(Received for publication, October 31, 1995; and in revised form, January 5, 1996)
From the
The insulin-like growth factor I receptor (IGF-IR) controls the
extent of cell proliferation in a variety of cell types by at least 3
different ways: it is mitogenic, it causes transformation, and it
protects cells from apoptosis. Previous reports indicated that certain
domains in the C terminus of the IGF-IR transmitted a transforming
signal that is additional to and separate from the mitogenic signal. We
have now mutated the four serine residues at 1280-1283 of the
IGF-IR, and transfected the mutant receptor into R cells. Cells expressing the mutant receptor are fully responsive
to IGF-I-mediated mitogenesis, but are not transformed (no colony
formation in soft agar). Several downstream signal transducers are not
affected by the mutation, again suggesting a separate pathway for
transformation. The mutant receptor can act as a dominant negative for
growth, but cannot induce apoptosis in cells with endogenous wild-type
receptors.
The IGF-IR ()belongs to the family of tyrosine kinase
growth factor receptors(1) , and is 70% homologous to the
IR(2) . For many years, the IGF-IR activated by its ligands
(IGF-I, IGF-II and insulin at supraphysiological concentrations) has
been known to transmit a mitogenic signal to cells in
culture(3, 4) . The importance of the IGF-IR in cell
growth has been confirmed in vivo by the finding that mouse
embryos with a targeted disruption of the IGF-IR and IGF-II genes have
a size at birth that is only 30% the size of wild-type
littermates(5, 6) . 3T3-like cells derived from these
mouse embryos devoid of IGF-IRs (R
cells) grow in 10%
serum, albeit at a rate 40% that of cells derived from wild-type
littermate embryos (W cells), and do not grow at all in SFM
supplemented by a variety of growth factors, which can sustain the
growth of W cells and other 3T3 cells(7) . R
cells are also refractory to transformation by SV40 large T
antigen, by an activated Ras or a combination of
both(7, 8) , by the E5 protein of bovine papilloma
virus(9) , and by overexpressed growth factor receptors, such
as the epidermal growth factor receptor(10) , the
platelet-derived growth factor
receptor(11) , and the
IR(12) , all conditions that readily transform cells from
wild-type littermate embryos or other 3T3-like cells with a
physiological number of IGF-IRs. Conversely, overexpression and/or
constitutive activation of the IGF-IR in a variety of cell types leads
to ligand-dependent growth in SFM and to the establishment of a
transformed
phenotype(7, 10, 13, 14, 15, 16, 17, 18) .
The important role of the IGF-IR in the establishment and maintenance of the transformed phenotype is also supported by other findings. Antisense oligodeoxynucleotides or antisense expression plasmids against IGF-II(19, 20) , IGF-I(21) , or the IGF-IR(8, 22, 23, 24, 25) , antibodies to the IGF-IR(26, 27) , and dominant negative mutants of the IGF-IR(28, 29, 30) , can all reverse the transformed phenotype, inhibit tumorigenesis, and induce loss of the metastatic phenotype(31) .
We have
recently reported that R cells, stably transfected
with an IGF-IR truncated at residue 1229, are fully responsive to
IGF-I-mediated mitogenesis but are not transformed(18) .
Subsequently, we reported that a point mutation at tyrosine 1251 (from
tyrosine to phenylalanine) also resulted in a receptor that could not
transform R
cells, but made them responsive to IGF-I
with growth(12) . Interestingly, a tyrosine to phenylalanine
mutation at residue 1250 had no effect, the receptor maintaining both
its mitogenic and transforming activities.
This dissociation between
mitogenicity and transforming activity is of considerable interest
because it suggests that, for transformation, the IGF-IR uses an
additional pathway which is not required for mitogenic signaling, and
that this pathway originates from the C terminus. In the case of the
Y1251F mutant, however, the transfectants, although they could not make
bona fide colonies in soft agar, were capable of generating tiny
clusters that did not attain the size traditionally acceptable for
scoring a colony(12) . These data, and those of Liu et al.(17) with chimeric Gag-IGF-IR constructs, suggested that
more than one residue in the C terminus was necessary to account for
the full transforming activity of the IGF-IR. In the present article,
we have investigated the role of a serine quartet, at residues
1280-1283, on the mitogenic and transforming activities of the
IGF-IR. Mutation at these four serines again results in the remarkable
dissociation between mitogenicity and transforming activity. And, as in
previous cases (12, 18) ()the tyrosyl
phosphorylation of IRS-1 and Shc, and the activities of several
downstream signal transducers are not affected, suggesting that the
transforming signal either follows an additional pathway totally
separate from the mitogenic signaling, or, alternatively, that it
causes in the known substrates more subtle changes that can be revealed
by Western blotting.
Tyrosyl phosphorylated IRS-1 and Shc were detected by the same method described above, except, cell lysates were subjected to immunoprecipitation with a polyclonal anti-IRS-1 antibody (Upstate Biotechnology Inc. (UBI), Lake Placid, NY), or a polyclonal anti-Shc antibody (Transduction Laboratories) before applying to a SDS-PAGE. To determine the amount of IRS-1, immunoprecipitates were blotted with anti-IRS-1 antibody.
Antibodies used in these experiments include: anti-Grb-2 monoclonal antibody and anti-Stat-1 monoclonal antibody from Transduction Laboratory; and anti-JAK polyclonal antibody from UBI.
Figure 1:
Effect of IGF-I on the proliferation of
cells overexpressing wild-type or mutant IGF-IR. Cells were plated in
serum-containing medium. After 24 h, the growth medium was removed and
replaced with SFM with or without IGF-I (20 or 50 ng/ml). Cell numbers
were determined after another 48 h. Each data point represents the mean
of triplicate cultures of the same condition. The data were
statistically analyzed and the growth of receptor-transfected cell
lines (wild-type or mutant), but not R cells, were
significantly increased when stimulated with IGF-1 (20 or 50 ng/ml). p < 0.05.
, SFM; &cjs2113;, IGF-1, 20 ng; &cjs2110;,
IGF-1, 50 ng.
Figure 2:
In vivo IGF-I stimulation of the
IGF-IR autophosphorylation. Tyrosine and serine phosphorylation of the
IGF-IR -subunit was determined in cells expressing the wild-type
and mutant IGF-IR after a 10-min IGF-I stimulation. The same amounts of
proteins from stimulated or unstimulated cells were immunoprecipitated
by an anti-IGF-IR antibody, separated by a SDS-PAGE, transferred to
nitrocellulose filters, and blotted with an anti-phosphotyrosine
antibody (panel a) or an anti-phosphoserine antibody (panel b) as described under ``Materials and
Methods.'' The size of the proteins were determined using a
molecular weight marker. The arrows indicate the position of
the IGF-IR
-subunit.
Although
there is no a priori reason why the tyrosyl phosphorylation of
IRS-1 and Shc should be affected (the mutant receptor is fully
mitogenic), we investigated it in selected clones. Fig. 3(panel a) shows that there is no impairment of
IRS-1 tyrosyl phosphorylation in the clones expressing the serine
mutant in response to IGF-1 stimulation; indeed, the signal is
increased in the 3 mutant clones in respect to R cells, but this could be accounted by the difference in the
number of receptors (see Table 1). The cell lines of Fig. 3had similar amounts of IRS-1, by Western blotting: in
arbitrary densitometry units, the values for R
,
R
/4A5, R
/4A17, and
R
/4A19 were, respectively, 3.8, 3.4, 3.3, and 3.1.
Similar results were obtained with Shc (Fig. 3b): after
immunoprecipitation with a Shc-antibody and blotting with an
anti-phosphotyrosine antibody, the signals were perhaps slightly
increased in the mutant receptor clones. These increases will be taken
into consideration under ``Discussion.''
Figure 3: Tyrosyl phosphorylation of IRS-1 and Shc in various clones. Cell lysates extracted from IGF-I stimulated or unstimulated cells were prepared as described under ``Materials and Methods.'' Lysates containing equal amounts of protein were immunoprecipitated with an anti-IRS-1 antibody, or an anti-Shc antibody, and then applied to the gel. Phosphotyrosine-containing proteins were detected as described under ``Materials and Methods.'' Panel a, phosphorylation of IRS-1; panel b, phosphorylation of Shc.
Figure 4: Association of Grb-2 with Shc. Cell lysates containing equal amounts of protein were immunoprecipitated with an anti-Shc antibody, and applied to the SDS-PAGE. The separated proteins were transferred to nitrocellulose and blotted with an anti-Grb-2 antibody. The arrow indicates the position of the Grb-2 protein.
We then examined the mitogen-activated protein (MAP) kinase activity, which is essentially the effector in the receptor tyrosine kinase/Ras/MAP kinase pathway. As shown in Table 2, we were again unable to identify any impairment in the MAP kinase activity in cell lines expressing the serine mutant receptor, when compared with cells expressing the wild-type receptor.
Since the Stat-JAK pathway has been implicated in the signaling by
the polypeptide growth
factors(36, 37, 38, 39) , we have
examined the status of Stat-1 in these cells. Immunoprecipitation with
an antibody to Stat-1 and blotting with anti-phosphotyrosine antibody
indicated no difference between R cells and the cells
expressing the mutant receptor (not shown). We also investigated the
association between the IGF-IR and JAK2, based on the reports which
have shown that JAK2, another component in the JAK/Stat pathway,
associated with the angiotensin II AT1 receptor (40) or the
growth hormone receptor (41) upon ligand binding. JAK2 was
detectable in lysates after immunoprecipitation with an antibody to the
IGF-IR, but the amount that co-precipitated was the same in
R
and mutant cells, and did not change whether the
cells were or not stimulated with IGF-I (data not shown).
Interestingly, though, the serine mutant receptor did not induce apoptosis, as an antisense expression plasmid to the IGF-IR can do (23) . The C6 cells of Table 3were tested in the diffusion chamber in vivo(23) . This assay, in which the cells are bathed in plasma and have to be anchorage-independent to grow, measures a totally different property from the mitogenic assay (in SFM, with cells attached to a plastic surface). The recovery of 4A3 cells (the clone that gave the lowest number of colonies in soft agar) from the chamber after 24 h was 92%, compared to 220% for the wild-type C6 cells, and 200% with the other C6 clone expressing the mutant receptor. When we measured recovery of 4A3 cells after 48 h, the recovery was 208%. Therefore, it seems that the clones 4A3 cells grew slower than wild-type C6 cells in vivo, or, alternatively, that only a fraction of them underwent apoptosis.
The IGF-IR activated by its ligands plays an important role in cell proliferation for several types of cells in at least 3 different ways: it participates in the mitogenic signaling, it is required for the establishment and maintenance of transformation, and it protects cells from apoptosis both in vitro and in vivo (for a review, see (42) ). Although the two receptors share many common responses, the IGF-IR is 10 times more mitogenic than the IR (43) , and is capable of transforming cells (see Introduction), whereas the IR, in the absence of an IGF-IR, cannot do so(12) . A number of mutations in the IGF-IR have been shown to affect both its mitogenicity and its transforming activity. These include mutations at tyrosine 950 (35) , in the 3 tyrosine residues of the kinase domain (29, 44) and at the ATP-binding site(10, 45) . These mutations, however, have profound effects on the various functions of the IGF-IR, and equivalent mutations in the IR have similar crippling effects (for a review, see (46) ). Conversely, there are mutations that have no effect on either the mitogenic or transforming signaling(12, 29) .
The C terminus of the IGF-IR is
a logical domain to investigate differences with the IR in terms of
mitogenesis and transformation because the homology with the IR, in
this region, is considerably lower than in the rest of the
subunits. Liu et al.(17) has shown that terminal
truncations of the IGF-IR affected its transforming activity, but they
used a chimeric receptor, with a Gag protein in lieu of the
subunit, that was constitutively activated, and did not test for
IGF-I-mediated mitogenicity (the IGF-IR is not obligatory for growth in
10% serum, as demonstrated by R
cells). Surmacz et al.(18) were the first to show that the
IGF-I-mediated mitogenic response and transforming activity of the
IGF-IR could be dissociated. They used for this purpose a receptor with
a deletion of the last 108 amino acids at the C terminus, transfected
into R
cells: this receptor transmitted a full
mitogenic signal, but could not make cells form colonies in soft agar.
Subsequently, Miura et al.(12) showed that a chimeric
receptor, where the C terminus of the IGF-IR had been replaced by the C
terminus of the IR(47) , had lost most of its transforming
activity. They also showed that a point mutation at tyrosine residue
1251 also caused abrogation of the transforming activity of the IGF-IR,
without affecting its IGF-I-mediated mitogenicity. This tyrosine
residue was chosen because tyrosines 1250 and 1251 are absent from the
IR: interestingly, a single point mutation at 1250 gave a receptor that
was both mitogenic and transforming(12) .
The dissociation
between IGF-I-mediated mitogenicity and transforming activity has
important implications (see below), and for this reason, we have
continued our investigations to define the domains in the IGF-IR C
terminus that are required for transformation. Among the candidates are
the region around residues 1293-1294 (absent from the IR), and
the serine quartet at 1280-1283 (there are only two serines in a
similar position in the IR). In this article, we present our results
with the serine mutations, and these are clear: mutation of the serine
quartet at 1280-1283 results in a receptor that is fully
mitogenic, but has lost its ability to transform R cells (colony formation in soft agar). A reasonable criticism
that can be leveled at the dissociation between mitogenicity and
transforming activity is that a slightly defective receptor (for
instance, the receptor truncated at 1229(18) , the Y1251F
mutant (12) and the serine mutant of this paper) can be enough
for mitogenicity but not enough for transforming activity; i.e. the dissociation is purely a question of quantitative signaling.
We have dealt with this criticism before(12) , and we confirm
here that two observations militate against this interpretation: 1)
some of the clones express extremely high numbers of receptors, up to
50 times the number of IGF-IRs that are sufficient to induce
transformation; and 2) both the Y1251F and the serine mutant act as
dominant negatives for transformation (see below), while transforming
receptors do not(30) .
Although the serine mutant receptor
acts as a dominant negative, reducing the number of colonies in soft
agar, when transfected into C6 cells, a very aggressive rat
glioblastoma cell line(22) , it failed to induce apoptosis of
C6 cells. We obtained the same results with other IGF-IR
mutants(30) , and, so far, the only mutant IGF-IR that inhibits
tumorigenesis seems to be the truncated receptor described by Prager et al.(28) . The modesty of the dominant negative
effect that several of the mutant IGF-IRs have can be explained in part
by the fact that growth factor receptors readily form hybrid receptors
or transactivate each
other(48, 49, 50, 51, 52, 53, 54) .
It is this problem that makes experiments with R cells (devoid of endogenous receptors) more informative. It
remains the fact that some mutant receptors that are dominant negative
for transformation do not induce apoptosis ( (30) and this
article), suggesting that the two processes can be clearly separated.
It is very important to realize, that mitogenicity, colony formation in
soft agar, and growth in vivo are radically different assays
that test diverse properties. In the mitogenic assay, cells are
attached to a plastic surface in SFM, and are asked to respond to
IGF-I. In soft agar (10% serum) and in vivo, cells have to be
anchorage-independent to grow: indeed, this is why the two properties
can be dissociated at the level of the receptor, they involve two
different cellular responses.
Tyrosyl phosphorylation of IRS-1 and Shc are not grossly impaired, and PI-3 kinase stimulation is perhaps increased, not surprisingly, since this receptor is fully mitogenic, and these substrates are part of the IGF-IR mitogenic signaling ((55, 56, 57) , and for review, see (58) ). There is in fact a suggestion that tyrosyl phosphorylation of IRS-1 and Shc and PI-3 kinase activity may be increased, as we had already observed with other non-transforming receptors(12, 18) . We would like to postpone a quantitative discussion of the signaling pathway: the yeast two-hybrid system is a better method to quantitate interaction between the IGF-IR and its substrates(59) . With the methods used in this article, which are the methods mostly used in the literature, and with the differences in the number of receptors, a precise quantitative assessment is not warranted. Our present experiments should simply be taken as indicating that there is no impairment of the known signaling pathway in our serine mutant.
To understand fully the implications
of these and previous findings, we should remember that the wild-type
IGF-IR, overexpressed, is fully mitogenic and transforming and that
neither IRS-1 (60) nor Shc, by themselves, or in combination,
can transform R cells. (
)Also, tyrosyl
phosphorylation of either IRS-1 or Shc, and PI-3 kinase activity are
not decreased in the receptor truncated at 1229(18) , in the
Y1251 mutant(12) , and in the serine mutant (this article),
again, not surprisingly, since these receptors are fully mitogenic.
It seems, therefore, that the C terminus of the IGF-IR sends a transforming signal that is additional to and separate from the mitogenic signal, and that can be localized between residues 1245 and 1310(61) . Within these boundaries, the serine quartet at 1280-1283 (this article), tyrosine 1251(12) , and the 1293-1294 region (61) act as the major sites for the transforming activity. It is possible that this signal is mediated through an unknown, or at least unidentified, substrate that interacts directly with the transforming domains of the IGF-IR. Alternatively, the C terminus may cause subtle changes in IRS-1 (or Shc) that are not detectable by a semi-quantitative assay for tyrosyl phosphorylation, changes that, in turn, could start a new pathway, still separate from the mitogenic pathway. It has to be, although, a pathway independent of the Ras pathway. Although the latter one is also required for mitogenesis and transformation(62, 63) , the additional transforming pathway of the IGF-IR is Ras-independent(7) . Other investigators have postulated a Ras-independent pathway for other growth factor receptors(38, 64, 65, 66) . For this reason, we have investigated, in this paper, Stat-1 and JAK-2, two components in one of the Ras-independent pathways(67, 68) , and we were unable to detect any differences between wild-type and mutant cells. A recent report by David et al.(69) has indicated that the MAP kinase activity is required for the Stats to be fully activated for regulation of gene expression. We, therefore, determined the MAP kinase activity in our cell lines and again found no impairment in the serine mutant cell lines. Apparently, the JAK-Stat pathway is not the transforming pathway we are searching for.
We have not dealt in this article with the problem of whether the four serines are or are not phosphorylated, and whether the abrogation of transforming activity is due to lack of phosphorylation or to a conformational change. It has been reported that serine 1293/1294 of the human IR are phosphorylated by a tightly associated tyrosine kinase (70) . Because of the complexity of the problem (for a review, see (46) ), we prefer, for the moment, to simply state that mutation of the serine quartet does cause abrogation of the IGF-IR transforming activity, but does not affect its mitogenicity. Also postponed, momentarily, is the question of which serines in the quartet are necessary and which are not. In the rat IGF-IR, the serine corresponding to serine 1282 in the human receptor, has been replaced by alanine (accession number L29232), and this would suggest that this serine may not be critical.
In conclusion, we have identified in the four serines at 1280-1283 a third, important domain of the human IGF-IR that is required for its transforming activity. The fact that this mutant receptor, like the Y1251F mutant (12) and the 1293-1294 mutant(61) , is fully mitogenic, clearly indicates that the IGF-IR has a transforming pathway, which is in addition to and separate from the mitogenic pathway. This dissociation has implications at both the basic and the applied levels. On one side, it suggests experiments to identify the additional transforming pathway, while at the same time it offers the possibility of interfering with transformed cells (apoptosis?) without greatly affecting the proliferation of normal cells.