel electrophoresis, and
immunoblotted with anti-Ras monoclonal antibody.
Alkaline Phosphatase Immunostaining of PC12 Cells and Neurite
Outgrowth Assay--
Transiently transfected PC12 cells were treated
with growth factors in serum-containing medium for 2 or 3 days. Cells
were fixed in 4% paraformaldehyde/PBS for 20 min and permeabilized in
0.5% Triton X-100/PBS for 5 min. After blocking in 10% bovine calf
serum/PBS for 1 h, cells were incubated with 0.3 µg/ml 9E10 anti-Myc for 1 h, with 1:5000 AP-conjugated secondary anti-mouse IgG1 for 0.5 h, and stained with nitro blue tetrazolium (440 µg/ml) + 5-bromo-4-chloro-3-indolyl phosphate (165 µg/ml) in
alkaline substrate buffer (0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 1 mM levamisole) for 15-60 min. Neurite outgrowth was
quantitated by scoring the percentage of stained cells with neurites
longer than the size of two cell bodies and, in some cases, normalized to a maximum of 100%. Generally 400 stained cells were counted per
condition in two independent experiments. Representative images were
photographed with a digital camera attached to a Leica inverted light microscope.
Sequential Double Immunofluorescent Staining of PC12
Cells--
PC12 cells were transiently transfected with HA-tagged
pSR An SNT/IRS Chimera Activated by Insulin Drives PC12 Neuronal
Differentiation--
FGFs and neurotrophins induce SNT tyrosine
phosphorylation, whereas insulin induces tyrosine phosphorylation of
multifunctional insulin receptor substrate (IRS) docking proteins. We
reasoned that a chimeric SNT/IRS protein bearing the SNT backbone, but containing the insulin receptor recognition domains of IRS, would undergo tyrosine phosphorylation in response to insulin. We constructed SNT1(IRS), a Myc epitope-tagged SNT1 protein in which the N-terminal 137 amino acid residues of SNT1, bearing a myristoylation motif and the
FGFR/Trk receptor-specific PTB domain, were replaced with the
N-terminal pleckstrin homology (PH) and PTB domains of IRS2 (residues
1-307) (Fig. 1A). When
SNT1(IRS) was stably transfected into NIH3T3 fibroblasts, insulin, but
not FGF, could induce SNT1(IRS) tyrosine phosphorylation (Fig.
1B). The behavior of SNT1(IRS) stood in contrast to
transfected SNT1-Myc, which was phosphorylated in response to FGF, but
not insulin (Fig. 1B).
To test whether SNT1(IRS) could promote neuronal differentiation,
Myc-tagged SNT1(IRS) or SNT1 were transiently transfected into PC12
cells, which were subsequently challenged for 2 days with the
neurotrophin nerve growth factor (NGF), FGF1, or insulin. Subsequently,
transfected cells, as identified by anti-Myc-tag immunostaining (Fig.
2A), were scored for neuronal
differentiation using neurite outgrowth as the morphological criterion
(23). Although PC12 cells transfected with SNT1 or SNT1(IRS) underwent neuronal differentiation in response to NGF or FGF (Fig.
2B), analogous to untransfected cells (data not shown),
insulin failed to induce differentiation of SNT1(IRS)-transfected cells
(Fig. 2B).
Biochemical and biological activities of SNT1 require its plasma
membrane attachment mediated by N-terminal myristoylation (5). The
failure of SNT1(IRS) to promote neuronal differentiation may have
resulted from reduced membrane association of the chimeric protein
lacking the myristoylation motif. Indeed, when SNT1(IRS) subcellular
distribution was compared with SNT1 by confocal immunofluorescence analysis of transfected PC12 cells, SNT1(IRS) was evenly distributed throughout the cytoplasm as opposed to the exclusively plasma membrane
localization of SNT1 (Fig. 2C).
To relocalize a chimeric SNT1(IRS) protein back to the plasma membrane,
we engineered the variant SNT1(IRS)CX (Fig. 1A) by fusing
the CAAX farnesylation motif of K-ras to
the C terminus (19). As anticipated, SNT1(IRS)CX was largely associated
with the plasma membrane in transfected PC12 cells (Fig. 2C)
and retained the ability to undergo insulin-induced tyrosine
phosphorylation in transfected NIH3T3 cells (Fig. 1B). When
SNT1(IRS)CX was transiently transfected into PC12 cells, it could
effectively mediate insulin-induced neuronal differentiation (Fig. 2,
A and B) and up-regulation of the VGF
differentiation-specific gene transcript (Fig. 2D). This result demonstrates that artificial stimulation of SNT effector functions is sufficient to recapitulate the biological response to
neuronal differentiation factors.
SNT1(IRS)CX-mediated Neuronal Differentiation Requires
Endogenous Ras, MEK, and Shp2 Activities and Is Accompanied by
Sustained Ras and ERK1/2 Activation--
Differentiation
factor-induced PC12 neuronal differentiation requires Ras, MEK, and
Shp2 activities (12, 13) and is accompanied by sustained ERK
activation. We sought to determine whether SNT1(IRS)CX promotes
neuronal differentiation through the same biochemical mechanisms. To
determine whether Ras and Shp2 are required for SNT1(IRS)CX-mediated
differentiation, Myc-tagged SNT1(IRS)CX was transiently cotransfected
with empty vector controls or with dominant-negative mutant forms of
Ras (RasN17) or Shp2 (Shp2C459S), and neurite
outgrowth was quantitated by scoring Myc-positive cells following 3-day
insulin stimulation. As shown in Fig. 3, either RasN17 (Fig. 3A) or Shp2C459S
(Fig. 3B) could significantly reduce SNT1(IRS)CX-mediated
differentiation induced by insulin.
Additional analyses made use of PC12 cells stably transfected with
SNT1(IRS)CX (see "Experimental Procedures"). PC12/SNT1(IRS)CX cells
were also capable of undergoing insulin-induced neurite outgrowth (Fig.
3C). Pretreatment of PC12/SNT1(IRS)CX with the MEK-specific
kinase inhibitor PD98059 dramatically reduced insulin-induced differentiation (Fig. 3C).
To determine whether SNT1(IRS)CX stimulation promotes sustained ERK
activation, parental PC12 and PC12/SNT1(IRS)CX cells were treated with
either NGF or insulin for 0.2 or 3 h, and total cell lysates were
analyzed for ERK activation by probing immunoblots with monoclonal
antibody specific for tyrosine/threonine dually phosphorylated ERKs. As
shown in Fig. 4, NGF-induced ERK1 and ERK2 activation in transfected or untransfected cells persists through
3 h, whereas insulin-induced ERK activation in parental PC12 cells
is transient and disappears by 3 h post-stimulation. By contrast,
ERK activity is strongly persistent in PC12/SNT1(IRS)CX cells
stimulated with insulin. The mechanism by which SNT1(IRS)CX mediates
sustained ERK activity may be through its ability to mediate sustained
Ras activity (Fig. 4).
The above results demonstrate that SNT1(IRS)CX promotes PC12 neuronal
differentiation through a repertoire of signaling intermediates indistinguishable from those employed by neuronal differentiation factors.
SNT1 Mutagenesis: Effects on Interactions with Grb2, Sos, and Shp2
in3T3 Cells--
We have engineered a series of potential effector
mutations in SNT1, guided by previous studies, consensus sequence
motifs, and sequence conservation between SNT1 and SNT2. As shown in
Fig. 5A, the 4YF mutation
converts tyrosines 196, 306, 349, and 392 to phenylalanines. These four
tyrosines all bear the Grb2-binding consensus motif YXN and
have been shown to mediate Grb2 binding and Ras activation in
transfected 293T cells (5). The 2YF mutation converts tyrosines 436 and
471 to phenylalanines. The sequences surrounding these two tyrosines
are identical in SNT2 and very similar in the two C-terminal tyrosines
of IRS1 which are known to recruit Shp2; indeed, SNT1 Y436 was shown to
be required for efficient Shp2 recruitment (11). The 6YF mutant
combines the mutations of 2YF and 4YF. The
Each mutant SNT was first assayed in stably transfected NIH3T3 cells
for FGF-induced tyrosine phosphorylation and target protein recruitment
(Fig. 5B). Consistent with previous studies (5, 11), 4YF
still undergoes FGF-induced tyrosine phosphorylation, but recruitment
of Grb2 and Sos are substantially diminished, whereas Shp2 recruitment
is unaffected. Reciprocally, 2YF is unaffected in Grb2 and Sos
recruitment, but Shp2 recruitment is abolished. Analogous to IRS·Shp2
interaction (24), Shp2 recruitment to SNT1 probably requires
simultaneous, cooperative interactions of Shp2's two SH2 domains with
SNT1 tyrosines 436 and 471, because the individual mutation of either
of these tyrosines virtually abolishes Shp2 interaction (Fig.
5C). 2YF, 4YF, and
Because neuronal differentiation correlates with sustained ERK
activation, we also tested whether each of the mutant SNT1(IRS)CX constructs would fail to sustain ERK activity. Transiently transfected cells were stimulated with insulin or NGF for 3 h, then fixed and
assayed by double-label immunofluorescence for both active ERK and
transfected epitope-tagged protein. Representative confocal images are
shown in Fig. 6, and the quantified data
are shown in Table II. Although wild-type
SNT1(IRS)CX promoted sustained ERK activation, SNT1(IRS)CX-
SNT1(IRS)CX-2YF failed to promote ERK activity (Fig. 6 and Table II),
strongly suggesting that Shp2 recruitment to SNT is essential for
sustained ERK activation in PC12 cells. To directly test the importance
of Shp2 for ERK activation, we transiently transfected PC12 cells with
epitope-tagged dominant-negative Shp2 (Shp2C459S) and
assayed the effect on NGF-induced sustained ERK activity. As shown in
Fig. 6 and Table II, inhibition of Shp2 activity dramatically impaired
NGF-induced sustained ERK activation. These findings are consistent
with data reported for other types of cells (25, 26).
Surprisingly, SNT1(IRS)CX-4YF, which lacks the major Grb2·Sos
recruitment sites, still induced sustained ERK activation in a
substantial percentage of transfected cells. However, the levels of
active ERK appeared consistently lower in insulin-stimulated SNT1(IRS)CX-4YF transfectants when compared with wild-type
SNT1(IRS)CX-transfected cells (see representative cells in Fig. 6).
Residual Grb2·Sos recruitment to SNT1(IRS)CX-4YF, which may be
mediated indirectly through tyrosine-phosphorylated Shp2 (11), may be
sufficient to mediate reduced, but readily detectable, ERK activation.
The more dramatic impairment of the 4YF mutation on neurite outgrowth (Table I) may reflect the mutant's failure to achieve a critical threshold of ERK activation. Alternatively, the 4YF mutation may substantially impair ERK activation beyond the 3-h time window analyzed here.
SNT1(IRS)CX-
Reduced SNT1(IRS)CX- Neurotrophins and FGFs promote neuronal differentiation by
coordinately activating Ras and Shp2 with resultant sustained
activation of ERK mitogen-activated protein kinases. SNTs were
suspected of playing a key role in the differentiation response,
because neurotrophins and FGFs specifically induce SNT tyrosine
phosphorylation, which is followed by SNT recruitment of Ras activators
and Shp2. We have shown here that artificial activation of SNT1 in PC12 cells recapitulates the differentiation response, which is preceded by
and is dependent on the same signaling pathways that are mediated by
differentiation factors. These findings suggest that SNT is a principle
mediator of growth factor-induced neuronal differentiation. We should
note that differentiation factors likely induce additional signaling
pathways, which act in concert with SNT. For example, although NGF and
FGF activate SNT to comparable extents in PC12 cells (4, 5), NGF more
potently induces their differentiation (30) (and Fig. 2). Reciprocally,
FGF is more potent than NGF at promoting early phase neuronal
differentiation of MAH sympathoadrenal progenitor cells (31, 32),
whereas both factors activate SNTs comparably.2
Although our data demonstrate that SNT phosphorylation leads to
sustained ERK activity through the activation of both Ras and Shp2, the
mechanism by which Ras and Shp2 coordinate sustained ERK activation is
unclear. Candidate targets for Shp2 may include Src-family nonreceptor
tyrosine kinases, because these kinases are activated by tyrosine
dephosphorylation and play essential roles in FGF-mediated biological
responses (33-36). It has been reported that NGF-induced sustained ERK
activation may also require and be more dependent on the activation of
the G protein Rap1 than on Ras; activated Rap1 was shown to form
complexes with RafB in NGF-treated cells, promoting the RafB Previous studies have shown that FGF induces tyrosine phosphorylation
of SNT and Shc, both of which recruit Grb2; nonetheless, virtually all
of the Sos in FGF-treated cells is recruited to SNT·Grb2 complexes at
the expense of Shc·Grb2 complexes (9). Our data offer a possible
mechanism for efficient SNT·Sos complex formation. In PC12 cells,
recruitment of Sos is dependent on the C-terminal domain of SNT in
addition to the phosphotyrosyl motifs that recruit Grb2 (Fig.
7A). Deletion of the SNT1 C-terminal motif in SNT1(IRS)CX
did not affect Grb2 or Shp2 recruitment but reduced Sos recruitment
with concomitant loss of sustained Ras/ERK activation and cell
differentiation. The highly conserved C termini of SNT1 and SNT2 may
contact Sos directly or through intermediary proteins, thereby acting
in concert with Grb2 to generate stable SNT·Grb2·Sos complexes.
), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and
Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor
Sos. To determine effector functions of SNT that promote neuronal
differentiation of PC12 pheochromocytoma cells, we engineered a
chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and
the insulin receptor recognition domains of IRS2. Insulin promoted
tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells
accompanied by sustained activation of ERK1/2 mitogen-activated protein
kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response
was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis
of SNT1(IRS)CX identified three classes of effector motifs within SNT
critical for both sustained ERK activation and neuronal
differentiation: 1) four phosphotyrosine motifs that mediate
recruitment of Grb2, 2) two phosphotyrosine motifs that mediate
recruitment of Shp2, and 3) a C-terminal motif that functions by
helping to recruit Sos. We discuss possible mechanisms by which three
functionally distinct SNT effector motifs collaborate to promote a
downstream biochemical and biological response.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) and SNT2
(FRS2
) are related membrane-anchored docking proteins (5, 6) that
are tyrosine-phosphorylated following specific interaction between
activated Trks or FGFRs and the phosphotyrosine binding (PTB) domain of
SNTs (6-8). Phosphorylated SNTs recruit the Grb2 adaptor protein in
complex with the Ras activators Sos1 and Sos2 (5, 9) and also recruit
the SH2 domain-regulated protein tyrosine phosphatase, Shp2 (10, 11).
These interactions have suggested the importance of SNTs in the
differentiation response, because experiments with dominant-negative
protein inhibitors have shown that Ras and Shp2 are required for
neurotrophin- and FGF-induced differentiation of PC12 neuronal
progenitor cells (12, 13). Furthermore, overexpression of SNT1 in PC12
cells potentiates the length of FGF-induced neurites in a Ras- and
Shp2-dependent manner (5, 11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-NGF was from R&D
Systems, and recombinant human insulin was from Sigma.
-mercaptoethanol.
mammalian cell expression vector bearing a triple Myc epitope tag sequence 3' of the cDNA cloning sites (6). Myc-tagged SNT1 point mutants (Y436F; Y471F; 2YF = Y436F/Y471F; 4YF = Y196F/Y306F/Y349F/Y392F; 6YF = 2YF + 4YF) in pSR
vector were
engineered by ligating together PCR-derived SNT fragments using primers
that incorporated Y
F mutations and either naturally occurring or
engineered restriction enzyme cleavage sites. Myc-tagged SNT1-
T
mutation was generated by PCR using sense primer 5' to the start codon
and antisense primer corresponding to the C-terminal region of SNT1
through Thr-495.
vector was constructed by PCR from
segments of human SNT1 and murine IRS2 (kindly provided by R. Kohanski)
and contained (N-terminal to C-terminal) IRS2-(1-307), ArgPro linker
segment, SNT1-(138-508), and triple Myc tag. pSR
-SNT1(IRS)CX was made by ligating annealed oligonucleotides encoding the
K-ras(B) C-terminal membrane-targeting sequence
(CAAX motif: KDGKKKKKKSRTRCTVM) (19) into the
position C-terminal to the triple Myc-tag sequence of pSR
-SNT1(IRS).
Myc-tagged SNT1(IRS)CX mutants (2YF, 4YF, 6YF, and
T) were
engineered by replacing restriction enzyme segments of
pSR
-SNT1(IRS)CX with corresponding mutant segments of SNT1 2YF, 4YF,
6YF, or
T.
-Myc-tagged SNT-derived constructs
or with corresponding HA-epitope-tagged constructs in which the triple
Myc tag was replaced with annealed oligonucleotides encoding two tandem
copies of the HA-epitope sequence (YDVPDYAS). For transient
transfection of single plasmids, ~1.2 × 106 cells
per 60-mm dish were transfected with 3 µg of expression plasmids and
16 µl of LipofectAMINE Reagent (Life Technologies, Inc.) in a total
of 3 ml.
-SNT1(IRS)CX
was mixed with 2.5 µg of pCEV29-RasN17 (kindly provided
by A. Chan), pCMV-HA-Shp2C459S (kindly provided by B. Neel), or corresponding empty vectors and transfected with
LipofectAMINE, as above.
-SNT1(IRS)CX plasmids or pCMV-HA-Shp2C459S. After
24 h, cells were split into Lab-Tek 4-well chamber slides (Nalge
Nunc International) precoated with poly-D-lysine (1 mg/ml for 2 h, Sigma) and laminin (10 µg/ml overnight, Sigma). 48 h post-transfection, cells were serum-starved for 2 h, and then treated with growth factors for 3 h. Cells were fixed in 4%
paraformaldehyde/PBS for 20 min, and permeabilized in 0.2% Triton
X-100/PBS for 5 min. All blocking or antibody incubation steps were
with 10% goat serum in PBT (PBS + 0.25% Tween 20), whereas all
washing steps were with PBT. After blocking for 1 h, cells were
incubated with 1:100 anti-phospho-ERK (IgG1) overnight at 4 °C, then
with 1:200 FITC-conjugated anti-mouse IgG1 for 3 h. After
extensive wash, cells were incubated with 1 µg/ml primary anti-HA
(IgG2b) for 2 h, 1:250 secondary biotin-conjugated anti-mouse
IgG2b for 1 h, and 1:5000 Texas Red-conjugated streptavidin for 30 min. Green and red fluorescence was examined under a Zeiss Axiophot 2 fluorescence microscope. The percentage of phospho-ERK-positive cells
was quantitated by scoring the number of cells with sustained ERK
activity (green) among the transfected cells
(red), with 200 transfected cells counted per condition. Representative images were scanned with a Leica TCS-CP confocal laser
scanning microscope.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Insulin-induced tyrosine phosphorylation of
SNT/IRS chimeric proteins in NIH3T3 cells. A, schematic
representation of SNT/IRS chimeric proteins. All chimeras bear the PH
and PTB domain of mouse IRS2 and the effector domain of human SNT1.
SNT1 sequences are in dark-shaded boxes, IRS2 sequences are
in the light-shaded box, and specific motifs
(myristoylation, Myc-tag, and CAAX) are indicated. Different
regions are not drawn to scale. B, NIH3T3 cells expressing
Myc-tagged full-length SNT1 or SNT/IRS chimeric proteins were treated
with different growth factors (GF) for 10 min: none ( ), 50 ng/ml FGF (F), and 5 µg/ml insulin (I). Lysates
were immunoprecipitated (IP) with anti-Myc, electrophoresed,
and immunoblotted (IB) with anti-Myc and
anti-phosphotyrosine (pY).
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Fig. 2.
SNT1(IRS)CX enables PC12 cells to terminally
differentiate in response to insulin. A, B,
and C, PC12 cells were transiently transfected with
Myc-tagged wild-type SNT1 or SNT/IRS chimeras. After 24 h, the
cells were treated with different growth factors for 48 h before
fixation: NGF (50 ng/ml), FGF (100 ng/ml), and INS (insulin, 5 µg/ml). A, detection of transfected cells. Fixed cells
were incubated with anti-Myc and AP-conjugated secondary antibodies,
and stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl
phosphate (purple). Representative stained cells are shown.
B, quantitation of neurite outgrowth. Neurite outgrowth was
quantitated by scoring the percentage of stained cells with neurites
longer than the size of two cell bodies. The data shown are from one of
two experiments, both of which yielded similar results. C,
subcellular localization of overexpressed proteins. Transfected cells
were incubated with anti-Myc and FITC-conjugated secondary antibodies.
Green fluorescence in representative confocal microscope images are
shown. D, induction of VGF expression. PC12 cells stably
transfected with Myc-tagged SNT1(IRS)CX and parental PC12 cells were
treated with different growth factors (GF) for 5 h:
None ( ), 50 ng/ml NGF (N), 5 µg/ml insulin
(I). Total RNA was isolated and subjected to analysis on a
Northern blot. The blot was probed with 32P-labeled VGF
full-length cDNA, and later reprobed with 32P-labeled
rat glyceraldehyde phosphate dehydrogenase cDNA.
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Fig. 3.
Ras, Shp2, and MEK are required for
SNT1(IRS)CX-mediated PC12 cell neurite outgrowth. Effect of
(A) RasN17 and (B)
Shp2C459S on PC12 cell neurite outgrowth. Myc-tagged
SNT1(IRS)CX was transiently cotransfected with indicated constructs or
corresponding empty vectors (as controls) into PC12 cells. After
24 h, the cells were treated with NGF (50 ng/ml) or INS (insulin,
5 µg/ml) for 72 h, then fixed and immunostained for Myc-tagged
proteins. The number of transfected cells displaying neurite outgrowth
was quantitated, and the data were expressed relative to a maximum of
100%. The data shown are from one of two experiments, both of which
yielded similar results. C, effect of PD98059 on PC12 cell
neurite outgrowth. PC12/SNT1(IRS)CX cells were pretreated with 20 µM PD98059 or 0.1% Me2SO ( , as control)
for 60 min prior to the addition of NGF or insulin for 48 h.
Neurite outgrowth was scored as described above.
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Fig. 4.
SNT1(IRS)CX-mediated PC12 cell neurite
outgrowth is accompanied by sustained Ras/ERK activity. Parental
PC12 and PC12/SNT1(IRS)CX cells were treated with different growth
factors (GF) for the indicated times: None ( ), 50 ng/ml
NGF (N), 5 µg/ml insulin (I). Total lysates or
lysates captured by GST-RafRBD agarose were electrophoresed, and
immunoblotted with anti-Ras, anti-phospho-ERK (p-ERK1/2) or
anti-ERK (ERK1/2) antibodies.
T mutation deletes the
C-terminal 13 residues of SNT1. The C terminus of SNTs bears no
previously described nor readily predictable function, but was targeted
for mutation because it is the region of highest sequence homology between SNT1 and SNT2 (Fig. 5A).
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Fig. 5.
Effect of SNT1 mutations on Grb2, Sos, and
Shp2 binding in NIH3T3 cells. A, description of
effector domain mutations on SNT1. Amino acid sequences of the
potential SH2 domain binding motifs and the C-terminal regions of SNT1
and SNT2 are aligned, with identical residues in SNT2 indicated by
asterisks. Tyrosines mutated to phenylalanines are in
boldface type. 4YF, Y196F/Y306F/Y349F/Y392F;
2YF, Y436F/Y471F; 6YF, 4YF + 2YF. Residues
deleted in T are underlined. B, effect of SNT1
mutations. NIH3T3 cells stably expressing Myc-tagged wild-type or
mutant SNT1 were left untreated or treated with FGF (100 ng/ml, 10 min). Lysates were immunoprecipitated (IP) with anti-Myc,
anti-Grb2, or anti-Sos1. Proteins were electrophoresed and
immunoblotted (IB) with anti-Myc, anti-pY, or anti-Shp2 as
indicated. C, Tyr-436 and Tyr-471 are needed for Shp2
recruitment. NIH3T3 cells expressing wild-type, Y436F, Y471F, or 2YF
SNT1 were treated with FGF, and lysates were analyzed for Myc-SNT
expression, Myc-SNT tyrosine phosphorylation, and Myc-SNT·Shp2
interaction (as in B).
T appeared unaffected in tyrosine phosphorylation
and recruitment of Grb2, Sos, and Shp2 in3T3 cells (Fig.
5B).
T Mutations Impair SNT1(IRS)CX-mediated Neuronal
Differentiation and Sustained ERK Activation in PC12 Cells--
For
functional studies, each of the mutations described above was shuttled
into the SNT1(IRS)CX construct. When expressed in NIH3T3 cells, the
recruitment potentials of these SNT1(IRS)CX mutants in response to
insulin recapitulated the behaviors of the corresponding mutations in
SNT1 (data not shown). We then tested all SNT1(IRS)CX mutants for their
ability to promote neuronal differentiation following transient
transfection of PC12 cells. As shown in Table
I, the 2YF, 4YF, 6YF, and
T mutations
dramatically reduced or abolished the ability of SNT1(IRS)CX to mediate
neurite outgrowth in response to insulin.
Effect of mutations on SNT1(IRS)CX-mediated neurite outgrowth
T). After
24 h, the cells were treated with NGF, insulin, or no growth
factors for 48 h, then fixed and immunostained for transfected
proteins. Neurite outgrowth among stained cells was quantitated, and
the ratio of insulin/NGF-mediated differentiation was calculated. The
data above are from one of two experiments, both of which yielded
similar results.
T had a
significantly reduced sustained ERK activation potential. The behavior
of SNT1(IRS)CX-
T establishes a previously unappreciated role for the
conserved SNT C-terminal domain in mediating biochemical and biological
responses in PC12 cells. SNT1(IRS)CX-
T has been subjected to further
biochemical analysis (see below).
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Fig. 6.
Detection of sustained ERK activation in PC12
cells expressing wild-type and mutant SNT1(IRS)CX. PC12 cells were
transiently transfected with HA-tagged SNT1(IRS)CX constructs (WT, 2YF,
4YF, 6YF, or T) or HA-tagged Shp2C459S. After 48 h,
the cells were treated with NGF, insulin, or no growth factors for
3 h, then fixed and analyzed by double immunofluorescence with
anti-phospho-ERK (p-ERK) (green) and anti-HA tag
(red). Representative confocal microscopic images of each
sample are shown.
Quantitation of sustained ERK activation in PC12 cells expressing
wild-type and mutant SNT1(IRS)CX
T Is Defective for Sos Recruitment in PC12
Cells--
To better understand the role of the SNT C terminus in
mediating sustained ERK activity and neuronal differentiation, we
established a stably transfected PC12 cell line expressing
SNT1(IRS)CX-
T. As shown in Fig.
7A, SNT1(IRS)CX-
T was
comparable to wild-type SNT1(IRS)CX in terms of levels of
insulin-induced tyrosine phosphorylation and association with both Grb2
and Shp2. However, association of Sos with insulin-activated
SNT1(IRS)CX-
T was significantly reduced. We interpret these results
to indicate that Sos is recruited to SNT by multiple contacts,
including the well-documented Grb2 SH3 domain interaction with a
proline-rich domain on Sos (14, 27-29) as well as SNT C-terminal
domain interaction with Sos by either direct or indirect mechanisms.
Impaired association of SNT1(IRS)CX-
T with Sos is cell
type-specific, because SNT1(IRS)CX-
T and wild-type SNT1(IRS)CX show
comparable association with Sos in3T3 cells (Fig. 7A).
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Fig. 7.
Biochemical analysis of PC12 cells stably
expressing SNT1(IRS)CX- T mutant.
A, effect of SNT1(IRS)CX-
T mutation on Grb2, Sos, and
Shp2 binding. Parental PC12 cells and cells from PC12/SNT1(IRS)CX
cloned lines (WT and
T) were
treated with 5 µg/ml insulin (INS) for the indicated
times. Lysates were immunoprecipitated (IP) with anti-Myc,
anti-Grb2, and anti-Sos1, electrophoresed, and immunoblotted
(IB) with anti-pY, anti-Myc, anti-Sos1 or anti-Shp2 as
indicated. NIH3T3 cells stably transfected with SNT1(IRS)CX-WT or -
T
were treated with insulin, and lysates were subjected to
immunoprecipitations and immunoblot detection. Sos recruitment to
SNT(IRS)CX is impaired by the
T mutation in PC12 cells.
B, effect of SNT1(IRS)CX-
T mutant on Ras/ERK activation.
Parental PC12 cells and cells from PC12/SNT1(IRS)CX cloned lines were
treated with different growth factors (GF) for the indicated
times: None (
), 50 ng/ml NGF (N), 5 µg/ml insulin
(I). Total lysates or lysates captured by GST-RafRBD agarose
were electrophoresed, and immunoblotted with anti-Ras, anti-phospho-ERK
(p-ERK1/2), or anti-ERK (ERK1/2).
SNT1(IRS)CX-mediated sustained Ras and ERK activation are impaired by
the
T mutation in PC12 cells.
T association with Sos in PC12 cells impairs
signaling through the Ras/ERK pathway. As shown in Fig. 7B,
insulin stimulation of SNT1(IRS)CX-
T failed to induce sustained Ras
activity above basal levels and reduced sustained ERK1/2 activation. Commensurate with these findings, the PC12/SNT1(IRS)CX-
T cells failed to undergo insulin-induced neuronal differentiation (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
MEK
ERK kinase cascade (37). However, the sublineage of PC12 cells used
in our studies showed no evidence of NGF-induced or SNT1(IRS)CX-induced
Rap1 activation.3
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ACKNOWLEDGEMENTS |
---|
We thank Rebecca Hardy and Scott Henderson for advice regarding confocal immunofluorescence, Moses Chao and Robert Krauss for providing PC12 cells, Mark Frankel and Ron Kohanski for IRS2 cDNA, Stephen Taylor for pGEX-RafRBD, Benjamin Neel for pCMV-HA-Shp2C459S, Andrew Chan for pCEV29-RasN17, and David Ornitz for IRESneo expression vector.
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FOOTNOTES |
---|
* This work was supported by United States Public Health Services Grants R21-GM55666 and R01-GM39906 (to M. G.). Confocal microscopy was conducted at the Mt. Sinai School of Medicine Microscopy Center, which is supported by National Science Foundation and National Institutes of Health institutional instrumentation grants.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Institute of Cancer Genetics, College of Physicians and Surgeons, Rus Berrie Pavilion, Rm. 313A, New York, NY 10032.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Box 1020, Mt. Sinai School of
Medicine, New York, NY 10029. Tel.: 212-241-3394; Fax: 212-860-9279;
E-mail: Mitchell.Goldfarb@mssm.edu.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M009925200
2 J. Hutchinson and M. Goldfarb, unpublished data.
3 H. Xu, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; RTK, receptor tyrosine-specific protein kinase; SNT, suc1-binding neurotrophic targets; PTB, phosphotyrosine binding; ERK, extracellular signal-regulated kinase; pY, 4G10 anti-phosphotyrosine; HA, hemagglutinin; AP, alkaline phosphatase; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; NGF, nerve growth factor; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; RBD, Ras-binding domain; PBS, phosphate-buffered saline; IRS, insulin receptor substrate; PH, pleckstrin homology; Trk, tropomyosin-receptor kinase.
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