(Received for publication, August 22, 1995; and in revised form, October 5, 1995)
From the
The tyrosines in the cytoplasmic domain of an oncogenic human
insulin-like growth factor I receptor (gag-IGFR) were systematically
mutated to phenylalanines to investigate the role of those tyrosines in
the enzymatic and biological function of the gag-IGFR. Our results
indicate that tyrosines 1131, 1135, 1136, and 1221 are important for
the receptor protein-tyrosine kinase (PTK) activity. However, mutation
of Tyr-1136 only slightly affects the kinase activity but dramatically
reduces the transforming ability and overall substrate phosphorylation,
in particular, annexin II, which is strongly phosphorylated by the
gag-IGFR but not by the Phe-1136 mutant. Single mutation of either
Tyr-943 or Tyr-950 resulted in significantly reduced phosphorylation of
the receptor but not on its PTK activity or transforming ability.
Tyr-950 together with its surrounding sequence is involved in mediating
the interaction between the gag-IGFR and insulin receptor substrate 1.
Our data also suggest that Tyr-1316 is involved in phosphorylation of
phospholipase C-, which is, however, not important for cell
transforming activity. Overall, our study has identified several
tyrosine residues of IGFR important for its PTK activity and substrate
interaction. The transforming potential of the gag-IGFR correlates well
with its ability to phosphorylate overall cellular substrates and to
activate phosphatidylinositol 3-kinase via insulin receptor substrate
1.
Receptor protein-tyrosine kinases (RPTKs) ()are
transmembrane glycoproteins with intrinsic protein kinase
activity(1, 2) . Ligand binding to an RPTK results in
its oligomerization, PTK activation, autophosphorylation, and
phosphorylation of cellular substrates leading to gene activation, DNA
synthesis, and eventual cell proliferation or differentiation.
Interactions between RPTKs and cellular substrates are mediated by
receptor tyrosine autophosphorylation(3) . Aside from
activating the kinase activity, phosphorylation of tyrosine residues
also provides binding sites for Src homology 2-containing signaling
proteins such as Grb2, Shc, Nck, PI 3-kinase p85 regulatory subunit,
GAP, PLC
, and Src family kinases(4, 5) . Mutation
of tyrosine residues in those binding sites often leads to a loss of
interaction with their corresponding substrates. Alternatively,
receptor-substrate interactions can be mediated through a recently
characterized phosphotyrosine binding domain interacting with a
NPXY motif (6) . IRS1, the major phosphorylation
substrate of insulin receptor, lacks a Src homology 2 domain but
interacts with insulin receptor at tyrosine 972 via such a
sequence(7, 8, 9) . It has also been shown
that Shc is phosphorylated in response to insulin stimulation and can
also interact with insulin receptor at tyrosine 972 through its
phoshpotyrosine binding domain(7, 10) .
The human insulin-like growth factor I receptor (IGFR) is an RPTK closely related to insulin receptor (IR). IGFR and IR share an 84% amino acid sequence identity in their kinase domains; however, their biological functions differ somewhat(11) . Cells treated with insulin rapidly increase glucose uptake, and lipid and glycogen synthesis but only increase DNA synthesis after a prolonged stimulation. IGF-I, however, appears to be a more potent stimulator of DNA synthesis and cell growth(12) . IGFR has been reported to be overexpressed in human breast cancers(13) . Dominant negative mutants and antisense mRNAs of IGFR can inhibit the growth of tumor cells and tumor formation in nude mice(14, 15) . Transfection of EGFR into IGFR knockout mouse cell lines cannot induce EGF-dependent cell transformation, while transfection of an exogenous IGFR can rescue EGF-induced cell transformation, suggesting that IGFR is required for EGFR-mediated transformation(16) . Collectively, they suggest that overexpression or constitutive activation of IGFR may play some role in cell transformation and tumorigenicity.
Our previous studies
on IGFR have shown that the cell-transforming activity of native IGFR
was significantly enhanced in an amino terminus truncated receptor,
coding for 36 amino acids of the extracellular domain, the entire
transmembrane, and cytoplasmic domains of IGFR subunit, fused to
the avian sarcoma virus UR2 gag sequence(17) . The gag-IGFR
encoding virus called UIGFR has an enhanced transforming potential over
that of native IGFR in cultured CEF but is not tumorigenic in
vivo. The gag-IGFR is a dimerized transmembrane
protein(17) . Further examination of this extracellular
36-amino acid sequences revealed that it has a negative modulating
effect on IGFR transforming and tumorigenic potential(18) .
Deletion of the entire 36 amino acids resulted in a strong transforming
and tumorigenic mutant called NM1(18) . Similar studies were
done on the carboxyl terminus of IGFR by deleting the carboxyl terminus
of the UIGFR fusion receptor(19) . A deletion of 27 amino acids
from the carboxyl terminus of the receptor, including a potential PI
3-kinase binding motif YXXM (amino acids 1316-1319),
resulted in a gag-IGFR that still retains its kinase activity and
transformation ability. Further deletion of 20 amino acids abolished
the kinase and transforming activities of the receptor. Surprisingly,
deletion of 67 amino acids restored both kinase activity and
transforming ability, and deletion of 88 amino acids abolished all the
activities. Overall, previous studies on the gag-IGFR indicate a strong
correlation between the receptor kinase activity and its transforming
ability. In this study, we further explored the mechanism of cell
transformation induced by this oncogenic gag-IGFR. Since
receptor-substrate interactions play important roles in RPTK signal
transduction and phosphorylated tyrosines on the receptor provide
binding sites for the substrates, tyrosine residues within the
cytoplasmic domain of NM1 gag-IGFR were systematically replaced by
phenylalanines. In addition, since IRS1 is a substrate of IGFR, a more
extensive study of tyrosine 950 and its neighboring sequence, the
presumed docking site for IRS1, was undertaken. Although substantial
studies on mutation of tyrosine residues in insulin receptor have been
carried out (see ``Discussion''), the role of tyrosine
residues in IGFR has largely been inferred from insulin receptor thus
far. Our study provides a direct systematic examination of the role of
tyrosine residues in an oncogenic form of IGFR.
The Tyr-950 mutation and the 13-amino acid deletion were generated by ligating two DNA fragments. A 5`-BglII site-containing oligonucleotide and a 3`-mutagenic oligonucleotide containing the Tyr-950 to Phe-950 mutation and a HgaI site were used with either pNM1 or pUIGFR as a template for polymerase chain reaction to generate the 5`-fragment. The 3`-DNA fragment used for both UF950 and Phe-950 was synthesized using a 5`-mutagenic oligonucleotide containing a HgaI site and a 3`-SphI site-containing oligonucleotide. The two fragments were digested with HgaI and ligated. The ligated product was used in a second round polymerase chain reaction with the BgllI and SphI oligonucleotides to amplify the ligated product. The final fragment was cloned back into pUIGFR or pNM1 using the BglII and SphI sites. The deletion mutant was constructed in a similar manner but using different mutagenic oligonucleotides containing a HgaI site and the sequence flanking the 13-amino acid deletion from tyrosine 943 to valine 956.
To prepare the polyclonal rabbit anti-IRS1 antibody, the 3`-sequence coding for rat carboxyl-terminal 271 amino acids. of IRS1 was cloned by polymerase chain reaction and inserted into a glutathione S-transferase bacterial expression vector. Glutathione S-transferase-IRS1 fusion protein was purified with glutathione-agarose beads followed by gel electrophoresis. 250 µg of purified protein was emulsified with an equal volume of complete Freund's adjuvant (Sigma) and used to immunize a New Zealand White rabbit (2.5 kg, female). For boost injections, 150 µg of protein was emulsified with an equal volume of incomplete Freund's adjuvant every 2 weeks. Bleeding was performed weekly starting 10 days after the second boost.
Figure 1: Schematic representation of UIGFR and NM1 chimeric receptors. The tyrosine to phenylalanine mutants are indicated by their amino acid positions(11) . The deletion in mutant d950 encompasses tyrosine 943 to valine 956. The CM2 mutant was reported previously(19) . Single-letter amino acid codes are used here and in subsequent figures. F3, Y1131F/Y1135F/Y1136F.
Figure 2: Anchorage-independent growth of transfected CEFs. Colony assays were set up 3 days after transfection, and the cultures were maintained at 41 °C. The pictures were taken 10 days after incubation. F3, Y1131F/Y1135F/Y1136F.
Figure 3:
In vitro kinase activity and
intracellular phosphorylation of mutant gag-IGFRs. Confluent
virus-infected cells were extracted with RIPA buffer, and equivalent
protein amounts of total lysates were used in each immunoprecipitation
using an anti-IGFR antiserum, anti-IB. Half of the immunoprecipitated
proteins was subjected to an in vitro kinase assay, while the
other half was Western blotted with anti-IB to monitor the amount of
IGFR. A parallel culture was treated with 200 µM
NaVO
for 4 h and then lysed and
immunoprecipitated similarly. The intracellular phosphorylation of the
receptor was determined by blotting with an anti-phosphotyrosine
antibody, RC20, coupled with alkaline phosphatase (panels a, b, and e). Alternatively, cells were labeled with
[
S]methionine before lysis, and the relative
amounts of gag-IGFR protein were determined by immunoprecipitation, gel
analysis, and autoradiography (panel d). The assays in panel c were done with transiently transfected cells, and the
protein amount of gag-IGFR was monitored by Western blot. The slower
mobility of UIGFR and UF950 proteins is due to the presence of an
additional 36 amino acids of the extracellular sequence in comparison
with that of NM1 (Fig. 1) and glycosylation of the UIGFR
proteins. F3,
Y1131F/Y1135F/Y1136F.
Figure 4:
Phosphorylation of mutants Phe-943 and
Phe-950 proteins. Mutant gag-IGFR-transfected CEFs were treated with
NaVO
as in Fig. 3and lysed. 200 µg
of total cellular proteins was immunoprecipitated with anti-IB and
subjected to Western blotting with either anti-IB or anti-P-Tyr (RC20). a, Phe-943 and NM1 proteins were detected by using a
goat-anti-rabbit secondary antibody conjugated with alkaline
phosphatase and followed by color reaction. b, Phe-950 and NM1
proteins were detected by
I-protein A labeling. The
extent of tyrosine phosphorylation was detected by alkaline
phosphatase-coupled anti-P-Tyr antibody, RC20, in both a and b.
Figure 5:
Phosphorylation of cellular proteins by
mutant gag-IGFRs. NaVO
pretreated cells were
lysed in the Western lysis buffer, and 10 µg of total cellular
proteins was resolved in the 10% SDS-PAGE gels. The filters were
blotted and reacted with anti-P-Tyr (RC20). F3,
Y1131F/Y1135F/Y1136F.
Figure 6:
Phosphorylation of annexin II in mutant
gag-IGFR transformed cells. a, NaVO
pretreated cells were lysed in the Western lysis buffer, and 10
µg of total cell lysate was analyzed in the SDS-PAGE gel. The
filter was first blotted with anti-annexin II to detect the annexin II
protein (lower panel). The same filter was stripped off the
antibody and further reacted with an anti-P-Tyr monoclonal antibody
(PT22) followed by
I-protein A labeling. b,
Na
VO
treated NM1-transformed cells were
fractionated into soluble (Sol.) and cytoskeletal fractions as
described under ``Experimental Procedures.'' The soluble
fraction was precipitated with 70% ethanol and redissolved in Western
lysis buffer. Both fractions were analyzed in the gel followed by
Western blotting with either RC20 or anti-annexin II. The far right
NM1 lane is the total cell extract. c, NM1-transformed
cells were treated with Na
VO
. In lanes 1 and 2, cells were lysed with RIPA buffer containing 0.1%
SDS, and the lysate was immunoprecipitated with the anti-annexin II. In lanes 3 and 4, cells were lysed in the Western lysis
buffer. The anti-annexin II precipitates and direct Western buffer
extracts were analyzed in the same gel and Western blotted. The filter
was reacted with RC20.
Figure 7:
Mutant gag-IGFR- and IRS1-associated PI
3-kinase activity and phosphorylation of IRS1. Cells pretreated or
untreated with NaVO
were lysed in RIPA or
Nonidet P-40 buffer and used for detecting IRS1 tyrosine
phosphorylation or PI 3-kinase assay, respectively, after
immunoprecipitation with either anti-IRS1 or anti-IB. For PI 3-kinase
assay, 300 µg of lysate was used, 1 mg was used for IRS1 tyrosine
phosphorylation, and 50 µg was used for monitoring the gag-IGFR
protein. The reduced IRS1-associated PI 3-kinase activity of Phe-1162
was due to experimental fluctuation, and repeated experiments showed no
difference from NM1.
Figure 8:
Tyrosine phosphorylation of PLC.
Various mutant gag-IGFR-transformed cells were treated with
Na
VO
and lysed with RIPA. 1 mg of total
cellular protein was used in each immunoprecipitation with
anti-PLC
. the immunoprecipitates were analyzed in the 10% SDS-PAGE
gel and followed by immunoblotting with
RC20.
Our results show that mutation of tyrosine residues of the
gag-IGFR fusion receptor can affect its kinase activity and
transforming ability. The cluster of the three tyrosines
(Y1131F/Y1135F/Y1136F) plays an important role in regulating the PTK
activity of the receptor. Those three tyrosines correspond to the
tyrosine residues 1158, 1162, and 1163 of insulin receptor shown to be
located at the gate of the kinase catalytic
center(24, 25) . It was reported earlier that the
autophosphorylation sites of the insulin receptor are tyrosines 965,
972, 1158, 1162, 1163, 1328, and 1334 with tyrosines 1158, 1162, and
1163 being the main regulators of the enzymatic activity for insulin
receptor(26, 27, 28) . Mutation at each site
reduces insulin-stimulated autophosphorylation by 45-60% of that
of the wild type receptor. Double mutation reduces autophosphorylation
by 70%, and replacement of all three tyrosines with phenylalanines
almost abolishes the kinase activity(29) . Our study indicates
that single mutation at position 1135 or 1136 produces little effect on
the kinase activity of the chimeric receptor. However, mutation at
position 1136 drastically reduced cell-transforming and
growth-promoting activities without affecting significantly the
receptor kinase autophosphorylation activity in vitro and
intracellularly. The Phe-1136 mutation does significantly affect its
ability to phosphorylate cellular substrates in general and the 36-kDa
annexin II protein in particular. This property correlates well with
its reduced biological function. These observations suggest that
mutation of this tyrosine may change the receptor conformation or that
phosphorylation of this residue is important for substrate recognition.
Interestingly, mutation of the Tyr-1136 corresponding tyrosines in the
two closely related RPTKs, IR and Ros, also results in significantly
reduced transforming activity without affecting their PTK activity. ()However, no global decrease in substrate phosphorylation
like Phe-1136 was observed in the IR and Ros mutants, although
reduction in interaction with specific substrates was observed. Double
mutation of Tyr-1135 and Tyr-1136 did not produce the drastic effect as
that of Tyr-1162 and Tyr-1163 in native IR. It is possible that the NM1
gag-IGFR is constitutively and highly activated such that it is less
sensitive to changes in those sites. Mutations at positions 943 and 950
result in faster mobility of the receptor proteins, indicating that
these two sites play important roles in the IGFR phosphorylation. The
downshift in the autophosphorylation products in vitro and
missing of the upper band in the anti-P-Tyr blot of those mutant
proteins suggest that Tyr-950 and Tyr-943 are the prominent
autophosphorylation sites. This is in agreement with an earlier study
showing that the juxtamembrane tyrosines of IR are the preferred
autophosphorylation sites in an in vitro assay at low ATP
concentrations(30) . The two corresponding tyrosines in IR are
tyrosines 965 and 972, which have been shown to be in vivo autophosphorylation sites(27) . The Tyr-972 mutation of a
gag-IR also resulted in a faster mobility of the protein in anti-P-Tyr
immunoblot. (
)Surprisingly, when the reported major
phosphorylation sites tyrosines 1135 and 1136 were substituted with
phenylalanines, no significant change in the receptor protein mobility
was observed. It is possible that tyrosine 943 and 950 mutations may
indirectly affect serine/threonine phosphorylation as well, resulting
in a more pronounced effect. Our observation suggests that, similar to
IR, IGFR also contains several potential tyrosine autophosphorylation
sites, and mutation of any of them, except Tyr-943 and Tyr-950, does
not significantly affect the overall phosphorylation of the receptor.
IRS1 is a key component in the IR signal transduction. It has been
shown that tyrosine 972 is critical for the binding of IRS1 to IR.
Point mutation of this tyrosine results in impaired phosphorylation of
IRS1 upon insulin stimulation(7, 8) . From sequence
alignment, tyrosine 950 in IGFR could be the interacting site for IRS1.
However, no clear experimental evidence has been provided(21) .
Mutation of tyrosine 950 in the highly activated and oncogenic NM1
gag-IGFR does not appear to affect its ability to induce IRS1
phosphorylation or to promote association of PI 3-kinase with IRS1, but
when the mutation is generated in the less active and moderate
transforming gag-IGFR, encoded by UIGFR, there is a significant
decrease in both cell-transforming ability and IRS1-associated PI
3-kinase activity, while tyrosine phosphorylation of IRS1 remains
unchanged. Deletion of 13 amino acids flanking tyrosine
950(943-956) of NM1 gag-IGFR, however, impacts on all three
activities, namely causing a decrease in transformation, IRS1
phosphorylation, and PI 3-kinase activity. While our result with UIGFR
is consistent with the notion that Tyr-950 of IGFR is involved in
interaction of IGFR with IRS1, the data with NM1 are not in complete
agreement. There are three possible explanations for these paradoxical
results. First, tyrosine 950 may not be the only site for the IRS1
interaction with IGFR, and IRS1 may have another redundant binding
site. Redundancy in substrate binding sites has been shown in other
RPTKs such as PDGFR(31) . In PDGFR, Shc has four binding sites,
which are tyrosines 579, 740, 751, and 771. The second explanation is
that IRS1 is phosphorylated by other tyrosine kinases activated by
IGFR. Interactions of RPTKs with non-receptor tyrosine kinase has been
reported. Src can be activated by PDGFR, and the binding sites for Src
in PDGFR have been identified as tyrosines 579 and 581(31) .
Furthermore, IGFR is activated in v-src transformed
cells(32) . The highly activated receptor, such as NM1 gag-IGFR
in particular, may have a higher potential of cross-activating other
PTKs. The third possibility is that this mutation can be compensated
for by either overexpression or hyperactivation of the gag-IGFR. Recent
studies suggested that the phosphotyrosine binding domain (6, 7) and the pleckstrin homology domain (33) of IRS1 may be involved in its interaction and tyrosine
phosphorylation with IR. However, the pleckstrin homology domain
apparently is not the direct binding site of IRS1 with IR(33) .
Phosphorylation of IRS1 can be abolished if the pleckstrin homology
domain is deleted; however, it can be restored by overexpression of IR. ()It has also been shown that overexpression of full-length
IR containing Phe-972 can rescue the phosphorylation of IRS1 despite
the mutated interaction site.
By using the yeast two-hybrid
system, a recent study showed that the amino-terminal region from amino
acids 160 to 516 of IRS1 is sufficient for its interaction with IGFR
and that phosphorylation of Tyr-950 of IGFR is essential for its
interaction with IRS1 and Shc but not with p85(34) . However,
it is not known from their study whether IRS1 can be phosphorylated by
the IGFR with the Phe-950 mutation. In our case, the constitutively
activated NM1 gag-IGFR may have compensated for the Tyr-950 mutation
with its high level expression and catalytic activity, which may result
in cross-activation of other IRS1-interacting PTKs as mentioned above.
Our result with d950 suggests that the sequence surrounding the site is
involved in the interaction with IRS1, but Tyr-950 is not critical in
the highly activated receptor. IRS1 is apparently phosphorylated to a
similar extent in all mutant-infected cells. However, it remains
possible that different sites are phosphorylated, which may account for
the reduced PI 3-kinase activity in d950 and UF950.
Annexin II is a
tetrameric Ca-dependent phospholipid binding protein
and is associated with the cytoskeleton(35) . However, other
reports showed that annexin II was mainly found in the
membrane-associated fraction(36) . Annexin II was first shown
to be a substrate of v-src in its transformed
cells(20) . Tyrosine phosphorylation of annexin II can also be
induced by PDGF receptor after treatment of PDGF(37) . Annexin
II in addition to being a substrate for protein-tyrosine kinases can
also be a substrate for protein kinase C(38) . Overexpression
of annexin II was reported in human pancreatic carcinoma cells and
primary pancreatic cancers, as well as in multidrug-resistant small
lung cancer(39, 40) . Study of the RAW large lymphoma
cells showed that depletion of cell surface annexin II could inhibit
RAW cells from adhering to the liver microvessel endothelial cells,
suggesting that annexin II might be involved in
metastasis(41) . Moreover, annexin II was shown to be the
receptor of tenascin-C, indicating that annexin II may mediate cellular
response to soluble tenascin-C in the extracellular
matrix(42) . Furthermore, annexin II is one of the fos target genes(43) . However, the exact function of annexin
II remains unknown. We have identified that annexin II is a substrate
for the gag-IGFR, and its phosphorylation correlates with the extent of
cell transformation by the mutant gag-IGFRs, raising the possibility
that it may play a role in the cell transformation.