(Received for publication, December 15, 1994)
From the
A temperature-sensitive mutant of the v-Abl protein has
previously been shown to exhibit tyrosine protein kinase activity in
Interleukin 3 (IL-3)-dependent IC.DP cells grown at the permissive
temperature (32 °C) but not at the restrictive temperature (39
°C). These IC.DP cells are dependent on IL-3 for suppression of
apoptosis at 39 °C, but at 32 °C cells will survive without
added growth factor. Both IL-3 and v-Abl stimulated the tyrosine
phosphorylation of SHC and GTPase-activating protein. However, while
IL-3 stimulated similar levels of tyrosine phosphorylation in
p46 and p52
, v-Abl preferentially
phosphorylated p52
, an event that occurred within 1 h of
temperature switch. v-Abl also differentially associated with
p46
in a temperatureindependent manner. In contrast, only
IL-3 stimulated detectable increases in both myelin basic protein
kinase and mitogen-activated protein (MAP) kinase kinase in in
vitro assays, although in more specific MAP kinase activity assays
a very slight increase in the activity of this enzyme was observed
after 6 h at the permissive temperature. Time course studies suggest
that phosphorylation and association of SHC with v-Abl is insufficient
to lead to significant activation of MAP kinase and that activation of
the MAP kinase kinase/MAP kinase pathway is not required for apoptotic
suppression.
Disregulated Abl PTKs ()are causative agents in human
leukemias. In chronic myeloid leukemia a chromosomal translocation,
generating the Philadelphia chromosome, results in the formation of a
chimeric bcr/abl gene, encoding the p210
protein (Daley et al., 1990, 1991; Elefanty et
al., 1990; Kelliher et al., 1990; Shtivelman et
al., 1987). The c-abl gene, which encodes a protein PTK,
is normally inactive. However, in the context of the
p210
fusion protein, the Abl PTK is
constitutively activated and resembles the leukemogenic
p160
gene product in its ability to
transform hemopoietic cells.
To investigate the effects of v-abl, a number of growth factor-dependent hemopoietic cell lines have been infected with Abelson murine leukemia virus, which can abrogate their growth factor requirements, and lead to autonomous proliferation without autocrine growth factor production (Cook et al., 1985, 1987; Hariharan et al., 1988; Mathey-Prevot et al., 1986; Pierce et al., 1985). Maintenance of IL-3 independence has been shown to require the continuous function of v-abl by the preparation of temperature-dependent mutants of the v-abl PTK (Kipreos et al., 1987; Kipreos and Wang, 1988). At the permissive temperature of 32 °C, these stimulate the survival and proliferation of some IL-3-dependent cell lines, while at restrictive temperatures, cells remain growth factor-dependent for their survival and proliferation (Kipreos and Wang, 1988). Such cell lines are not representative of the true in vivo situation, however, as expression of transfected abl oncogenes in murine multipotent hemopoietic progenitor cells does not lead to factor-independent growth and bone marrow progenitor cells from patients with chronic myeloid leukemia are not able to form colonies in the absence of hemopoietic growth factors (Elefanty et al., 1990; Gishizky and Witte, 1992; Metcalf, 1989; Pierce et al., 1985; Silver, 1990; Vogt et al., 1987). We have used an IL-3-dependent murine mast cell line, IC2.9 (Koyasu et al., 1987), transfected with constructs encoding the temperature-sensitive Abl mutant (DP) previously described (Kipreos et al., 1987; Kipreos and Wang, 1988), as a model system in which to investigate the biochemical mechanisms whereby abl activation leads to the survival of hemopoietic cells.
Previous results have shown that
activation of the v-Abl PTK by switching from the restrictive (39
°C) to the permissive temperature (32 °C), leads to suppression
of apoptosis without proliferation, along with increased phospholipid
breakdown and increases in diacylglycerol (Owen et al., 1993).
Abl is a member of the Src family of non-receptor PTKs, and it has been
demonstrated that such proteins contain conserved non-catalytic Src
homology domains, SH2 and SH3. These domains are extensively involved
in protein-protein interactions; for example, SH2 domains interact with
phosphorylated tyrosine residues on their target proteins, leading to
the formation of multiprotein signaling complexes (Kishimoto et
al., 1994; Koch et al., 1991; Pawson and Gish, 1992). We
have continued our previous studies to further identify the signaling
pathways stimulated by v-Abl. We report the effects of v-Abl on the
SHC-coupling protein, the Ras-GTPase activating protein GAP,
phospholipase C-, and MAP kinase, and compare them with the
effects of IL-3. These studies allow us to begin to discriminate
between possible proliferative and survival signals.
Tyrosine phosphorylation of individual
proteins was assessed by immunoprecipitation of 200 µg of total
cell lysate with either 2 µg of anti-phosphotyrosine or
alternatively with a panel of antibodies to known signaling proteins
(GAP and PLC- antibodies from Upstate Biotechnology Inc., MAP
kinase antibodies from Santa Cruz Biotechnology Inc. and 2 different
SHC antibodies, one was the kind gift of Dr. T. Pawson, University of
Toronto and the other was obtained from Santa Cruz) and 30 µl of
protein G-agarose. Immunoprecipitates were collected by centrifugation
and washed (four times) with lysis buffer, before being resolved by
SDS-PAGE and Western blotting. Immunoblotting was carried out as
indicated.
The association of SHC protein with v-Abl was assayed by
immunoprecipitation using 1 µg of polyclonal anti-Abl antibody
(Oncogene Science, Uniondale, NY) and immunoblotting for SHC as
described above. Control experiments showed that the IC.DP and the
IC2.9 cell lines express equal levels of p46 and p52 as
determined by immunoblotting of total cell lysates.
Immunoprecipitates were also analyzed to determine the tyrosine phosphorylation status of the MAP kinase proteins, ERK-1 and ERK-2, by immunoblotting with anti-phosphotyrosine antibodies as described above.
MAP kinase
kinase activity was assayed (as described previously (Traverse et
al., 1992)) by a two-step assay, where, in the first step, total
cell lysates were preincubated with the MAP kinase kinase substrate,
recombinant MAP kinase protein (kindly supplied by Prof. P. Cohen,
Dundee, United Kingdom). The consequent activity of this protein was
then determined in the second step by its ability to stimulate the
incorporation of P from [
P]ATP into
a substrate, myelin basic protein (MBP) (Traverse et al.,
1992).
Figure 1:
The effect of temperature and IL-3 on
the growth of IC2.9 and ICDP cells. IC.DP, previously maintained
overnight at 39 °C, were cultured, at 2 10
cells/ml, under the conditions shown for 24 or 48 h before cell
viability and percent apoptosis were assessed. Results show data from
one representative experiment of four.
We have previously demonstrated that in contrast to IL-3, activation of the v-Abl PTK leads to phosphatidylcholine and phosphatidyl 4,5-bisphosphate hydrolysis to generate phosphocholine and inositol 1,4,5-trisphosphate. These results indicate that the actions of v-Abl may be mediated through alternative signaling pathways from those utilized by IL-3, which has no such effects. As IL-3 addition has been shown to lead to tyrosine phosphorylation of several cellular proteins (Alai et al., 1992; Carroll et al., 1990; Cutler et al., 1993; Duronio et al., 1992; Quelle et al., 1992), we have now compared the PTK cascades activated by v-Abl and IL-3.
Figure 2: The effects of IL-3 on protein tyrosine phosphorylation in IC2.9 and IC.DP cells. IC.DP (A and B) or IC2.9 (C) cells were maintained at 39 or 32 °C, in low IL-3, for 18 h prior to washing to remove IL-3. Cells were then incubated for an additional 3 h at 39 (A and C) or 32 °C (B and C) before stimulating with 200 units/ml IL-3 for the times shown. Results shown are typical of three experiments.
These results suggest that activation of v-Abl PTK activity leads to phosphorylation of a subset of the proteins that are phosphorylated in response to IL-3; however, they provide only a crude assessment of the possible differences between the two signaling pathways. We therefore undertook a more detailed analysis of the proteins which are phosphorylated by activation of the v-Abl PTK and/or IL-3, in order to discern signaling pathways that may be associated with cell survival and apoptotic suppression.
Results shown in Fig. 3(A and B) confirm that PLC- is
indeed tyrosine phosphorylated in a temperature-dependent manner within
6 h of temperature switch and that this effect is rapidly reversible by
switching back to the restrictive temperature (Fig. 3, C and D). No temperature-dependent phosphorylation of
PLC-
was observed in the control IC2.9 cell line, and IL-3 did not
stimulate tyrosine phosphorylation at either temperature in either cell
line (results not shown). Thus, at least part of the diacylglycerol
formed as a consequence of v-Abl activation may be derived from
PLC-
-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate.
Figure 3:
The effect of temperature switch on the
phosphorylation state of PLC-. Cell lysates from cells prepared as
described under ``Materials and Methods'' after temperature
switch, from 39 to 32 °C or vice versa, for the times
shown were immunoprecipitated with anti-phosphotyrosine (A and C) or anti-PLC-
(B and D).
Immunoprecipitates were analyzed by SDS-PAGE and Western blotting for
the presence of PLC-
(A and C) or
phosphotyrosine-containing proteins (B and D). Data
shown are from a representative experiment of
three.
Another protein that has been shown to associate with PTKs through
SH2-phosphotyrosine interactions is the GTPase-activating protein GAP.
Activation of the v-Abl PTK resulted in a an increase in the amount of
GAP protein that was associated with the tyrosine-phosphorylated
fraction of the total cell line (Fig. 4A). Further
results show that GAP is tyrosine-phosphorylated within 4 h of
temperature switch (Fig. 4B). No changes in the level
of GAP phosphorylation were observed in the control cell line (results
not shown). IL-3 stimulated tyrosine phosphorylation of GAP, but only
in cells maintained at 39 °C (Fig. 4, B and C); no increase in tyrosine phosphate levels was observed in
cells maintained at 32 °C (Fig. 4C) (i.e. this phosphorylation is non-additive). Thus, in contrast to
PLC-, which is only tyrosine phosphorylated by v-Abl, both IL-3
and v-Abl stimulate phosphorylation of GAP.
Figure 4: The effect of temperature switches or IL-3 on tyrosine phosphorylation of GAP. IC.DP cell lysates from cells prepared as described under ``Materials and Methods'' after temperature switch, from 39 to 32 °C (A and B), or IL-3 stimulation (C and B) (in cells maintained at the starting temperature), for the times shown were immunoprecipitated with anti-phosphotyrosine antibody (A and C) or anti-GAP antibody (B). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting for the presence of phosphotyrosinecontaining proteins (B) or GAP protein (A and C). Data shown are from a representative experiment of three.
The effect of activation of the Abl PTK or IL-3 on
SHC was studied with two different antibodies (see ``Materials and
Methods''), and similar results were obtained with both antibodies
when either the amount of SHC associated with the phosphotyrosine
fraction of cell lysates (Fig. 5) or direct assessment of the
phosphotyrosine content of SHC was determined (Fig. 6).
Activation of v-Abl PTK by temperature switch, from 39 to 32 °C,
resulted in an increased association between p46 and
p52
and the tyrosine-phosphorylated fraction of the IC.DP
cell lysates, and this correlates with an increase in phosphotyrosine
content of the protein (Fig. 5A and Fig. 6, A and B). Phosphorylation of SHC associated with a
temperature switch from 39 to 32 °C was not observed in the control
IC2.9 cell line under the same conditions. v-Abl PTK-mediated
phosphorylation of SHC was observed within 1 h of temperature switch
and was maintained for a period greater than 6 h. This response was,
however, also rapidly reversible by switching back to the restrictive
temperature (Fig. 5B and 6C).
Figure 5: The effects of temperature switch and IL-3 upon the levels of SHC protein associated with the tyrosine phosphorylated fraction of IC.DP cells. Cell lysates from cells prepared as described under ``Materials and Methods'' after either temperature switch, from 39 to 32 °C (A), or vice versa (B), or stimulation with IL-3 (with cells maintained at the starting temperature), for the times shown were immunoprecipitated with anti-phosphotyrosine antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting for the presence of the SHC protein (using the antibody supplied by Tony Pawson). Data shown are from a representative experiment of five.
Figure 6: The effects of temperature switch and IL-3 upon the tyrosine phosphoryation of SHC protein. Cell lysates from cells prepared as described under ``Materials and Methods'' after either temperature switch, from 39 to 32 °C (A and B), or vice versa (A and C), or stimulation with IL-3 (with cells maintained at the starting temperature) (A and D), for the times shown were immunoprecipitated with anti-SHC antibody (two different antibodies were used, the first was a gift from Tony Pawson (A) and the second from Santa Cruz (B-D). Immunoprecipitates were analyzed by SDS-PAGE and Western blotting for phosphotyrosine-containing protein. Data shown are from a representative experiment of three.
IL-3 stimulated tyrosine phosphorylation of both p46 and p52 isoforms of SHC in IC.DP cells maintained at 39 or 32 °C within 10 min of addition (Fig. 5C and Fig. 6D).
As it has
been reported that SHC associates directly with PTKs, we performed
experiments to determine whether this linker protein associated
directly with v-Abl. Immunoprecipitation with anti-Abl antibody and
immunoblotting with SHC indicates that the v-Abl protein is associated
with SHC (Fig. 7A); immunoprecipitates from the IC2.9
cell line show that there is no association between the normal cellular
homologue c-Abl and SHC (results not shown). This association of SHC
with v-Abl has two interesting features. First, it is not
temperature-dependent, i.e. there is a substantial amount of
SHC associated with v-Abl at the restrictive temperature and movement
of cells to the permissive temperature does not alter this (Fig. 7, A and B). Thus, association between
v-Abl and SHC is not dependent on v-Abl activation or SHC
phosphorylation. The second interesting feature is that the p46 isoform
of SHC is preferentially associated compared to p52, as
we observed no detectable association of the latter isoform with v-Abl,
despite the fact that immunoblotting experiments showed that there was
equivalent amounts of these two isoforms in the IC.DP cells. Similarly,
in IC.DP cells maintained at 39 or 32 °C, increased phosphorylation
of SHC stimulated by IL-3 addition did not lead to increased
association between v-Abl and either isoform of SHC (Fig. 7C).
Figure 7: The effect of temperature switch and IL-3 upon the association of SHC with the v-Abl protein. Cell lysates from cells prepared as described under ``Materials and Methods'' after temperature switch or IL-3 stimulation for the times shown were immunoprecipitated with anti-Abl antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting for the presence of the SHC protein. Data shown are from a representative experiment of five.
Initially, in vitro assays were performed to measure incorporation of phosphate into MBP, in total cell extracts, as an assessment of MAP kinase activity. IL-3 stimulated a small transient increase in MBP kinase activity within 10 min (23 ± 3% increase above controls with no IL-3, p < 0.001, n = 5). The base-line resting MBP kinase activity was 0.78 ± 0.06 units/mg protein. In contrast, no significant changes in myelin basic protein phosphorylation were observed, in response to temperature switch from 39 to 32 °C, even after 12 h at the permissive temperature. Thus, v-Abl activation did not lead to a detectable increase in MBP kinase activity in these cells.
In order to further clarify these results, a more sensitive and specific assay for MAP kinase activity was employed. ERK1 (p44) and ERK2 (p42) specific antibodies were used to immunoprecipitate MAP kinase from total cell lysates and the immunoprecipitates were used in an in vitro assay of MAP kinase activity (see ``Materials and Methods''). Using this assay, IL-3 stimulated a large increase in ERK1 and ERK2 activity, at both the restrictive and the permissive temperature. This response was maximal within 10 min of IL-3 addition (Fig. 8, B and C). Also, a small increase in the MAP kinase activity of both the ERK1 and ERK2 fractions was consistently observed after v-Abl activation, but only after 6 h at the permissive temperature (Fig. 8A).
Figure 8:
The effect of temperature switch or IL-3
on the activity of MAP kinase. IC.DP cell lysates from cells prepared
as described under ``Materials and Methods'' after either
temperature switch, from 39 to 32 °C (A) or IL-3
stimulation in cells maintained at 39 (B) or 32 °C (C) for the times shown were immunoprecipitated with anti-ERK1
or anti-ERK2 antibodies. Immunoprecipitates were assessed for MAP
kinase activity as described under ``Materials and Methods.''
Results were analyzed by autoradiography to determine the presence of
[P]myelin basic protein. Data shown are from a
representative experiment of four.
MAP kinase is activated by phosphorylation of both tyrosine and serine/threonine sites on the enzyme by the dual specificity kinase, MAP kinase kinase (Kosako et al., 1992; Seger et al., 1992). IL-3 stimulated tyrosine phosphorylation of MAP kinase (Fig. 9B) in the IC.DP cells at both 39 and 32 °C. In contrast, temperature switches, from the restrictive to the permissive temperature for v-Abl PTK activity, showed no detectable change in the phosphorylation status of ERK1 and ERK2 over 6 h. However, when cells from steady state incubations (i.e. 24 h) at 39 or 32 °C were compared, there was a small increase in phosphotyrosine content of ERK1 and ERK2 (Fig. 9A).
Figure 9: The effect of IL-3 stimulation or steady state incubation at 39 or 32 °C on the tyrosine phosphorylation of MAP kinase. IC.DP cell lysates were prepared as described under ``Materials and Methods'' from either, cells which had been maintained continuously at 39 or 32 °C for 24 h (15 h in low growth factor medium (see ``Materials and Methods'') followed by 9 h in the absence of growth factor), or from cells after IL-3 stimulation (B) (in cells maintained at 39 or 32 °C) for the times shown. Cell lysates were immunoprecipitated with either anti-ERK1 or anti-ERK2 antibodies and immunoprecipitates were analyzed by SDS-PAGE and Western blotting for the presence of phosphotyrosine-containing proteins. Data shown are from a representative experiment of three.
Further experiments were carried out to examine the activity of the enzyme MAP kinase kinase after both IL-3 and temperature switches in the IC.DP cell lines. The resting MAP kinase kinase activity was measured as 0.8 ± 0.3 unit/mg protein (mean ± S.E., n = 3) (Traverse et al., 1992). Temperature switches from 39 to 32 °C did not result in any detectable changes in MAP kinase kinase activity even after 6 h at the permissive temperature. In agreement with the results of the MAP kinase activity measurements, however, IL-3 stimulated an increase in MAP kinase kinase activity (3.1 ± 0.8 units/mg, mean ± S.E., n = 3 at 10 min). This response was observed in cells that had been maintained at either 39 and 32 °C, showing that the presence of activated v-Abl does not substitute for IL-3 in this pathway.
It is difficult to divorce cytokine-stimulated proliferation
signals from survival signals in hemopoietic growth factor-dependent
cell lines. For example, we have found that low concentrations of IL-3
do not simply induce suppression of apoptosis but also engage
relatively slow cell cycling associated with increased
[H]thymidine uptake. This means that
distinguishing between cellular signaling events associated with IL-3-mediated survival and proliferation is impossible as a
proportion of the cells will exhibit a proliferative response even at
low IL-3 concentrations. Alternatively, however, temperature-sensitive
mutants of the v-abl PTK expressed in IL-3-dependent cells
provide us with a model system in which to study the molecular
mechanisms associated with survival in hemopoietic cells. In the
absence of serum, the temperature-sensitive mutant of the v-Abl PTK
suppresses apoptosis (at the permissive temperature) but does not
stimulate proliferation. Thus, a comparison of v-Abl and IL-3-mediated
signaling events should allow us to partially differentiate between
survival and proliferative responses.
Recently there has been a
large increase in our knowledge of the cellular signaling events
elicited by PTK receptors leading to a mitogenic response and cellular
proliferation. Many growth factors are known to stimulate a generic
signaling pathway, the MAP kinase pathway, in which one of the initial
events is autophosphorylation of the receptors on tyrosine residues,
interaction between these tyrosine residues and SH2 domains in other
signaling proteins (e.g. SHC and Grb-2) followed by tyrosine
phosphorylation of these signaling proteins (Burns et al.,
1993; Cutler et al., 1993; Damen et al., 1993;
Matsuguchi et al., 1994; Rozakis-Adcock et al., 1993;
Skolnik et al., 1993a, 1993b). The IL-3 receptor is a member
of a family of cytokine receptors where the ligand binding component
lacks intrinsic PTK activity, but forms a heterodimer with a second
subunit resulting in autophosphorylation of the
subunit
followed by association and tyrosine phosphorylation of SHC (Cutler et al., 1993). Similarly, IL-3 has been shown to stimulate Ras
activation leading to the activation of the p42 and p44 isoforms of MAP
kinase (Okuda et al., 1992; Welham et al., 1992).
Activation of this enzyme may then lead to its translocation to the
nucleus, where it stimulates the phosphorylation of transcription
factors (Lenormand et al., 1993; Traverse et al.,
1992).
We have shown that in the IC2.9 and IC.DP mast cell lines, IL-3 leads to tyrosine phosphorylation of the SHC linker protein, RasGAP and also MAP kinase, events that culminate in activation of the latter enzyme. These results suggest that IL-3 can activate the recently characterized signaling cascade leading from receptor complexes with SHC and Grb-2, through activation of Ras, Raf, and MAP kinase kinase to activation of MAP kinase and proliferation signals.
SHC has been shown to be constitutively phosphorylated in cells that
express p210 and exists in a complex with Grb-2,
which binds to tyrosine 177 of the Bcr region of the fusion protein
(Pendergast et al., 1993; Puil et al., 1994; Tauchi et al., 1994). We have shown here that v-Abl PTK also tyrosine
phosphorylates and associates with SHC, suggesting that the Abl region
of the fusion protein may also couple to SHC. One interesting point to
emerge from these studies is that v-Abl preferentially associates with
the p46 isoform of the protein despite the fact that Western blots of
cell lysates show that there is equal expression of both SHC isoforms
in these cells. However activation of the v-Abl PTK leads to tyrosine
phosphorylation of both isoforms with p52
showing the
largest increase. In addition, association of p46
with
v-Abl is not dependent on temperature, i.e. SHC association
with v-Abl is not dependent on tyrosine phosphorylation of either SHC
or the non-receptor PTK. It is therefore possible that this association
is mediated through the SH3 and not SH2 interactions. This differential
association of p46
and/or the preferential
phosphorylation of p52
with v-Abl may form the basis of
signaling specificity and account for the differential effects of IL-3
and v-Abl on the MAP kinase pathway, although this hypothesis needs
further investigation.
In contrast to IL-3, v-Abl PTK had no detectable effect on MAP kinase tyrosine phosphorylation status or MAP kinase kinase activity. Sensitive assays to determine the activity of the MAP kinase enzyme showed that v-Abl stimulated a small increase in MAP kinase activity after 6 h at the permissive temperature. The time course of SHC phosphorylation by IL-3 correlates with the increase in MAP kinase activity observed; however, the time course of phosphorylation of SHC by v-Abl (occurs within 1 h of temperature switch) does not match that of activation of MAP kinase, which requires a minimum of 6 h. These results suggest that association and phosphorylation of SHC with v-Abl is insufficient to lead directly to activation of MAP kinase, although it may in the long term lead to the slight activation observed.
The above results show that v-Abl does not simply usurp the IL-3-stimulated signaling pathway and suggest that the principal mode of action of the v-Abl PTK is not through the MAP kinase kinase/MAP kinase pathway. This result fits the hypothesis that activation of MAP kinase is linked (through nuclear phosphorylation events) to proliferation and differentiation events. In the IC.DP cell system, in common with the observed in vivo effects of abl oncogenes, activation of v-Abl stimulates survival but does not stimulate proliferation. These results suggest therefore that MAP kinase activation is not associated with cell survival or suppression of apoptosis.
In contrast to the phosphorylation of SHC
and GAP, the onset of phosphorylation of PLC- is slow. However,
tyrosine phosphorylation of PLC-
occurs at a rate in keeping with
activation of this enzyme leading to the increase in inositol
1,4,5-trisphosphate levels within IC.DP cells after temperature switch
which we have observed (Owen et al., 1993). Similarly, as
breakdown of phosphatidyl 4,5-bisphosphate by PLC-
also generates
another second messenger, diacylglycerol, the observed increases in
this molecule (after temperature switch) may be partially derived from
PLC-
action. The observed phosphorylation of GAP and PLC-
agrees with a recently published report where increased tyrosine
phosphorylation of PLC-
and GAP was observed in cell lines
expressing p185
compared to normal controls (Gotoh et al., 1994), although no analysis of the temporal
relationships between these signals was possible.