(Received for publication, August 10, 1995; and in revised form, September 19, 1995)
From the
Immunoprecipitates of metabolically labeled PC12 cells consistently contained a 43-kDa protein that was associated with Shc, a signal-transducing protein with a single SH2 domain. Following affinity chromatography with immobilized recombinant glutathione S-transferase (GST)-Shc fusion protein, the 43-kDa protein was identified as actin by mass spectrometry and immunoblotting. Cosedimentation experiments using purified actin and GST-Shc showed that Shc binds directly to F-actin, confirming Shc-actin interaction in vivo. Various GST-truncated Shc fusion proteins were prepared and used in actin cosedimentation assays. Constructs containing the SH2 and collagen homology domains were not precipitated, and those containing the amino-terminal domain were. Thus, Shc-actin interactions do not occur in the region of tyrosine phosphorylation and leave the SH2 domain free to bind to other tyrosine-phosphorylated molecules. Although the major pool of Shc in unstimulated PC12 cells is soluble, two other pools are associated with the cytoskeleton and the submembranous cytoskeleton. Upon nerve growth factor stimulation, approximately 50% of the soluble Shc translocates to both cytoskeleton environments within 2 min, decreasing thereafter. When cells were pretreated with cytochalasin D, a drug that disrupts actin filaments, Shc translocation to the cytoskeleton was abolished. However, in the submembranous fraction, the Shc level was elevated in resting cells following cytochalasin D treatment. The kinetics of translocation, compared to mitogen-activated protein kinase activation, and the nature of the Shc-actin interaction suggest that the cytoskeletal association of Shc, induced by growth factors, may be related to membrane ruffling and actin fiber reorganization.
Shc ()protein is part of a family of adapters
involved in the mitogenic and differentiation signaling of a variety of
receptor tyrosine kinases including those for EGF(1) ,
NGF(2, 3) , PDGF(4) , and
insulin(5, 6) . Shc also transduces the signal of
other receptor classes such as those for interleukin 2 (7) and
the T-cell(8) . In addition, FGF, growth hormone,
thrombopoietin, interleukin 3, granulocyte-macrophage
colony-stimulating factors, erythropoietin, and hepatocyte growth
factor induce Shc phosphorylation, but the direct interaction of Shc
with their respective receptors has not yet been
observed(9, 10, 11, 12, 13, 14, 15) .
The Shc adapter protein is composed of one SH2 domain at the
carboxyl-terminal end, one collagen homology (CH) domain and one
amino-terminal domain. It exists in two main forms of 46 and 52 kDa,
resulting from alternative translation start sites, and a 66-kDa
isoform whose origin is less clear(1) . When a receptor such as
that for NGF is activated, Shc binds rapidly to a specific
phosphotyrosine of the receptor and becomes phosphorylated on a
tyrosine residue(1, 16, 17) . The adapter
protein Grb-2 then associates with Shc which allows p21 activation via the guanine nucleotide release protein, Sos,
leading to the activation of the MAPK
pathway(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) .
Initially, the Shc SH2 domain was presumed to be responsible for the
binding of Shc to the activated growth factor receptor, although a
discrepancy existed between Shc recognition site and Shc SH2 domain
specificity(2, 27, 28, 29) . Several
recent studies have shown that Shc binds the activated receptor in the
NPXpY sequence by its amino-terminal or phosphotyrosine
binding
domain(30, 31, 32, 33, 34, 35) .
This mechanism suggests that Shc SH2 domain remains free to interact
with other yet unidentified phosphotyrosine-containing proteins. In
addition, it has been shown that the Shc phosphotyrosine binding domain
is able to recognize several other non-receptor
phosphoproteins(36) . Recent studies indicate that when a
growth factor receptor contains a binding site for Shc and Grb-2, the
Shc pathway is dominant(5, 20) . This could be
explained by the higher affinity of the phosphotyrosine binding domain
for the NXXpY motif compared with the affinity that the SH2
domains display for their proper binding sites (32) . Besides
its binding to activated receptors, Shc has been shown to associate
with intracellular molecules such as phosphatidylinositol
3-kinase(37) , Sos(38) ,
c-Abl(39, 40, 41) , and the tyrosine
phosphatase PEST (42) as well as to several unidentified
phosphotyrosine-containing
proteins(10, 39, 43) . However, the
physiological relevance of these associations is still unclear.
Shc has oncogenic properties since its overexpression induces transformation in fibroblasts and differentiation in PC12 cells(1, 16) . Differentiation of PC12 cells induced by NGF requires Shc activity. When Tyr 490 of TrkA, the binding site for Shc, is mutated to phenylalanine, NGF can no longer promote PC12 cell differentiation (3) .
In the present study, Shc synthesis in PC12 cells stimulated by NGF and other growth factors was examined. It was observed that a 43-kDa protein was always found in Shc immunoprecipitates. This protein was identified as actin. This suggests a relationship between Shc and the cytoskeleton that was confirmed by the fact that, after NGF stimulation, Shc rapidly translocates from a soluble compartment to the cytoskeleton in an actin-dependent manner.
Figure 1: Schematic representation of Shc domains expressed as GST-fusion proteins. Full-length human Shc cDNA and cDNAs fragments were cloned by reverse transcription-PCR of HepG2 cell total RNA and subcloned in the pGEX-3X expression vector. The arrow indicates the two translation initiation starts. N, Shc amino-terminal domain (hatched area); CH, collagen homology Shc domain (filled area); SH2, Shc Src-homology type 2 domain (open area).
Figure 2:
Shc immunoprecipitate of PC12 cells
stimulated by growth factors. The cells were grown to subconfluence,
serum-starved for 48 h, and stimulated for the indicated periods of
time with EGF (10 ngml
), basic FGF (10
ng
ml
), and NGF (100
ng
ml
). Lysates, prepared as described in the
text, were incubated with an anti-Shc polyclonal antibody and
precipitated with protein A-agarose. Immunoprecipitated material was
separated on 10% SDS-PAGE. The gel was fixed, dried, and exposed to
x-ray film. Positions of p46/p52
and p43 are indicated by
an arrow.
Figure 3:
Identification of the 43-kDa protein found
in Shc immunoprecipitates. GST-Shc fusion protein bound to
glutathione-agarose was used to precipitate p43 from a PC12 cell. The
bound material was eluted and separated by SDS-PAGE, blotted onto
Immobilon CD, and negatively stained. The p43 bands were excised,
digested with endoproteinase Lys-C (A), or sequentially
extracted with 30% acetonitrile, 2.5% trifluoroacetic acid (B)
or 60% acetonitrile, 2.5% trifluoroacetic acid (C). 10% of the
Lys-C digest and the acetonitrile extract were analyzed by MALDI-MS.
The three panels show reflectron spectra internally calibrated
with oxidized bovine insulin B chain. The numbers in square
brackets represent the Lys-C fragments of /
-actin
numbered from the amino terminus. Xtrct =
membrane-extractable.
Figure 4: Sequence information on one of the Lys-C fragments. Sequence of specific fragment ions were obtained from PSD-MALDI-MS analysis of the selected ion 1961.7 ({*}) from the sample shown in Fig. 3B. The fragment ions consistent with b series ions are labeled, and the b series ions expected from the given sequence are shown as well. The strong ion at m/z 124 represents the methylated histidine ammonium ion.
The presence of actin in Shc immunoprecipitates was
further analyzed by Western blots using an anti-actin monoclonal
antibody. As shown in Fig. 5, a 43-kDa band comigrating with
actin was found in the immunoprecipitates confirming the result from
mass spectrometry. In contrast to the metabolically labeled cells, the
amount of actin precipitated was almost identical for resting cells and
cells stimulated with NGF for 10 min and 4 h. These data suggested that
the increasing level of [S]methionine-labeled
actin associated with Shc in growth factor-stimulated PC12 cells (Fig. 2) was due to the induction of actin synthesis that occurs
following PC12 cell growth factor stimulation and especially when these
cells are committed to differentiate(56) .
Figure 5:
Confirmation by immunoblotting of the
presence of actin in Shc immunoprecipitate. To further assess the
result obtained by mass spectrometry, Shc was immunoprecipitated from
PC12 cells stimulated with 100 ngml
NGF for
the indicated periods of time. Blot of Shc-immunoprecipitated materials
was probed with an anti-actin monoclonal antibody. 50 µg of protein
from a PC12 cell total extract was run in
parallel.
Figure 6:
Cosedimentation experiments of actin and
GST-Shc and -truncated Shc fusion proteins. GST-Shc and -truncated Shc
fusion proteins were used in the sedimentation assay alone (GST-p46Shc* and GST-p52Shc*) or in the presence of
purified muscle G-actin. 10 µg of G-actin in a buffer containing 2
mM Tris-HCl (pH 7.4), 0.2 mM CaCl, 0.5
mM ATP were mixed with 2 µg of GST-p46
,
GST-p52
, GST-NCH, GST-CH, GST-SH2, GST, or a mixture of
purified proteins containing
-galactosidase (116 kDa) plus bovine
serum albumin (66 kDa) plus carbonic anhydrase (29 kDa) and lysozyme
(14.3 kDa). Polymerization of G-actin was induced by adding 2 mM MgCl
and 75 mM KCl. After 1 h at room
temperature, F-actin was pelleted by centrifugation at 100,000
g
1 h. The pellet (P) and the concentrated
supernatant (S) were separated by SDS-PAGE and stained with
Coomassie Brilliant Blue. Lanes T correspond to the starting
material. When GST-CH was used, proteins contained in each fraction
were analyzed in Western blot with an anti-GST monoclonal antibody and
the enhanced chemiluminescence detection system. Note that the film was
overexposed to ensure that GST-CH fusion protein was absent from the
pellet. The double arrow indicates the position
GST-p46
and GST-p52
. The arrow marked with an A indicates the position of
actin.
Because GST-Shc was found in the supernatant with unpolymerized
actin, the possibility that Shc also binds G-actin was considered. In
order to determine whether Shc could bind G-actin, agarose bound
GST-p46, GST-p52
, and GST-NCH fusion
proteins were incubated with G-actin. Agarose beads were then washed
twice with or without 1% Triton X-100, and proteins were separated by
SDS-PAGE. As shown in Fig. 7, after Coomassie Brilliant Blue
staining of the gel, no 43-kDa protein corresponding to actin could be
detected in association with GST-Shc fusion proteins or GST alone even
when washes were performed without Triton X-100. This result indicates
that in our sedimentation assay, Shc did not bind to unpolymerized
actin and that the fact it was found in the supernatant after actin
polymerization and sedimentation was probably due to incomplete actin
polymerization and/or nonoptimal experimental conditions. This result
also indicates that the actin detected in Shc immunoprecipitates ( Fig. 2and Fig. 3) was bound to Shc as F-actin and not as
G-actin.
Figure 7:
Shc/G-actin binding assay. Shc binding to
unpolymerized G-actin was assessed by incubating 2 µg of
agarose-bound GST-Shc fusion protein with 10 µg of G-actin in the
same buffer as for the cosedimentation assay except that KCl and
MgCl were omitted. After 2 washes in the same buffer with
(+) or without(-) 1% Triton X-100, proteins were separated
on SDS-PAGE and stained with Coomassie Brilliant Blue. The A lane and the arrow correspond to the position of
actin.
Figure 8:
Subcellular distribution of Shc in PC12
cells upon NGF stimulation. PC12 cells treated with NGF (100
ngml
) for the indicated times were lysed and
sequentially extracted in 1% Triton X-100 and/or 0.5 M NaCl
plus 0.3% deoxycholic acid to obtain Triton X-100-soluble proteins (TS), the cytoskeleton (Triton X-100-insoluble proteins, TI), and the submembranous cytoskeleton (SM). Shc was
immunoprecipitated as described in the text and analyzed on Western
blots using anti-Shc (Transduction Laboratories) and the 4G10
anti-phosphotyrosine monoclonal antibodies. The immunoblots were
analyzed by peroxidase-conjugated antibodies and enhanced
chemiluminescence. Cytochalasin D was added to the culture medium 2 h
prior to NGF stimulation at a final concentration of 2 µM.
Data are representative of 3 independent
experiments.
Because Shc acts as an actin-binding protein and is transiently translocated to the cytoskeleton upon growth factor stimulation, its redistribution in cells with a disrupted actin cytoskeleton was examined. For this purpose, resting PC12 cells were treated with 2 µM cytochalasin D for 2 h prior to growth factor stimulation and Shc distribution as well as tyrosine phosphorylation were analyzed as above. Fig. 8(right panel) shows that, in resting cells, Shc was found in the soluble (TS) and the submembranous (SM) fractions but was not detectable in the cytoskeletal fraction (TI) even after a longer exposure (data not shown). Upon NGF stimulation, Shc concentration in the soluble fraction remained unchanged. However, the kinetics and amount of Shc tyrosine phosphorylation were not significantly affected by cytochalasin D treatment. In the cytoskeletal fraction, Shc was not detectable even after 2 min of NGF stimulation, indicating that Shc could not translocate in cytochalasin D-treated cells. In the submembranous fraction of cytochalasin D-treated cells, Shc was found present at a level comparable to the soluble fraction. The concentration of Shc in this fraction did not change following NGF stimulation. Although Shc tyrosine phosphorylation occurred, it was weak compared with the amount of Shc present. This result indicates that growth factor-induced Shc translocation depends on the integrity of the actin cytoskeleton.
Shc is a member of a diverse group of adaptor molecules that link many tyrosine kinase-containing receptors to downstream signaling entities. In PC12 cells, it is rapidly bound to Tyr-490 of the TrkA receptor following NGF stimulation and, following phosphorylation, provides the assembly site for the formation of the ``activated'' Ras complex that is necessary for the activation of MAPK cascades and their translocation to the nucleus(2, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . Recent evidence has established that the interaction with TrkA is through the amino-terminal region rather than the SH2 domain located in the carboxyl-terminal domain of the molecule(30, 35) .
The observation that Shc can also bind to filamentous actin and that this interaction is rapidly stimulated by growth factors suggest a new role for this adaptor. As with the receptor interaction, binding occurs through the amino-terminal domain. However, it does not interact with phosphotyrosine residues on actin and does not require self-phosphorylation. This suggests that the binding to receptor and actin would be eventually exclusive and that the cytoskeletal bound Shc is also able to bind additional phosphotyrosine-containing proteins through its SH2 domain as is presumably the case for the receptor-bound form(30, 31, 35) . Although we have demonstrated that in vitro Shc can bind directly to F-actin, the possibility that in vivo Shc could also associate indirectly with the actin cytoskeleton cannot be ruled out.
The
large increase in actin associated with Shc detected after 4 h of NGF
and FGF stimulation in PC12 cells appears to be a function of the long
term response of these cells to produce neurites. EGF-treated cells do
not show this response and do not go on to produce neurites. However,
this peak in apparent actin association appears to be due primarily to
an increase in synthesis (determined by an increase in
[S]methionine labeling) as judged by the
concentration of actin detected by Western blot (Fig. 5). The
rapid mobilization (within 2 min) of soluble Shc to cytoskeleton
appears to represent the early growth factor response. Although Shc
translocation was observed when PC12 cells were stimulated with NGF, we
do not think that this phenomenon reflects any NGF-specific effect. The
reason we did not perform our experiments with EGF is that the EGF
receptor is an actin-binding protein and that an important percentage
of the total EGF receptor population is linked to the actin
cytoskeleton(52) . Therefore, we think it would not have been
easy to discriminate Shc translocated to the cytoskeleton from Shc
bound to the activated EGF receptor present in the different
cytoskeletal preparations.
Shc redistribution upon growth factor stimulation has previously been described in cells stimulated by interleukin 3 (11) or PDGF (57) where phosphorylated Shc was found in membrane preparations supposedly in association with activated receptors. Our findings suggest that a part of Shc found in membrane preparations could be associated with the actin cytoskeleton.
What induces Shc translocation? It is interesting to note that the
level of tyrosine-phosphorylated Shc in the cytoskeletal fractions
parallels the amount of Shc that translocates to these compartments.
This could suggest that tyrosine phosphorylation of Shc increases its
affinity for F-actin and consequently induces its translocation to the
actin cytoskeleton. However, if true, a decrease in the amount of
tyrosine-phosphorylated molecules should also have been detected in the
soluble fraction or at least there should have been an accumulation of
tyrosine-phosphorylated Shc in the cytoskeleton. Instead, the amount of
tyrosine-phosphorylated Shc in the soluble fraction increases
continuously up to 10 min of NGF stimulation while the translocation
phenomena is transitory. Therefore, it seems more likely that Shc
translocation is independent of Shc phosphorylation as the in vitro actin cosedimentation assay suggested. One hypothesis is that
there are two different Shc subpopulations that are independently
phosphorylated and that another early intracellular event which follows
TrkA activation and is independent of Shc binding to the receptor and
of its tyrosine phosphorylation, is responsible for Shc translocation.
It is tempting to suggest that the rapid and transitory intracellular
calcium influx that follows the activation of a growth factor receptor (58, 59, 60, 61, 62) could
be responsible for Shc translocation. Consistent with this view,
phospholipase C activation, leading to the formation of the second
messengers diacylglycerol and inositol triphosphate, which in turn
activate protein kinase C and mobilize intracellular calcium,
respectively(59, 60) , could modify Shc affinity for
F-actin, resulting in the translocation of a Shc subpopulation
(phosphorylated or not) to the actin cytoskeleton. Once Shc has been
translocated, it could then be phosphorylated in situ by a
cytoskeletal associated non-receptor tyrosine kinase. Such a
non-receptor tyrosine kinase could be represented by the Src family of
tyrosine kinases, c-Abl or Syk, which are known to associate and/or to
translocate to the cytoskeleton under some circumstances, and Shc is
known to be a substrate for them (17, 41, 63, 64, 65, 66, 67, 68) .
What is the function of Shc translocation? It is now well established that Shc phosphorylation by the activated receptor tyrosine kinase is the first step of a cascade of events that leads eventually to MAPK activation(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . As shown here, Shc translocates to two different cytoskeletal fractions and may become phosphorylated on tyrosine by other non-receptor tyrosine kinases present on the cytoskeleton. Thus, in addition to soluble Shc, it is possible that the bound Shc also functions in initiating the activation of the Ras/MAPK pathways. In support of this idea, Grb-2 is found in Shc immunoprecipitates at a level that correlates with the amount of phosphorylated Shc (data not shown). However, the kinetics of MAPK activation, which coincides in PC12 cells with the level of tyrosine-phosphorylated Shc found in the soluble fraction (data not shown), argues against this interpretation. MAPK activation reaches a maximum 10 min after NGF stimulation and remains at a high level up to 1 h. In contrast, in the cytoskeletal fraction, phosphorylated Shc concentration is maximal after 2 min following NGF stimulation and decreases quickly thereafter. This difference may in fact reveal the involvement of cytoskeleton-associated Shc in other functions.
Shc is an adapter protein which has been found recently to associate with several identified(37, 38, 39, 40, 41, 42) and unidentified phosphotyrosine-containing proteins(10, 36, 43) . In addition, Shc has several potentially phosphorylatable tyrosine residues that could be targets for Grb-like molecules and is, in some circumstances, phosphorylated on serine residues(17, 69) . Thus, Shc is clearly complex, and it is likely that it can initiate pathways other than the Ras/MAPK pathway, occurring at different times and in different locations inside the cell. One such role is the reorganization of actin stress fibers and membrane ruffling that occur in response to growth factor stimulation. Both phenomena are under the control of the small GTP-binding proteins, Rho (70) and Rac(71) , respectively. Recently, Grb-2 has been linked to the formation of membrane ruffles probably by modulating Ras and Rac signaling pathways(72) . In addition, it has been reported that a GST-Grb-2 fusion protein, microinjected into cells, localizes to membrane ruffles(73) . Although this localization seems to be mediated by the Grb-2 SH3 domain, the presence of tyrosine-phosphorylated Shc in the membrane ruffles may account in part for the Grb-2 co-localization. Therefore, Shc may also participate in membrane ruffling and the early actin reorganization following growth factor stimulation by coupling Grb-2 and small GTP-binding proteins to the actin cytoskeleton.
A direct role for Shc in the regulation of actin polymerization should also be considered. Shc does not have any known catalytic activity, and, therefore, it is unlikely to be directly involved in the regulation of actin polymerization or the reorganization of the cytoskeleton that follows growth factor stimulation(74, 75, 76, 77) . However, the possibility that Shc binding to the actin-cytoskeleton could regulate actin polymerization by competing with agents like gelsolin, which are inhibitors of actin polymerization, is a possibility since this function has been envisaged, for example, for the non-receptor tyrosine kinase, c-Abl, another actin-binding molecule(66) .
Many other proteins involved in the signal
transduction of growth factors can also be found associated with the
cytoskeleton. Grb-2 has been localized specifically with the membrane
ruffles(73) , and phospholipase C, phosphatidylinositol
3-kinase, c-Src, and c-Abl whose enzymatic activities or presence have
been detected in the cytoskeletal fraction in various
situations(63, 64, 65, 66, 73, 78, 79, 80) .
Interestingly, all these proteins contain an SH3 domain, which plays an
important role for subcellular compartmentalization and cytoskeletal
localization as has been shown for Grb-2 and phospholipase
C
(73, 81) . However, this is not always true. The
catalytic and the SH2 domains of v-Src are required for its binding to
the cytoskeleton(65) , and c-Abl contains a specific F-actin
binding sequence(66) . In this study, Shc has been shown to
bind F-actin directly through its amino-terminal domain in the absence
of any other protein. Experiments are now in progress to determine more
precisely the F-actin binding of Shc amino terminus.