©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
v-Abl-mediated Apoptotic Suppression Is Associated with SHC Phosphorylation without Concomitant Mitogen-activated Protein Kinase Activation (*)

(Received for publication, December 15, 1994)

P. Jane Owen-Lynch (§) Amanda K. Y. Wong Anthony D. Whetton

From the Leukaemia Research Fund Group, Department of Biochemistry and Applied Molecular Biology, University of Manchester, Institute of Science and Technology, Sackville Street, Manchester M60 1QD, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Disregulated Abl PTKs (^1)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^v 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.


MATERIALS AND METHODS

Cell Culture

IC2.9 and ICDP cells were routinely cultured in Fischer's medium, supplemented with 10% (v/v) horse serum and 5% (v/v) medium conditioned by the X63-Ag-653 cell line transfected with the IL-3 gene (mIL-3 CM) (Karasuyama and Melchers, 1988) and were maintained at 37 °C in a gassed incubator at 5% CO(2).

Measurement of Growth Characteristics

Cellular viability was determined by trypan blue exclusion, and the number of apoptotic cells was assessed by acridine orange staining. Cells were incubated overnight at 39 °C, washed to remove the growth factor, and then plated at 0.5 times 10^5 cells/ml, in a total volume of 100 µl, with appropriate additives. Viability and percent apoptosis were determined at set time points. Recombinant murine IL-3 used was purified to homogeneity as described (Miyajima et al., 1987) and was a gift from DNAX, Palo Alto, CA.

Detection of Tyrosine-phosphorylated or v-Abl-associated Proteins

Prior to experiments investigating the effects of temperature switches, cells were maintained for 18 h at 39 °C or 32 °C in Fischer's medium containing a low concentration of m-IL-3 (0.5%, v/v) and horse serum (5%, v/v). Cells were washed and resuspended in Fischer's medium to remove the IL-3 and then maintained for an additional 3 h at 39 or 32 °C, before switching to the alternative temperature for the times indicated or stimulating by addition of IL-3 (200 units/ml). Cells were harvested by centrifugation and lysed as described previously in a buffer containing the following inhibitors: 1 mM EDTA, 1 mM EGTA, 1 mM Na(3)VO(4), 50 mM NaF, 10 mM phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, benzamidine, antipain, aprotinin, trypsin inhibitors, N-tosylphenylalanine chloromethyl ketone, and N-tosyllysine chloromethyl ketone, all at 10 µg/ml. Cleared total cell lysates were prepared before tyrosine phosphorylation of total intracellular proteins was analyzed by immunoblotting with monoclonal anti-phosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY) as described previously (Owen et al., 1993).

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.

Measurement of MAP Kinase and MAP Kinase Kinase Activity

Cell lysates prepared as described above were immunoprecipitated with anti-ERK1- or anti-ERK2-specific antibodies (Santa Cruz Biotechnology, Inc., CA). MAP kinase activity in these immunoprecipitates was assayed as described by Bashey et al. (1994), using a myelin basic protein kinase assay. In initial confirmatory experiments, the immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with anti-ERK1 or anti-ERK2. Results showed that equal amounts of ERK protein were immunoprecipitated for each point and that the antibodies were specific for ERK1 or ERK2 in immunoprecipitation assays. Immunoprecipitates were incubated with [P]ATP and a MAP kinase substrate, myelin basic protein, and the incorporation of P into myelin basic protein was assessed by SDS-PAGE and autoradiography (Bashey et al., 1994).

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).


RESULTS

Growth Characteristics of the IC.DP Subclone in Response to IL-3

We have demonstrated previously that transfection of the temperature-sensitive mutant of the v-Abl PTK into the IC2.9 cell line results in abrogation of the IL-3 requirement for cell survival at the permissive temperature (Evans et al., 1993; Owen et al., 1993). Viability data show that long term proliferation is not observed when cells are grown at the permissive temperature (in the absence of added growth factor and serum) but that, in contrast to cells grown at the restrictive temperature, no apoptosis is observed: i.e. activation of the v-Abl PTK mediates cell survival not proliferation (Fig. 1). Similarly, there is no effect of v-Abl on the proliferation induced by maximal concentrations of IL-3 (in the absence of added serum): i.e. v-Abl does not synergize with the IL-3 to elicit supramaximal levels of proliferation.


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 times 10^5 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.

IL-3-mediated Tyrosine Phosphorylation

We have shown previously that activation of the v-Abl PTK (by switching from the restrictive to the permissive temperature) results in an increase in total tyrosine phosphorylated proteins within 1 h, reaching a new equilibrium by 2 h. These phosphorylation events were reversible as switching back to the restrictive temperature resulted in a rapid decline in tyrosine phosphorylation levels. No such changes in phosphotyrosine content were seen in the control, IC2.9, cell line (Owen et al., 1993). Stimulation of IC.DP cells, maintained at the restrictive temperature with IL-3 for 5 min, results in tyrosine phosphorylation of a range of proteins (Fig. 2A). However, in cells previously maintained for 18 h at the permissive temperature for v-Abl PTK activity, the control level of tyrosine phosphorylation is already increased, but IL-3 addition still resulted in phosphorylation of a subset of the bands phosphorylated at 39 °C (Fig. 2B). In the control cell line, IC2.9, IL-3 stimulation resulted in tyrosine phosphorylation of a similar range of proteins, as in IC.DP at 39 °C, at both the restrictive and the permissive temperature (Fig. 2C).


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.

Tyrosine Phosphorylation of PLC- and GAP

It has been shown previously that v-Abl PTK, but not IL-3 stimulates the breakdown of inositol phospholipids in IC.DP cells (Owen et al., 1993). As the related PTK, Src, stimulates the phosphorylation and activation of phospholipase C- (Liao et al., 1993; Nakanishi et al., 1993), we assessed whether some of these previously observed responses to the v-Abl PTK could be a result of activation of this phospholipase.

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.



Phosphorylation of the SHC Linker Protein by IL-3 and v-Abl PTK

Activation of both receptor-associated PTKs and PTK receptors has been shown to lead to activation of signaling cascades initiated by the formation of multiprotein complexes involving SH2/phosphotyrosine interactions (Kishimoto et al., 1994). One of the earliest effects to be identified so far is the association, and tyrosine phosphorylation, of a linker protein SHC with tyrosine-phosphorylated sites on activated PTK receptors (Damen et al., 1993; Pelicci et al., 1992; Rozakis-Adcock et al., 1992; Sato et al., 1993). Indeed, IL-3-stimulated tyrosine phosphorylation of SHC has been reported (Cutler et al., 1993).

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.



Activation of the MAP Kinase Signaling Cascade by IL-3 and v-Abl

Phosphorylation and association of SHC with PTKs has been shown in several systems, including Drosophila, to be the link between receptor activation and the GTP-binding protein Ras, through Grb-2 binding and the guanine nucleotide exchange factor SOS (Egan et al., 1993; Rozakis-Adcock et al., 1992, 1993; Skolnik et al., 1993a, 1993b). This signaling cascade then leads to the activation of a protein kinase cascade pathway culminating in activation of MAP kinase and proliferation (see, for example, Lenormand et al.(1993) and Okuda et al.(1992)). We examined whether this signaling cascade was activated in response to IL-3 or temperature switch in the IC2.9 and IC.DP cell lines.

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.


DISCUSSION

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 [^3H]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 beta subunit resulting in autophosphorylation of the beta 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.


FOOTNOTES

*
This work was supported by the Leukaemia Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

(^1)
The abbreviations used are: PTK, protein tyrosine kinase; IL, interleukin; ERK, extracellulary regulated kinase; GAP, GTPase-activating protein; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein.


ACKNOWLEDGEMENTS

We thank Jean Wang (University of California, San Diego) for providing the IC2.9 and IC.DP cell lines, Dr. T. Pawson for SHC antibodies, and S. Slack for technical support. We also thank Professor P. Cohen for help with the MBP kinase assays and helpful discussion of this document.


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