(Received for publication, August 7, 1995; and in revised form, November 17, 1995)
From the
The role of Shc as a substrate of receptors for growth factors
and cytokines is well established. To gain further insight into the
function of Shc in signal transduction, we used an affinity method to
identify potential Shc-binding proteins. Incubation of bovine brain
lysates with a glutathione S-transferase (GST)-Shc fusion
protein immobilized on glutathione-Sepharose beads resulted in the
binding of cellular proteins of 115, 110, and 100 kDa as well as
those of 50 and 17 kDa. Amino acid sequencing of tryptic peptides
revealed that the 100-kDa protein was almost identical to
-adaptin
and that the 110- and 115-kDa proteins were almost identical to
-adaptin. Using immunoblot analysis,
anti-
-adaptin antibody recognized several proteins of 100
115
kDa, and anti-
-adaptin antibody recognized a 100-kDa protein,
suggesting that
-,
-, and
-adaptins are bound to the GST-Shc fusion protein. Immunoblot
analysis with anti-
-adaptin antibody revealed that
-adaptin
was coimmunoprecipitated with Shc from PC12, KB, and COS cell lysates,
suggesting a specific interaction of Shc and adaptins in intact cells.
A binding study using mutant GST-Shc fusion proteins revealed that the
collagen homologous region (amino acids 233-377) of Shc was
required for adaptin binding. Conversely, the collagen homologous
region of Shc inhibited the binding of adaptins to GST-Shc. In
addition, adaptin was able to bind mutant fusion proteins containing
amino acids 233-369, 233-355, 346-369, and
346-355 of Shc, but failed to bind a mutant containing amino
acids 233-345, suggesting that amino acids 346-355
(RDLFDMKPFE) in the collagen homologous region of Shc are required for
adaptin binding. Thus, this study indicates the specific interaction of
Shc with
- and
-adaptin components of plasma membrane adaptor
proteins that are thought to be involved in receptor endocytosis.
Many receptor tyrosine kinases such as those for epidermal
growth factor (EGF), ()platelet-derived growth factor, and
insulin transmit intracellular signals for cell proliferation and
differentiation(1) . The binding of ligands and the subsequent
conformational alteration of the extracellular domain induce receptor
oligomerization, which results in elevated protein-tyrosine kinase
activity of the receptor and leads to increased autophosphorylation and
intracellular substrate phosphorylation(1, 2) .
Receptor autophosphorylation is an important structural feature of the
association of the receptor with proteins containing Src homology 2
(SH2) domains(1, 3, 4) . A number of proteins
that contain SH2 domains have been identified, including phospholipase
C-
1, Ras GTPase-activating protein, the 85-kDa regulatory subunit
of phosphatidylinositol 3-kinase, Grb2, and Shc(5) . Many of
these proteins also contain one or more SH3 domains, which specifically
interact with proline-rich sequences, thus serving as linker molecules
to bring other proteins into the signaling complex(5) .
Shc
protein consists of three overlapping polypeptides of 46, 52, and
66 kDa. Shc proteins of 46 and 52 kDa encoded by a 3.4-kilobase mRNA
are ubiquitously expressed, whereas a 66-kDa Shc protein is likely to
be encoded by a distinct shc transcript and is absent in some
hematopoietic cells(6) . Shc is composed of a single SH2 domain
at the C terminus, an adjacent glycine/proline-rich region that is 50%
homologous to human
1-collagen, and a distinct N-terminal
phosphotyrosine-binding domain (PTB domain)(6, 7) .
Although Shc lacks apparent catalytic activity, overexpression of Shc
protein induces malignant transformation in 3T3 cells (6) and
neurite outgrowth in PC12 cells(8) . Shc has been shown to be
involved in Ras activation by a number of receptors for growth factors
as well as cytokines(9, 10, 11) . Shc is
phosphorylated on tyrosine upon stimulation of these receptors (8) and subsequently interacts with Grb2, which forms a complex
with Sos, a Ras guanine nucleotide exchange protein(12) . Shc
is also a good substrate for Src family kinases, and in v-Src- and
v-Fps-expressing cells, Shc is constitutively tyrosine-phosphorylated
and forms a complex with Grb2(13) . Thus, tyrosine 317 within
the collagen homologous region of Shc is suggested to be the binding
site of Grb2 (6) , and Shc interacts with
tyrosine-phosphorylated growth factor receptors such as EGF receptors,
TrkA, and ErbB-2(14, 15, 16) . Recent reports
have shown that the N-terminal PTB domain as well as the SH2 domain
interact with tyrosine-phosphorylated growth factor receptors (7, 17, 18) , suggesting the assembly of
different signaling complexes. Thus, Shc seems to have multiple
functions in signal transduction. The role of the collagen homologous
domain remains to be defined. To gain further insight into the function
of Shc in signal transduction, we tried to identify Shc-binding
proteins using glutathione S-transferase (GST)-Shc fusion
proteins.
Bacterial cultures expressing pGEX-Shc were
grown in Luria-Bertani medium containing 50 µg/ml ampicillin, and
the expression of fusion protein was induced by adding
isopropyl--D-thiogalactopyranoside to a final
concentration of 0.25 mM. After a 4-h incubation at 37 °C
or a 16-h incubation at 25 °C, induced bacteria were lysed by
sonication in 50 mM Tris-HCl (pH 7.4) containing 1% Triton
X-100, 1% Tween 20, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin.
Glutathione-Sepharose beads preloaded with GST-Shc fusion protein
were incubated with bovine lysates for 2 h at 4 °C. After extensive
washing, proteins were released from the beads by boiling in SDS sample
buffer. Samples were subjected to SDS-PAGE, transferred onto
nitrocellulose, and blotted with antibodies to - and
-adaptins as described above.
Figure 1:
Identification of GST-Shc-binding
proteins from bovine brain lysates and amino acid sequencing of these
proteins. A, proteins bound to GST-Shc-(4-473) fusion
protein. Bovine brain lysates were incubated with GST or
GST-Shc-(4-473) coupled to glutathione-Sepharose beads. The beads
were washed extensively, and associated proteins were released by
boiling in SDS sample buffer. Released proteins were resolved by
SDS-PAGE on a 6% gel and visualized by silver stain. B,
alignment of tryptic peptides of 115, 110, and 100-kDa proteins
with
- and
-adaptin sequences. Peptide sequences obtained
from lysyl endoproteinase digestion (FR) are indicated in boldface above the appropriate sequence of adaptins. Tryptic
peptides of p115 and p110 are aligned with mouse
- and
-adaptins(31) , and those of p100 are aligned
with human
-adaptin(27) . Dashes indicate
identities to
-adaptin.
To determine the identity of the binding
proteins, these proteins were purified in quantities sufficient for
protein sequencing by high resolution methods(20) . Bovine
brain lysates (1 ml) were incubated with 20 µg of GST-Shc
immobilized on glutathione-Sepharose. After washing, proteins bound to
the resin were subjected to SDS-PAGE and visualized by Coomassie stain.
The bands of Shc-binding proteins were excised and stored at -20
°C. This was repeated until the amounts of Shc-binding proteins
reached
5 µg. Affinity-purified Shc-binding proteins were then
digested in the gel with lysyl endoproteinase. Peptides were resolved
by high pressure liquid chromatography and subjected to protein
sequence determination. Amino acid sequencing of the fragments of the
100-kDa protein yielded the sequences KEYATEVDVDFVRK, KMERQVVLRTDK,
KGLEISGTFTHRQGHFYMEMN, KLVYLYLVNYAK, and KNSFGVIPSTPLAI. Comparison
with the sequences in the GenBank
Data Bank revealed
almost complete identity to rat and human
-adaptins, a component
of the plasma membrane adaptor complex of coated vesicles (Fig. 1B) (22, 23) . Amino acid
sequencing of the fragments of the 110-kDa protein yielded the
sequences KSEFRQNLGRMYLFYGNK, KTSVQFQNFSPTVVHPGDL, and
KYLQVGHLLREPNAQAQMYRLTLRTK, and that of the 115-kDa protein yielded
KTSVQFQNFSPTVVHPGDL. These amino acid sequences were almost identical
to the mouse
-adaptin, but not
-adaptin, of the adaptor complex (Fig. 1B) (24) . These results indicate that
- and
-adaptins bind to Shc expressed as fusion
protein with GST.
The Shc binding activity of adaptins was confirmed
by immunoblot analysis with specific antibodies to - and
-adaptins. Anti-
-adaptin antibody (AC1-M11) recognizes both
- and
-adaptins, but not
-adaptin. Anti-
-adaptin antibody (B1-M6) has no
cross-reactivity to
-adaptins(21) . Affinity-purified
Shc-binding proteins were subjected to SDS-PAGE, transferred onto
nitrocellulose, and blotted with antibodies to
- and
-adaptins. Antibody to
-adaptin recognized several proteins
of 100
115 kDa that probably correspond to
-,
-,
-, and
-subunits (data not shown). Anti-
-adaptin
antibody recognized a single protein with an apparent molecular mass of
100 kDa (data not shown). Nonimmunized mouse globulin did not
recognize these bands. These results indicate that Shc-binding proteins
include the
-,
-, and
-adaptins
of the adaptor complex.
Figure 2:
Association of adaptins with Shc in intact
cells. PC12, KB, and COS cells untreated or treated with 10 nM EGF for 2 min were lysed in 0.5 M Tris-HCl (pH 7.4)
containing 0.5% Triton X-100 and protease inhibitors. Clarified lysates
were adjusted to 50 mM Tris-HCl (pH 7.4) containing 0.5%
Triton X-100, 100 mM NaCl, and protease inhibitors and then
incubated with anti-Shc antibody coupled to protein A-Sepharose.
Immunoprecipitates (IP) were subjected to SDS-PAGE,
transferred onto nitrocellulose, and immunoblotted (IB) with
anti--adaptin antibody. Lysates equal to 2% of that used for
immunoprecipitation were also electrophoresed and blotted with
anti-
-adaptin antibody. Arrowheads indicate
-adaptin. NRS, nonimmunized rabbit
serum.
Figure 3: Association of adaptins with mutant GST-Shc proteins. Various GST-Shc fusion proteins corresponding to the indicated residues were immobilized on glutathione-Sepharose beads and incubated with bovine brain lysates for 2 h at 4 °C. Associated proteins were released in SDS sample buffer, resolved by SDS-PAGE, and stained with silver. Gly/Pro, glycine/proline-rich collagen homologous domain.
Since -
and
-adaptins are components of the plasma membrane adaptor
complex of clathrin-coated vesicles and the adaptor complex also
contains two other subunits of
50 and 17 kDa, we then investigated
whether two other subunits could be identified in affinity-purified
proteins with GST-Shc-(4-473). When affinity-purified proteins
were released by boiling in SDS sample buffer or by elution with 1 M Tris-HCl (pH 7.4), proteins with lower molecular masses of
50, 47, 45, 35, 19, and 17 kDa as well as
115, 110, and 100
kDa were identified (Fig. 4, B and C). Among
these lower molecular mass proteins, proteins of 47, 45, and 35 kDa
bound to GST (Fig. 4A), indicating that proteins of
50, 19, and 17 kDa as well as those of 115, 110, and 100 kDa
specifically bind to Shc. Association of these proteins with
GST-Shc-(4-473) was inhibited by the collagen homologous domain
of Shc-(233-369) prepared by thrombin digestion of the GST fusion
protein (Fig. 4, B and C). In contrast,
Shc-(233-345) failed to inhibit the association of these proteins
with GST-Shc-(4-473) (Fig. 4D). These results
support the finding that the collagen homologous region of Shc is a
binding site for adaptins and also suggest that adaptins bind to Shc as
a plasma membrane adaptor complex.
Figure 4: Association of proteins from bovine brain lysates with GST-Shc fusion protein and inhibition of binding by the collagen homologous region of Shc. A, GST or GST-Shc-(4-473) fusion protein immobilized on glutathione-Sepharose beads was incubated with or without bovine brain lysates for 2 h at 4 °C. After extensive washing, associated proteins were released by elution with 1 M Tris-HCl (pH 7.4). B-D, brain lysates were first incubated with or without the collagen homologous region of Shc prepared by thrombin digestion of GST-Shc-(233-369) or GST-Shc-(233-345) and then added to GST-Shc-(4-473) immobilized on glutathione-Sepharose beads. Associated proteins were released either by boiling in SDS sample buffer (B) or by elution with 1 M Tris-HCl (pH 7.4) (C and D). Released proteins were resolved by SDS-PAGE and stained with silver.
An affinity chromatography approach was used to identify
Shc-binding proteins from bovine brain lysates. This resulted in the
identification of three major proteins of 115, 110, and 100 kDa
and several bands of lower molecular mass including
50 and 17 kDa.
Microsequencing of 100
115-kDa proteins showed them to be almost
identical to the previously characterized proteins
-
and
-adaptins. These adaptins are components of adaptor proteins,
also referred to as assembly proteins or clathrin-associated proteins,
which anchor the clathrin lattice on the surface of coated pits and
coated vesicles(25, 26) . Two structurally related
classes of adaptor proteins are present. Plasma membrane adaptor
HA2-AP2 consists of
- and
-adaptins and two smaller subunits
of 50 and 17 kDa, and Golgi adaptor HA1-AP1 consists of
- and
`-adaptins and two smaller subunits of 47 and 19
kDa(27, 28) . In addition, there are two isoforms of
-adaptin (
and
) encoded by
distinct but highly homologous genes(24) . Two alternative
transcripts of
-adaptin have been identified: one is
expressed in brain(24) , and a smaller isoform is expressed
ubiquitously(29) . The smaller isoform of
-adaptin migrates on SDS-polyacrylamide gels with a
mobility similar to that of
-adaptin, which is also
expressed ubiquitously(24) . In the present study, amino acid
sequencing revealed that the 100-kDa protein is
-adaptin, whereas
immunoblot analysis with antibodies to
- and
-adaptins
disclosed that the 100-kDa protein consists of
- and
-adaptins. The anti-
-adaptin antibody recognizes both
- and
-adaptins, but not the
-subunit(21) . Both isoforms of
-adaptin are present
in brain, and a smaller isoform of
- and
-adaptins migrates very closely to
-adaptin on
SDS-PAGE. In addition, we could detect the association of
-adaptin
with Shc in intact cells derived from tissues other than those of
neural origin such as human epidermoid carcinoma KB cells or monkey
kidney COS cells. These findings suggest that
-,
-, and
-adaptins bind to Shc. The reason why we
failed to detect peptide fragments of a smaller isoform of
- and
-adaptins is probably due to
the relatively low amount of these isoforms. Both
- and
-adaptins are present in cells as an HA2-AP2 complex with two
smaller subunits. The analysis on gradient gels showed the association
of 50- and 17-kDa proteins with Shc. Therefore, it seems likely that
the HA2-AP2 complex binds to Shc.
HA2-AP2 is implicated in functions
essential for the dynamic cycle of clathrin-coated pits and vesicles.
Sorting of membrane proteins and receptors into clathrin-coated
vesicles is thought to require recognition of their cytoplasmic domains
by adaptors. In vitro binding of adaptors to the cytoplasmic
domain of transmembrane proteins such as low density lipoprotein,
cation-independent mannose 6-phosphate/insulin-like growth factor II,
asialoglycoprotein receptors, and lysosomal acid phosphatase has been
demonstrated(30, 31, 32, 33) .
Furthermore, structural analysis of protein sequence motifs around tail
residues shown to be critical for rapid internalization has indicated
that the signal for clathrin-mediated internalization contains a
tyrosine residue, which is exposed in a -turn conformation. The
amino acid sequences required for rapid internalization of transferrin,
cation-independent mannose 6-phosphate/insulin-like growth factor II,
cation-dependent mannose 6-phosphate, asialoglycoprotein receptors, and
lysosomal acid phosphatase are YTDL, YSKV, YRGV, YQDL, and YRHV,
respectively, where the tyrosine residue has been shown to be critical
for rapid internalization (reviewed in (34) ). An in vitro study of lysosomal acid phosphatase has shown that the HA2-AP2
adaptor fails to bind the peptide derived from the cytoplasmic domain
of lysosomal acid phosphatase in which the tyrosine residue has been
changed to alanine (33) , suggesting that the tyrosine residue
is required for HA2-AP2 binding. Another group of internalization
recognition sequences is seen in the cytoplasmic domains of low density
lipoprotein (FDNPVY) and cation-dependent mannose 6-phosphate (FPHLAF)
receptors (reviewed in (34) ). Mutagenesis analysis of these
receptors revealed that both the phenylalanine and the tyrosine
residues of the low density lipoprotein receptor and both phenylalanine
residues of the cation-dependent mannose 6-phosphate receptor are
required for rapid internalization(35, 36) ,
suggesting that the complete internalization signal spans a 6-amino
acid region with critical aromatic residues at positions 1 and 6.
However, there is no direct evidence indicating that this region is an
HA2-AP2-binding site. In the present study, we have shown that amino
acids 346-355 of Shc are required for adaptin binding. The amino
acid sequence of this region of Shc is RDLFDMKPFE, which contains a
6-amino acid segment with aromatic residues on both sides. It is
noteworthy that the adaptin-binding site of Shc contains an amino acid
sequence motif similar to that which has been shown to be required for
rapid internalization of membrane proteins.
Recent progress in
protein-protein interaction studies has revealed the specificity of
binding by certain modular domains. SH2 domains specifically interact
with phosphorylated tyrosines, and SH3 domains with proline-rich
sequences(5) . For example, with respect to EGF receptors, the
SH2-containing proteins phospholipase C-1, Shc, and Grb2 directly
bind to phosphorylated tyrosine residues of EGF receptors (14, 37, 38) . Shc becomes
tyrosine-phosphorylated after EGF receptor activation, and the SH2
domain of Grb2 binds to EGF receptors indirectly via the phosphorylated
tyrosine 317 of Shc. Grb2 possesses two SH3 domains, which bind to
proline-rich sequences of Sos. Recently, the SH3 domains of Grb2 and
phospholipase C-
1 were found to bind proteins other than Sos, such
as dynamin, synapsin I, and 145-kDa
protein(39, 40, 41) . Dynamin has extensive
similarity to the product of the Drosophila gene shibire(42) and possesses intrinsic GTPase activity. It has been
demonstrated that point mutations in the GTP-binding domain of human
dynamin specifically inhibit early events in receptor-mediated
endocytosis(43) . In fact, mammalian dynamin was recently shown
to have a role in clathrin-coated vesicle
function(44, 45) . In NIH/3T3 cells, phospholipase
C-
1 associates with dynamin, and dynamin associates with
platelet-derived growth factor receptors in a platelet-derived growth
factor-dependent manner(41) , suggesting a role for dynamin in
ligand-induced receptor endocytosis. In addition, adaptins associate
with the C-terminal tail of EGF receptors in an EGF-dependent
manner(46) , (
)although adaptins constitutively
associate with Shc. Thus, it is interesting that components implicated
in receptor endocytosis, dynamin and HA2-AP2, associate with substrates
of receptors for growth factors and cytokines.
Recently, novel types of association of Shc with other proteins have been reported. Shc was shown to associate with the PEST tyrosine phosphatase(47) . Two serine residues at positions 5 and 29 in the N-terminal 45-amino acid region of 52-kDa Shc are suggested to be a binding site for the PEST tyrosine phosphatase(47) . Shc has also been shown to bind a tyrosine-phosphorylated protein of 145 kDa in Balb/3T3 cells and L6 myoblasts via the PTB domain(7) . Using mutant GST-Shc fusion proteins, the N-terminal region of Shc-(46-232) was shown to be responsible for the binding of tyrosine-phosphorylated p145. In addition, Shc-(46-209) binds to tyrosine-phosphorylated growth factor receptors such as EGF receptors and TrkA(17) . In the present study, we have shown that the collagen homologous region of Shc (amino acids 346-355) is implicated in the association with the HA2-AP2 complex. From our data, it is unclear which subunit of the HA2-AP2 complex binds to Shc. In addition, the role of a Shc-HA2-AP2 complex in signal transduction remains unclear. One possibility is that Shc may play a role in ligand-induced receptor internalization since HA2-AP2 complexes are implicated in receptor endocytosis. However, it is possible that this complex plays some other role in signal transduction. It will be important to answer this issue.