Cellular signaling events elicited by v-abl associated with growth factor independence in an interleukin-3-dependent cell line.

A temperature-sensitive mutant of the v-abl oncoprotein has previously been shown to have markedly reduced tyrosine protein kinase activity in interleukin 3 (IL-3)-dependent cells grown at restrictive (39 degrees C), compared to permissive (32 degrees C) temperatures. Transfection of this mutant v-abl into the IC2.9 cell line, generated the IC.DP subclone which was dependent on IL-3 for survival at 39 degrees C, but not at 32 degrees C. Furthermore, IC.DP cells cultured at 32 degrees C exhibited IL-3-independent thymidine incorporation, which was not apparent at 39 degrees C. Switching cells from the restrictive to the permissive temperature resulted in an increase in cellular inositol-1,4,5-trisphosphate, choline phosphate and diacylglycerol levels in the IC.DP cell line. These increases were only observed after a lag period of 4 h. Within 2 h of switching IC.DP cells previously maintained at 32 to 39 degrees C, there was a significant decrease in all three metabolites. Temperature switches had no effect upon these metabolites in the parent IC2.9 cell line. Down-regulation of protein kinase C inhibited v-abl-stimulated DNA synthesis in IC.DP cells cultured at 32 degrees C. IC.DP cells cultured at 32 degrees C were found to have a constitutively activated Na+/H+ antiport, although this activation was inhibited by the down-modulation of protein kinase C. These data indicate a role for phospholipid hydrolysis and protein kinase C activation in V-ABL-mediated abrogation of IL-3 dependence.

A temperature-sensitive mutant of the v-ab1 oncoprotein has previously been shown to have markedly reduced tyrosine protein kinase activity in interleukin 3 (IL-3)-dependent cells grown at restrictive (39 "C), compared to permissive (32 "C) temperatures. Transfection of this mutant u-ab1 into the IC2.9 cell line, generated the IC.DP subclone which was dependent on IL-3 for survival at 39 "C, but not at 32 "C. Furthermore, IC.DP cells cultured at 32 O C exhibited IL-3independent thymidine incorporation, which was not apparent at 39 "C. Switching cells from the restrictive to the permissive temperature resulted in an increase in cellular inositol-1,4,5-trisphosphate, choline phosphate and diacylglycerol levels in the IC.DP cell line. These increases were only observed after a lag period of 4 h. Within 2 h of switching IC.DP cells previously maintained at 32 to 39 "C, there was a significant decrease in all three metabolites. Temperature switches had no effect upon these metabolites in the parent IC2.9 cell line. Down-regulation of protein kinase C inhibited v-abl-stimulated DNA synthesis in IC.DP cells cultured at 32 "C. IC.DP cells cultured at 32 "C were found to have a constitutively activated Na+/H+ antiport, although this activation was inhibited by the down-modulation of protein kinase C. These data indicate a role for phospholipid hydrolysis and protein kinase C activation in V-ABL-mediated abrogation of IL-3 dependence.
The ab1 oncogene product is one of several tyrosine kinases that can transform hemopoietic cells in uitro and stimulate their autonomous proliferation (Cook et al., 1985;Mathey et al., 1986;Pierce et al., 1985). It was first identified as being responsible for the transforming activities of the Abelson murine leukemia virus, and there is now considerable evidence to suggest that activated ab1 genes have an important role in the initiation of human leukemias. A chromosomal translocation which occurs in over 95% of patients with chronic myeloid leukemia generates a chimeric bcrlabl gene. This encodes the ~2lO&'/~'protein (Shtivelman et al., 1985), which may have a causal role in leukemia (Daley et al., 1990Elefanty et al., 1990;Kelliher et al., 1990). The c-ab1 gene also encodes a protein tyrosine kinase, which in the context of the ~2 1 0~~'~' fusion protein, resembles the transforming ~1 6 0 " -~' gene product in exhibiting disregulated tyrosine kinase activity (Clark et al., 1987;Kelliher et al., 1990;Lug0 et al., 1990).
* 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 be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
While the biological effects of ~2 1 0~' /~' are becoming clearer, there is, as yet, little known about the biochemical effects of the ab1 gene product.
The mechanisms whereby u-ab1 and u-src stimulate cellular transformation may be related, since both belong to the src family of homologous cytoplasmic, non-receptor tyrosine kinases. The activities of u-src have been more extensively studied, and some of the biochemical events elicited by activated src tyrosine kinase include its autophosphorylation, association with signal transducing proteins containing src homology 2 domains ( e g . phosphatidylinositol-phospholipase Cy and phosphatidylinositol-3-kinase) and stimulation of phospholipid hydrolysis to generate second messengers e.g. sn-1,2 diacylglycerol (DAG),' and inositol-1,4,5-trisphosphate (IP3). In addition, u-src activates the raf-1 serine/threonine kinase (Morrison et al., 1988;Qureshi et al., 1991), through a mechanism that may involve activation of ras and tyrosine phosphorylation of the ras GTPase activating protein, ras-GAP (Brott et al., 1991;Moran et al., 1991).
To investigate the effects of u-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;Mathey et al., 1986;Pierce et al., 1985). Maintenance of IL-3 independence has been shown to require the continuous function of u-ab1 by the preparation of temperaturedependent mutants of the u-ab1 tyrosine kinase (Kipreos et al., 1987). At the permissive temperature of 32 "C, these stimulate the survival and proliferation of IL-3-dependent cell lines, while at restrictive temperatures, cells remain dependent on this growth factor for their survival and proliferation (Kipreos and Wang, 1988). We have used the IL-3dependent murine mast cell line, IC2.9 (Koyasu et al., 19871, 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 ab1 activation leads to the survival and proliferation of hemopoietic cells.

Signal
Transduction by the v-ab1 Oncogene 15697 the effects of temperature switches, cells were generally maintained for the preceding 18 h at 39 or 32 "C. Measurement of Expression and Phosphorylation of V-ABL-Expression of V-ABL was detected by Western blotting using a monoclonal anti-ah/ antibody (Oncogene Science). Cells maintained for 18 h a t either 39 or 32 "C were lysed as described below. Identical amounts of cellular protein were then separated by SDS-polyacrylamide gel electrophoresis before transferring onto nitrocellulose and processing as described previously (Kan et a/., 1992).
Phosphorylation of V-ABL was detected by immunoprecipitation. Cells (3 X 10fi/ml) were labeled in phosphate free Dulbecco's modified Eagle's medium, supplemented with 3% dialyzed mIL-3 CM, 5% dialyzed horse serum, and 100 pCi/ml [:'2P]orthophosphate, and incubated for 18 h a t 39 or 32 "C. Aliquots (1 ml) were then transferred a t various time points to the alternative temperature. Control cells, i.e. time 0 h, were kept a t 39 or 32 "C throughout, so that all cells were subjected to an equivalent labeling period. Cells were harvested by centrifugation, lysed by resuspending them in 0.5 ml of ice-cold lysis buffer (50 mM Tris acetate buffer, pH 7.5, 1 mM EDTA, 1 mM EGTA, 120 mM NaCI, 1 mM NasVO,, 50 mM NaF, 10 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 10 pg/ml pepstatin A, 10 pg/ml leupeptin, 10 pg/ml benzamidine, 10 pg/ml antipain, and 1% (v/v) Nonidet P-40), and kept on ice for 30 min. Lysates were cleared by centrifugation and then incubated overnight with 1 pg/ml rabbit polyclonal anti-c-ab1 antibody (Oncogene Science) and 30 pl/ ml of protein G-agarose (Calbiochem), at 4 "C. Immunoprecipitates were collected by centrifugation and washed (four times) with lysis buffer, before being resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography.
Tyrosine phosphorylation of intracellular proteins was analyzed using immunoblotting with monoclonal anti-phosphotyrosine antibody (UBI). Proteins were resolved by SDS-polyacrylamide gel electrophoresis, and Western blotting was carried out as ahove except that membranes were blocked for 3 h with phosphate-buffered saline containing 3% bovine serum albumin (Fraction V).
Measurement of Growth Characteristics-Cellular viability was determined by trypan blue exclusion. Cells were cultured a t 39 "C overnight in IL-3 containing medium, washed twice to remove the IL-3, and then cultured at 1 X lo5 cells/ml in Fischer's medium supplemented with horse serum (10%). Cells were then incubated a t either 32 or 39 "C and viability determined at set time points.
['HH]Thymidine incorporation was used as a measure of DNA synthesis. Cells were prepared as above and then plated a t 1 X lo" cells/ml, in a total volume of 100 pl, with appropriate additives. After 16 h, [methyl-'Hjthymidine (1 pCi/well) was added, and 4 h later incorporation into acid-insoluble material was assayed (Whetton et al., 1988b). The recombinant murine IL-3 added to pHi and ['HI thymidine incorporation assays was purified to homogeneity as described (Miyajima et al., 1987) and was a gift from DNAX, Palo Alto, CA.
Measurement of Intracellular Signaling Euents-Mass measurement of IPa levels was carried out as described (Palmer and Wakelam, 1990). DAG levels were determined by loading cells with [2-:'H] glycerol (10 pCi/ml) or [9,lO-'Hlpalmitic acid (1 pCi/ml) in normal culture medium for 48 and 24 h, respectively. Cells were shifted to 39 or 32 "C for the final 18 h of the loading period. Lipids were extracted as described (Bligh and Dyer, 1959) and separated by thin layer chromatography on Silica Gel 150 plates, along with appropriate standards, using hexane/diethylether/acetic acid (5050:1, v/v). This method will separate the 1,2 and 1,3 isomers of diacylglycerol.
Breakdown of phosphatidylcholine was analyzed using cells labeled to equilibrium (24 h) with [methyl-'Hlcholine chloride (2 pCi/ml) in normal culture medium. Water-soluble [:'H]choline metabolites were extracted and resolved by ion-exchange chromatography as described (Cook and Wakelam, 1989).
Assay of Protein Kinme C Actiuity-The partial purification and assay of PKC activity were performed essentially as previously described (Shearman et al., 1989). Enzyme activity was assayed by measuring the incorporation of '?Pi, from [y-"'P]ATP, into histone Determination of Intracellular pH (pHJ-Cells were cultured as described ahove, except that hicarbonate-buffered Fischer's medium was replaced hy HEPES-buffered (25 mM) Fischer's, pH 7.2. Cells were incubated a t either 32 or 39 "C as described in the relevant figure legends. pHi was then determined at 32 or 39 "C as appropriate using the fluorescent indicator his-(carboxyethy1)-carboxyfluorescein (BCECF, Calbiochem) (Tsien et al., 1982), as described previously (Vallance et al., 1990).

H-111s.
Measurement of Rate of pH, Recouep-Ammonium chloride prepulsing experiments were performed essentially as described (Bierman et al., 1987;Vallance et al., 1990), except that assays were performed a t 32 "C. In some cases a sodium-free buffer was employed for these experiments, and this consisted of 140 mM choline chloride, 2.68 mM KCI, 8.1 mM K2HP04, 1.47 mM KH,PO,, 10 mM glucose, and 0.01% bovine serum albumin, pH 7.2.

u-abl Expression and
Activity-The IC.DP cell line expresses a temperature-sensitive u-ab1 protein tyrosine kinase.
Fig. l . 4 confirms that the V-ABL protein is expressed in these cells and that its expression is not temperature dependent, with equal levels of the protein being observed at 39 and 32 "C. Previously, the temperature-sensitive u-ab1 has been shown to undergo tyrosine phosphorylation upon switching Lane I, IC.DP, 39 "C. Lane 2, IC.2.9, 39 "C. Lane 3, IC.DP, 32 "C. Lane 4, IC.2.9.32 "C. B and C, temperature-sensitive activation of V-ABL in the IC.DP cell line. An autoradiograph of polyclonal anti-ab1 antibody immunoprecipitates from cell lysates labeled with ["PI orthophosphate is shown. Cells were maintained at 39 ( B ) or 32 "C (C) for 18 h before switching them to 32 and 39 "C, respectively, for the times shown. Control cells were maintained a t a constant temperature throughout. D, and E, temperature-sensitive tyrosine phosphorylation in the IC.DP cell line. IC.DP cells were maintained at 39 or 32 "C for 18 h prior to washing to remove IL-3 and incubated for Signal Transduction by the u-ab1 Oncogene from the restrictive (39 "C) to the permissive (32 "C) temperature (Kipreos and Wang, 1988). Fig. 1B confirms that V-ABL is phosphorylated within 1 h of a temperature switch from 39 to 32 "C, with phosphorylation reaching a new steady state at 4 h. The phosphorylation of the DP ~1 6 0 " -~' mutant is reversible, since switching cells from 32 to 39 "C results in a rapid dephosphorylation of V-ABL (Fig. 1C).
Immunoblotting experiments with anti-phosphotyrosine antibodies on whole cell lysates demonstrate that a temperature switch to the permissive temperature results in an increase in tyrosine phosphorylation of several cellular proteins. Similarly, the reverse switch resulted in a rapid fall in protein tyrosine phosphate levels. In the parent cell line, no changes in phosphotyrosine levels were observed with either temperature switch. One of the proteins which is tyrosine phosphorylated on switching from 39 to 32 "C has a molecular mass of 160 kDa which corresponds to the molecular mass of the V-ABL protein. Thus, at 32 "C the u-ab1 tyrosine kinase is active, whereas at 39 "C, this enzymatic activity is severely depleted.
Growth Characteristics of the IC.DP Subclone-In the absence of IL-3, IC2.9 cells rapidly lost viability, at both 39 and 32 "C ( Fig. 2 A ) . However, IC.DP cells cultured at 32 "C survived and proliferated in the absence of IL-3, although viabil- ity was lost at 39 "C (Fig. 2B). The IL-3 independence of IC.DP cells cultured at 32 "C was reversible; switching cells to 39 "C resulted in a loss of viability which occurred at the same rate as observed in IC2.9 cells.
A marked increase in IL-3-independent [3H]thymidine incorporation was observed when IC.DP cells were cultured at 32 "C (see Fig. 2 0 ) . This effect was absent in IC2.9 cells, and addition of IL-3 (100 units/ml) to IC.DP cells at 32 "C resulted in levels of DNA synthesis similar to those observed at 39 "C, (Fig. 2, C and D). Thus, activation of the u-ab1 tyrosine kinase abrogates the IL-3 dependence of the IC.DP subclone.
Having confirmed the expression of a temperature-sensitive p160"-d' gene product in IC.DP cells, we examined the intracellular signaling events that might be associated with their IL-3-independent survival and proliferation when cultured at the permissive temperature for V-ABL activity.
Effect of V-ABL on Phospholipid Signaling Pathways-In order to determine the mechanism whereby V-ABL abrogates the IL-3 dependence of IC.DP cells, we have assessed the effects of V-ABL upon the breakdown of both phosphatidylinositol-4,5-bisphosphate (Ptd-Ins4,5-P2) and phosphatidylcholine. Fig. 3 illustrates the effects of temperature switches upon levels of IP3 (a product of Ptd-Ins4,5-P* hydrolysis) in the IC.DP subclone. Cells were maintained at 39 or 32 "C for 18 h before IL-3 was removed, and cells were switched from 39 to 32 "C or vice uersa, for the times shown. Temperature switches were found to have no effect upon mass IP3 levels in IC2.9 cells in the absence of IL-3. IC2.9 cells maintained at 39 "C for 18 h contained 0.43 k 0.13 ( n = 12) pmol of IP3/106 cells compared to 0.48 f 0.13 pmol ( n = 12) in cells at 32 "C. However, when IC.DP cells were shifted from the restrictive to the permissive temperature, there was a 5.55-fold increase in mass IP, following a lag phase of 4 h (see Fig. 3). Conversely, switching cells from 32 to 39 "C resulted in a rapid fall in mass IPS, which returned to the levels observed in cells cultured at 39 "C within 4 h. Activation of the v-ab1 tyrosine kinase therefore stimulates a reversible rise in mass IPS levels.
Similar experiments were carried out to examine the effects of temperature switches upon phosphatidylcholine breakdown (Fig. 4). The results presented indicate that [3H]choline phosphate levels remained constant in the parent cell line, but as was the case with IPS levels, activation of the v-ab1 tyrosine kinase was found to stimulate an increase in [3H]choline phosphate levels after an initial lag of 4 h (Fig. 4A) switch resulted in a corresponding increase (Fig. 4B). Temperature shifts had no effect upon the levels of [3H]choline in the IC2.9 cell line.
Measurement of [3H]glycerophosphocholine levels illustrated that they were temperature sensitive in both the IC2.9 and IC.DP cell lines and were not specifically affected by the activation of the v-ab1 tyrosine kinase (Fig. 4C). Switching cells from 39 to 32 "C resulted in a decrease in [3H]glycerophosphocholine levels in both IC2.9 and IC.DP cells, with the reverse switch stimulating a similar percentage increase. These changes would therefore appear to be dependent upon temperature rather than activation of V-ABL.
These experiments suggest that the temperature-induced activation of V-ABL results in an increase in phospholipase C-mediated breakdown of phospholipids. Since DAG is also formed as a product of both inositol lipid and phosphatidylcholine hydrolysis, the effects of temperature switches upon DAG levels were assessed (Fig. 5). The results shown illustrate that activation of V-ABL (by a 39-32 "C temperature switch) resulted in a 1.89-fold increase in [3H]DAG levels in [3H] palmitate-labeled IC.DP cells, after a lag of 4 h. The reverse temperature switch stimulated a rapid decrease in [3H]DAG levels in these cells (Fig. 5 A ) . Temperature shift experiments on the parent cell line resulted in no significant variation in [3H]DAG levels (Fig. 5A). In [3H]glycerol-labeled IC.DP cells, no significant changes in t3H]DAG were observed.

Involvement of Protein Kinase C in the Effects of the v-ab1
Tyrosine Kinase-DAG can activate the calcium, phospholipid-dependent protein kinase, PKC (Nishizuka, 1984). The Signal Transduction by the u-ab1 Oncogene role of this kinase in u-ubl-stimulated IL-3-independent proliferation was therefore assessed, Chronic treatment of cells with phorbol esters results in down-modulation of PKC (Rodriguez-Pena and Rozengurt, 1984). Long term (18 h) treatment of 1C.DP cells with phorbol esters TPA and phorbol dibutyrate resulted in a reduction of total PKC activity to 2.74 f 2.74% and 6.06 f 3.95% (S.E. n = 3)) respectively, relative to untreated control cells. The effect of PKC down-modulation upon [3H]thymidine incorporation in 1C.DP cells cultured at 32 "C, in the absence of IL-3, is shown (Fig. 6A). Down-regulation of PKC resulted in an inhibition of thymidine incorporation. Similar effects were obtained with phorbol dibutyrate, but analogues of TPA which neither activate nor down-modulate PKC (P-phorbol, 4a-phorbol didecanoate, and 4-methoxy TPA) were found to have no effect upon [3H]thymidine incorporation (Fig. 6A).
We have also assessed the ability of the protein kinase C inhibitor, calphostin C, to inhibit the IL-3-independent proliferation of IC.DP cells cultured at 32 "C (this inhibitor is reported to have little effect upon the activity of members of the src tyrosine kinase family) (Tamaoki, 1991;Tamaoki and Nakano, 1990). There was a marked dose-dependent inhibition of [3H]thymidine incorporation under these conditions (see Fig. 6B). The dose at which half-maximal inhibition of [3H]thymidine incorporation occurred, 140 f 10 nM ( n = 3), was similar to that resulting in a 50% inhibition of protein kinase C activity in vitro (50 nM) (Tamaoki, 1991;Tamaoki and Nakano, 1990). Antiport Activation in IC.DP CelLs-Protein kinase C can activate the Na+/H+ antiport, resulting in an increase in pHi (Bierman et ul., 1987;Whetton et al., 1988b). In order to determine whether V-ABL activation can stimulate an increase in the activity of the Na+/H+ antiport, we have assessed the effects of temperature shifts upon the pHi of the IC2.9 and IC.DP cell lines (see Fig. 7 and Table I). Within 2 h of switching IC.DP cells from 39 to 32 "C, there was a small but detectable increase in pHi, which then continued over the next 4 h (Fig. 7B). A reverse temperature shift from 32 to 39 "C was found to result in a rapid decrease in pHi (Fig. 7A). Temperature shifts were found to have no such a effects upon pHi in the IC2.9 cell line (see Table I).

The Role of Protein Kinase C in V-ABL-stimulated Na+/H+
In order to confirm that the increases in pHi associated with activation of V-ABL were due to increased Na+/H+ antiport activity, we examined the recovery of pHi following NHrC1-induced acidification (Vallance et al., 1990). Despite a near identical degree of acidification, the pHi of IC.DP cells maintained at 32 "C prior to the removal of ammonium ions recovered significantly faster than that of cells preincubated at 39 "C. Consistent with the data presented in Table I, these cells were also found to re-equilibrate to a higher steady state pHi (Fig. 8B). In contrast, temperature shifts can be seen to have no such effects upon the rate of pH recovery in IC2.9 cells (Fig. 8A). Importantly, the recovery in pH; observed under these conditions was found to be both 5-N-(methyl-N-isobuty1)amiloride (5-MNIA)-sensitive and dependent upon the presence of extracellular sodium ions (data not presented) and can therefore be attributed to the activity of the Na+/H+ antiport (Simchowitz and Cragoe, 1986).
Constitutive antiport activation in IC.DP cells maintained at 32 "C was further illustrated by their response to two agents that stimulate acute increases in pH; in the IC2.9 cell line. Both IL-3 and TPA were found to stimulate a rapid increase in pHi of IC2.9 cells maintained at 39 and 32 "C (see Table   I). However, while IL-3 and TPA stimulated corresponding increases in IC.DP cells at 39 "C, neither were found to stimulate any significant increase in the pHi of IC.DP cells that had been maintained at 32 "C for 18 h (Table I) The effect of temperature switches upon pH' of IC.DP cells. IC.DP cells were washed free of 1L-3 and maintained at either 32 "C ( A ) or 39 "C ( B ) before being switched to 39 and 32 "C, respectively, for the times indicated. Cells were loaded with BCECF, and pH, was determined. Results shown represent the mean ( n = 3) of changes in pHi in temperature switched cells relative to control cells maintained at a constant temperature throughout. S.E. values did not exceed 10% of the data points shown. A similar experiment with IC2.9 cells showed no significant changes in pHi.

TABLE I
Effect of temperature switch on resting pH; and responsiveness to IL-3 and TPA in IC2.9 and IC.DP cells Following culture at 32 and 39 "C for 6 and 18 h, cells were incubated for a further 2 h in the absence of IL-3. Cells were loaded with BCECF prior to challenging with IL-3 (100 units/ml) or TPA (100 ng/ml) and pHi determined. Results are from a single experiment typical of three. Standard deviation values did not exceed 10% of the mean value of any of the data points shown. 39  compared to an increase of 0.11 f 0.02 in cells maintained at 39 "C (Table I).
To investigate whether V-ABL exerts its effects upon the activity of the antiport via PKC, the effect of PKC downmodulation was assessed. Cells were switched from 39 to 32 "C, and the increase in pHi over the next 6 h was determined. PKC down-modulated cells showed a rise in pH< which was 0.09 f 0.04 (mean f S.E., n = 3) pH units lower than that observed in control cells (which exhibited an increase in pHi of 0.125 over the same 6-h period). Furthermore, during the first 4 h after a 39-32 "C temperature switch, PKC-downmodulated cells showed no significant increase in pHi compared to the 0.05 pH unit increase observed in non-downmodulated control cells. It would therefore appear that V-ABL stimulates constitutive activation of the Na+/H+ antiport in IC.DP cells, and this is partially mediated by PKC.

DISCUSSION
Transfection of IL-3-dependent hemopoietic cells with Abelson murine leukemia virus leads to an abrogation of the requirement for growth factor via a non-autocrine mechanism (Cook et al., 1985;Pierce et al., 1985). Furthermore, temperature-sensitive mutants of the V-ABL tyrosine kinase have demonstrated a correlation between tyrosine kinase activity INTRACELLULAR pH and IL-3-independent proliferation (Kipreos and Wang, 1988). The rapid and reversible effect of temperature upon the tyrosine kinase activity of these conditional mutants makes them ideal as model systems with which to study the biochemical role of u-ab1 in promoting autonomous survival and proliferation.
Using the IC2.9 mast cell line transfected with the DP conditional mutant of u-ab1 (Kipreos et al., 1987;Kipreos and Wang, 1988), we have found that a temperature shift from 39 to 32 "C leads to the autophosphorylation of V-ABL, an event associated with the activation of its tyrosine kinase activity, and an increase in cellular phosphotyrosine levels. The rate at which a new equilibrium of V-ABL phosphorylation was achieved upon switching cells from the restrictive to the permissive temperature was comparable to that previously reported for the DP mutant (Kipreos and Wang, 1988). An analysis of the changes in second messengers in IC.DP cells upon temperature switch has revealed some of the cellular signaling events associated with the activation of this tyrosine kinase, and which may play a role in abrogating IL-3 dependence.
IL-3 is known to activate protein kinase C, probably by stimulating the generation of the second messenger DAG (Farrar et al., 1985;Whetton et al., 1986Whetton et al., , 1988aWhetton et al., , 1988b. The main source of mitogen-stimulated increases in DAG was originally thought to be phospholipase C-mediated breakdown of PtdIns-4,5-Pz which also generates IPS. However, evidence indicates that IL-3 does not stimulate this pathway in IC2.9 or other IL-3-dependent cell lines (Hamilton et al., 1989;Whetton et al., 1988aWhetton et al., , 1988b.2 Alternative sources of DAG have recently been characterized, e.g. phosphatidylcholine hydrolysis by phospholipases C and D (Billah and Anthes, 1990). There is some evidence to suggest that IL-3-stimulated DAG production occurs as a consequence of phosphatidylcholine hydrolysis (Duronio et al., 1989;Ruggiero et al., 1991). However in [3H]choline-labeled IC2.9 cells, IL-3 had no effect on the levels of any of the water-soluble metabolites of phosphatidylcholine breakdown (over 60 min) suggesting that V-ABL is able to recruit alternative signaling pathways which can lead to the activation of protein kinase C.
Constitutive activation of v-ab1 in fibroblasts and in the hemopoietic 32D cell line indicated that DAG levels were elevated compared to those in the non-transfected parent cell lines (Fry et al., 1985;Ruggiero et al., 1991). In the 32D cell line, this rise in DAG was thought to result from phosphatidylcholine breakdown (Ruggiero et al., 1991). In IC.DP (but not IC2.9) cells, the switch from 39 to 32 "C resulted in an elevated level of choline phosphate in [3H]choline-labeled

cells. A similar increase in DAG levels was observed in [3H]
palmitate-labeled IC.DP cells (Fig. 5A), an effect which occurred after a lag of 4 h. As phosphatidylcholine contains relatively high proportions of palmitate fatty acid, it is likely that the main source of this DAG is phosphatidylcholine. The decrease in choline levels in IC.DP cells switched from 39 to 32 "C is probably due to recruitment of choline for the resynthesis of phosphatidylcholine.
All of the above changes in phospholipid metabolites occur with a lag phase of 4 h, and it is possible that V-ABL affects these pathways via the induction of protein synthesis. However, in the presence of the protein synthesis inhibitor cycloheximide (5 PM) the increase in [3H]DAG levels (after an 18 h switch from the restrictive to the permissive temperature) is 97 f 15% (mean k S.E., n = 3) of that observed in its absence. These results indicate that the changes in phospholipid metabolism are a direct effect of the u-ab1 tyrosine kinase and are not mediated through an induction of enzyme synthesis.
Preliminary data indicate that phosphatidic acid formation is not stimulated by a switch from the restrictive to the permissive temperature, suggesting that phospholipase D is not activated by the u-ab1 tyrosine kinase. Together these results suggest that activation of the u-ab1 tyrosine kinase stimulates phospholipase C-mediated breakdown of phosphatidylcholine, generating DAG, which may then activate protein kinase C.
When IC.DP cells were labeled with [3H]glycerol, in contrast to [3H]palmitate-labeled cells, no significant increase in DAG was observed as glycerol labeling techniques are less sensitive in detecting changes in DAG levels within a particular pool of lipid. Although there is an increase in the level of IPS in IC.DP cells when V-ABL is activated, further experiments will be required to establish whether Ptd-Ins4,5-Pz hydrolysis contributes to any significant increase in the mass of DAG in IC.DP cells cultured at the permissive temperature. Preliminary experiments with [3H]inositol-labeled cells suggest that V-ABL activation does not increase the rate of inositol phospholipid t u r n~v e r .~ Thus, elevated IP3 levels may be a consequence of decreased rates of degradation of this second messenger in IC.DP cells cultured at 32 "C and not due to the increased breakdown of Ptd-Ins4,5-Pz.

C. A. Evans and
Our data indicate that part of the mechanism whereby u-ab1 stimulates the abrogation of IL-3 dependence results from its ability to modulate phospholipid metabolism, thereby generating the second messenger DAG. It would be anticipated that this would subsequently activate protein kinase C. Downmodulation of protein kinase C by chronic treatment with phorbol esters (Rodriguez-Pena and Rozengurt, 1984), or inhibition of its activity with the specific inhibitor calphostin C (Tamaoki, 1991;Tamaoki and Nakano, 1990), certainly decreases the rate of IL-3-independent thymidine incorporation observed in IC.DP cells cultured at the permissive temperature (see Fig. 6). Furthermore, we have used protein kinase C-mediated activation of the Na'/H+ antiport as a means of discerning whether some of the actions of the u-ab1 tyrosine kinase are mediated by PKC.
We have found that the proliferative stimulus of both V-ABL (activated by a 39-32 "C temperature switch) and IL-3 result in an increase in pHi of IC.DP cells. Parallel studies with the parent IC2.9 cell line demonstrated that IL-3, but not temperature switches, stimulate increases in pHi. Thus, the changes in pHi observed in IC.DP cells do not arise from nonspecific effects of the temperature change employed.
Confirmation that the effects upon pHi of IC.DP cells are associated with increased Na+/H' antiport activity comes from studies of the response of these cells to acute acidification. In bicarbonate-free medium, IC.DP cells cultured at 32 "C recover from acidification faster, and reach a higher steady-state pHi, than those cultured at 39 "C, an effect dependent on the presence of extracellular sodium and inhibited by the amiloride analogue, 5-MNIA (Simchowitz and Cragoe, 1986). Together, these experiments infer that v-ab1 can constitutively activate the Na+/H' antiport. Down-modulation of protein kinase C partially inhibits the increase in pHi observed when IC.DP cells are switched from 39 to 32 "C. As IL-3 is known to activate the Na'/H+ antiport and stimulate cellular proliferation via a PKC-dependent mechanism (Whetton et al., 1988a(Whetton et al., , 1988b, it is likely that u-abl-mediated PKC activation is part of a cascade of events associated with the abrogation of growth factor dependence in IL-3-dependent cell lines. Thus, the effect of the u-ab1 tyrosine kinase on phospholipid breakdown is important in cell proliferation as a means of activating protein kinase C. How do the effects of u-ab1 compare to those defined for other non-receptor tyrosine kinases? Most of the work in this area has employed the u-src oncogene, where the use of cell lines transfected with this oncogene has revealed remarkably similar effects to those reported here. The activation of u-src stimulates hydrolysis of phosphatidylcholine to generate the second messenger DAG (Diaz et al., 1990;Song et al., 1991), thereby activating protein kinase C (Durkin et al., 1990).
Furthermore, both u-ab1 and u-src have been reported to stimulate the de novo synthesis of DAG (Chiarugi et al., 1987(Chiarugi et al., , 1989, although our data concerning phosphatidylcholine breakdown suggest that this is not the case in IC.DP cells in the short term. Plainly, the substrates for V-SRC and V-ABL will not simply include enzymes which can influence phospholipid metabolism, although both can associate with phosphatidylinositol-3-kinase (Fukui and Hanafusa, 1989;Varticovski et al., 1991). We envisage that there will be major differences in the substrate specificity of these kinases, which may govern the biological response elicited in transfected cells. To date, the only defined protein, other than phosphatidylinositol-3kinase, shown to be phosphorylated as a consequence of V-

Signal Transduction by the u-ab1
Oncogene 15703 ABL activation is C-RAF (Carroll et al., 1990), which is also phosphorylated by V-SRC and necessary for SRC function (Morrison et al., 1988;Qureshi et al., 1991). Using conditional mutants it will now be possible to identify some of the major proteins phosphorylated as a consequence of V-ABL activation and determine how these relate to the early signaling events associated with leukemic transformation by the ab1 oncogene.