Eur. J. Immunol. 1992. 22: 817-821 IL-2 regulation of p2lraS 817

Manuel Izquierdo, Julian Downward*, Hiroki Otanioo, Warren J. LeonardoA and Doreen A. Cantrell

Lymphocyte Activation Laboratory and Signal Transduction Laboratory*, Imperial Cancer Research Fund, London, Cell Biology and Metabolism Branch., Laboratory of Molecular Geneticso, National Institute of Child Health and Human Development, Section on Pulmonary and Molecular ImmunologyA, National Health Lung and Blood Institute, Bethesda

Interleukin (1L)-2 activation of p2lraS in murine myeloid cells transfected with human IL-2 receptor p chain

The T cell growth factor interleukin-2 (IL-2) induces ~21‘“’ activation in T lymphocytes. To determine whether the IL-2 receptor (IL-2R) can regulate p2lraS when expressed in a non-Tcell environment we have examined the ability of IL-2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL-2R fi chains. These cells are denoted 853 cells. 32D cells normally proliferate in response to IL-3 but the expression of the IL-2R fi chain confers IL-2 responsiveness to the cells. Our data show that IL-3 is able to activate p21ra5 in the parental 32D cells and both IL-2 and IL-3 can stimulate ~21‘”‘ in the IL- 2R-expressing 853 clone of 32D. InT lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate ~21‘”’. However, in 32D and 853 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC-mediated pathway for ~21‘”’ regulation exists in T lymphocytes but not in 32D cells. The IL-2R can couple to p2lra’ independently of the concomitant presence of the PKC pathway for p21ra5 regulation.These data imply that multiple intracellular mechanisms may exist to regulate ~21‘”‘ and that cells of different lineages may differ with regard to p21raS regulation.

1 Introduction

The protein products of rus protooncogenes (H, N, and K-rus) bind GTP and catalyse its hydrolysis to GDP. The GTP-bound form of the protein is “active” and the GDP bound form “inactive” in terms of biological activity [l]. Ras oncogenes result from mutations of cellular rus genes that allow p21ras to accumulate in the GTP-bound active state [2-51. Ras oncogenes, which are able to transform cells in culture, have been identified in a high proportion of human tumors and are thought to play a role in the control of cell growth and differentiation [6].

It is now recognized that activation of p21TaS proteins (i.e. accumulation of p21raS-GTP complexes) is a rapid response to triggering of many growth regulatory receptors. In T lymphocytes, p2lraS is activated by receptors such as the T cell receptor for antigen (TcR) and the CD2 antigen [7,8] which control G,+G1 T cell cycle progression. The IL-2R that controls Gl+S phase transition in T cells, and hence controls T cell mitosis, is also able to stimulate ~ 2 1 ‘ ~ ’ [9, 101. Finally, in myeloid cells and fibroblasts p2lraS proteins are regulated by signals generated by cytokines such as IL-3 and granulocyte-monocyte (GM)-CSF [lo] and

[ 100711

Correspondence: Manuel Izquierdo, Lymphocyte Activation Laboratory, ICRF Laboratories, PO Box 123, Lincoln’s Inn Fields, London WC2A 3PX, GB

Abbreviations: SLO: Streptolysin-0 GAP: GTPase-activating protein PBu2: Phorbol 12,13-dibutyrate PDGF Platelet- derived growth factor EGF Epidermal growth factor GM- CSF Granulocytelmacrophage CSF Psi: PKC pseudosubstrate inhibitor peptide WEHI3B CM: WEHI3B cell-conditioned medium

by growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin [11-131. It is thought unlikely that all the different receptors that modulate p21ras activation do so via a common intracellular pathway. In T cells for example, PKC can regulate ~21‘”‘. However, although theTcR and CD2 antigens can stimulate PKC [14] the IL-2Rapparently cannot [lS, 161 and must use a non PKC-dependent mechanism to couple to ~21‘”’. Similarly, in fibroblasts insulin does not appear to stimulate PKC but can activate p21ra5, it is moreover, unclear from studies with phorbol esters whether the PKC-mediated pathway for ~21‘”‘ regulation exists in fibroblasts or myeloid cells [lo, 131. One possibility emerging from these prelim- inary studies is that cells of different lineages may use different intracellular mechanisms to regulate the function of p2l‘dS.

The high-affinity IL-2R that regulates T cell growth is a heterodimer comprising a SS-kDa a subunit and a 70-75- kDa fi subunit. The IL-2R a chain binds IL-2 with low (Kd=lO nM) affinity, the IL-2R 8 chain binds with interme- diate affinity (&=I nM) and the high-affinity (&=lo pM) IL-2R is generated by the physical association of IL-2R a and 8 chains [17]. It is the IL-2R fi chain that is thought to be important in IL-2R signal transduction [18]. The trans- fection of myeloid cells with human 1L-2R chain results in the expression of intermediate- and high-affinity receptors and confers IL-2 responsiveness to these cells; the prolifer- ative response to IL-2 is not only mediated by 8 chains alone but also by a high-affinity IL-2R formed by com- plexes of the transfected IL-2R 8 chain and endogenous murine a chains [19]. There is little known regarding the immediate biochemical responses generated by these chim- eric IL-2R in myeloid cells. The object of the present study, therefore, was to examine p21ra’ regulation in myeloid cells expressing IL-2R and determine whether IL-2R can couple to the intracellular signaling pathways that activate ~21‘”‘ proteins in a non-T cell environment.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992 0014-2980/92/03O3-0817$3 S O + .25IO

818 M. Izquierdo, J. Downward, H. Otani et al.

2 Materials and methods

Eur. J. Immunol. 1992. 22: 817-821

2.4 Evaluation of PKC activity in a cell permeabilization system

2.1 Materials and cells

Purified reduced streptolysin-0 (SLO) and PHA were obtained from Wellcome Diagnostics (Dartford, GB). The peptide GS has the sequence Pro-Leu-Ser-Arg-Thr-Leu- Ser-Val-Ala-Ala-Lys-Lys and its properties as a selective substrate for PKC have been previously reported [20]. The PKC pseudosubstrate peptide inhibitor has the sequence Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Glu-Lys-Asn-Val and is a potent inhibitor of the enzyme in vitro and in permeabilized cells [20]. Human recombinant IL-2 was obtained from Cetus (Emeryville, CA). HumanT lympho- blasts were prepared as described [21] by stimulating peripheral blood mononuclear cells ( 106/ml) in RPMI 1640/10% (v/v) FCS with 5 pg of PHA/ml for 72 h. After washing, cells were maintained in exponential growth in RPMI 1640/10 % FCS supplemented with 20 ng/ml recom- binant IL-2. 32D is an IL-3-dependent murine myeloid progenitor cell line [22]. 853 is a clone of 32D cells obtained by expression of human IL-2R chain cDNA into 32D cells using a recombinant retrovirus [19]. 32D cells were cultured in RPMI 1640/10%0 FCS (v/v) supplemented with 10% (v/v) of WEHI3B cell conditioned medium (WEHI-3B CM) as a source of IL-3. p53 cells were grown either using the same conditions or in presence of 20 ng/ml recombinant IL-2 instead of IL-3-containing conditioned medium.

2.2 Proliferation assays

Cells (2 x lo4 in 200 pl) were cultured for 24 h in duplicate in 96-well-flat-bottom plates with the indicated level of recombinant human IL-2 or WEHI-3B cell-conditioned medium followed by a 4-h pulse with 1 pCi of [3H] dThd. IL-Zresponsive human T lymphoblasts were used as a positive control for IL-2-induced proliferation

2.3 p2lmS activation

p21rd3 proteins were immunoprecipitated with antibody Y13-259 from unstimulated or stimulated cells in which guanine nucleotides were labeled himynthetically with [32P] orthophosphate as described [7]. For p21rd' immuno- precipitations, a modification of the protocol previously described [7] was used: cell lysis was performed in 50 mM Hepes, pH 7.4, 100 mM NaCl, 1 '%, Triton X-114, 5 mM MgC12, 1 mg/ml BSA, 10 p~ benzamidine, 10 pg/ml leu- peptin, 10 pg/ml aprotinin (lysis buffer). Nuclei were removed by centrifugation at 15 000 x g for 4 min and 0.5 ~ N a c l wa5 added to the lysate. Lysates were incubated at 37°C for 2 min to induce precipitation of the detergent. Phase separation was performed by centrifugation at 15 000 x g for 2 min. The detergent-containing pellet was resuspended in ice-cold lysis buffer containing 0.5 M NaCl, 0.5 % sodium deoxycholate, 0.05 % SDS and Triton X-100 instead of Triton X-114. Immunoprecipitation with p21ra5 antibody Y13-259 and analysis of guanine nucleotides bound to p21ra' were performed as previously described [7]. Labeled nucleotides, separated by thin-layer chromatogra- phy, were quantitated by direct scanning for fi radiation using an Ambis 8 Scanner. Results are expressed as proportion of ~21'"' bound to GTF!

Peptide GS phosphorylation assays in cells permeabilized with streptolysin 0 (SLO) were performed as described [20]. Briefly, 5 x lo6 cells were permeabilized with or without stimuli in 250 p1 of permeabilization medium containing 0.4 I.U./ml SLO, 150 mM KCl, 10 mM Pipes, 10 mM EGTA, 150 nM free Ca2+, 5 mM free Mg2+, 100 pM Y - [ ~ ~ P ] ATP and 100 p~ peptide GS substrate, in the presence or absence of 100 pM PKC pseudosubstrate inhibitor. Evaluation of the GS peptide phosphorylation was performed as previously detailed [20]. Results are expressed as pmol of phosphate incorporated into the peptide.

3 Results

3.1 Proliferative response induced by IL-2 and IL-3 in IL-3-dependent myeloid progenitor cells

Initial experiments were designed to determine whether human IL-2Rp chain-expressing myeloid progenitor cells (853 cells) growing in conditioned medium containing IL-3 (WEHI-3B CM) were able to proliferate in response to recombinant IL-2. p53 cells proliferated identically to the 32D parental cells in WEHI-3B CM (Fig. 1, left panel). Recombinant IL-2 was able to induce a strong proliferative response (usually 70 %-SO '70 of the maximal dThd incorporation obtained with IL-3) in 853 cells, while the parental 32D cells did not respond to IL-2 at the concen- trations of IL-2 tested (Fig. 1, right panel). It is noteworthy that the half maximal proliferative response to IL-2 was obtained in the 0.05-0.25 ng/ml (3-15 PM) range, corre- sponding to the occupancy of high-affinity (Kd = 10 pM) receptors rather than the occupancy of intermediate- affinity (Kd= 1 nM) receptors. These results are consistent with previous data concerning the 853 cells. The p53 clone expressed low levels of endogenous murine 1L-2R a chain, and hence express high-affinity hybrid IL-2R consisting of endogenous murine ct chains and human IL-2R p chains ~ 9 1 .

3.2 IL-2 can induce p2lmS activation in p53 cells

Since the expression of IL-2R 8 chain conferred IL-2 responsiveness to p53 cells, the ability of IL-2 to activate p2lraS in the cells was tested. Experiments were performed either in 853 cells that were grown with IL-3 or in cells that were cultured in medium containing IL-2 for 1 week. The titration curve for IL-2 responsiveness was identical in p53 cells maintained in culture by IL-3 or IL-2 (data not shown). The activation state of p21rd' was measured by metabolic labeling of endogenous guanine nucleotides with [32P] orthophosphate followed by cell lysis and immunoprecipi- tation of p21ras. As an external control normal T lympho- blasts were used since it has been shown that IL-2 is able to induce p21ras activation in these cells [9]. The proportion of p21ra5 bound to GTP (i.e. ratio between GTP and GTP+GDP-labeled nucleotides on ras) was evaluated by autoradiography and direct 8 emission scan of the resolved nucleotides eluted from p21rd5 proteins. Fig. 2 shows an autoradiography (upper panel) and a histogram (lower

Eur. J. Immunol. 1992. 22: 817-821 IL-2 regulation of ~ 2 1 ' " ~ 81Y

WEHI-36 CM (K) [IL-2] (nglml)

Figure 1 . IL2- and IL-3 induced proliferation of 32D and 653 cells. (353 (-m-) and 32D (-U-) cells were cultured in microtiter wells as indicated in Sect. 2.2 with different concentrations of IL-2 (right panel) or different dilutions of WEHI-3B CM (left panel). [3H] dThd uptake was determined after 24 h of culture, labeling the cells during the last 4 h. Each data point is the average of duplicate determinations and the data are representative of at least four experiments.

T lym. - panel) corresponding to the analysis of the guanine nucleo- tides bound to p2lrds in activated and unstimulated cells in a

32 D B 53 -- GDP 3

GTP 3

40 -

s m 30- z Y

c

20 - '? n n

p t o -

I-

representative-experiment. The data show that >95 % of the p21ra' in unstimulated 32D, (353 and T lymphoblasts was in GDP-bound (inactive) state. Stimulation of T lympho- blasts with IL-2 induced the accumulation of GTP on p2lraS, increasing the amount of GTP up to 30 % of total labeled nucleotide (range in three experiments: 25 YO-36 %). IL-3 induced a significant accumulation of ~21'"' in a GTP- bound state in both 32D parental cells (range 15 %-20 %), (353 cells growing with IL-3 (range 20 %-22 %) and 853 -- cells growing with IL-2 (range 18 YO-23 %). IL-2 was not

0 - - - - able to direct p21ras activation in 32D parental cells (range 3 %-5 YO of p21raS-GTP complexes). In contrast, IL-2 was

a - 3 GTP-bound state in the 853 cells that were grown either with IL-3 (range 12%-25%0) or IL-2 (range

- 7 ' able to induce a significant accumulation of p21ra' in a

20 Yo-23 Yo).

32D 053 c e l l s

T lym

3.3 PBu2 is not able to activate p21ras in 32D and p53 cells

Control IL-2 IL-3

In T lymphocytes, stimulation of PKC with PBuz results in p21ra5 activation. The effect of PBu2 on p2lTaS in 32D and 853 cells is shown in Fig. 3.The data show that PBu2 did not influence GTP levels on ~21'"' in 32D or (353 cells. In contrast, as described previously, treatment of human T lymphoblasts with PBu2 resulted in large stimulation of ~21'"'. The inability of PBu2 to regulate p21ra5 could reflect that 32D and 853 cells lack functional PKC. Alternatively,

Figure2. Effcct of cell stimulation with IL-2 and IL-3 on the nucleotide bound to ~21 '"~ . Upper panel, autoradiography corre- sponding to the thin-layer chromatogram of the nucleotides eluted from immunoprecipitates of p21ras from 32D, p53 and T lympho- blasts (T lym) labeled with [32P] orthophosphate. Immunoprecipi- tation was performed as indicated in Sect. 2.3 using monoclonal antibody Y13-259 in all lanes. No nucleotides were visualized using an irrelevant antibody as control (not shown). Twenty million cells were unstimulated (control) or stimulated for 20 min with human recombinant IL-2 (20 ng/ml) or murine recombinant IL-3 (20 U/ml). The positions which GTP and GDP standards run are indicated. Lower panel, histogram of the chromatogram was performed by direct scanning for f3 radiation. Data are expressed as percentage of GTP bound to p21TdS with respect to the total amount of nucleotide on ~ 2 1 ' " ~ and are representative of three experi- ments.

the intracellular mechanism that enables PKC to regulate ~ 2 1 ' " ~ may be absent from the cells.

To examine whether 32D and 853 cells express functional PKC the ability of the PKC activator PBu2 to induce the phosphorylation of a PKC GS peptide substrate was tested in a permeabilized cell system. The specificity and sensitiv- ity of this assay have been previously reported [18]. As shown in Fig. 4 PBu2 was able to induce a significant phosphorylation of the GS peptide in both cell types as well as inT lymphoblasts used as an external control. A specific PKC pseudosubstrate inhibitor (Psi) strongly inhibited this response. Similar results were obtained using dioctanoyl- glycerol as a PKC stimulus (data not shown). Peptide GS is a substrate for all known isozymes of PKC. These data

820 M. Izquierdo, J. Downward, H. Otani et al. Eur. J. Immunol. 1992. 22: 817-821

60 60

- 50 ae - t 40

C

Control IL-2 PDBu

0

32D B53 T lyrn

cells

Figure3. Effect of cell stimulation with PBu2 (PDBu) on the nucleotide bound to p21raS.The experiment was performed and the results were expressed as indicated in the legend of Fig. 2. Cells were unstimulated or stimulated with IL-2 (20 ng/ml) or PBuz (100 nglml) for 20 min. Data are representative of three experi- ments.

demonstrate that 32D and (353 cells express functional PKC at a level similar to that present in T lymphocytes.

4 Discussion

In the present report, the data show that IL-2R can transduce a signal that activates p21ras, not only in T lym- phocytes, but also when expressed in a heterologous cell, the IL-2R-expressing (353 clone of 32D cells. IL-2 respon- siveness of pS3 cells is conferred by expression of human IL-2R p chains which form a complex with endogenous murine IL-2R a subunits to generate a high-affinity IL-2R. The present data show that the levels of IL-2 that induce proliferation in (353 cells correspond to those leading to occupancy of high-affinity IL-2R. Similarly, p2lraS activa- tion was induced by low levels of IL-2 indicative that this response was also mediated by high-affinity IL-2 receptors. In addition, IL-3 can induce p21ras activation; thus, p2lraS activation is a common response to triggering of both the IL-2R and IL3R.

In T lymphocytes, the best characterized mechanism for p21ras activation involves PKC-mediated inhibition of a p21ras GTPase-activating protein (GAP); the resultant inhibition of p21rd\ GTPase activity allows p21raS-GTP complexes to accumulate [7 ] . However, previous studies have indicated that PKC stimulation is not involved in IL-2 signal transduction [ 15, 161. Accordingly, the simplest hypothesis is that IL-2 regulates p21ras via a non PKC- mediated mechanism; the details of this mechanism and the involvement of GAP protein remain to be established.

In 853 and 32D cells, unlike in T cells, activation of PKC with PBu2 is not sufficient to stimulate p2lraS.This failure of PBu2 to stimulate p21ra5 does not reflect its inability to stimulate PKC since as judged by a sensitive PKC assay, 32D and pS3 cells express functional PKC. Accordingly, the failure of PKC activation to result in p2lraS stimulation in pS3 and 32D cells must reflect that the mechanism that couples PKC to p21ra5 inT cells is absent in 32D cells. Since IL-2R can couple to p2lraS in both (353 cells and T cells the

z c s - n E

T lyrn

Control Control+PSi PDBu PDBu+PSi

3 2 D

- n

853

Figure 4. Peptide GS phosphorylation in permeabilized cells. Assays were carried out in p53,32D and T lymphoblasts for 5 min with 100 PM peptide GS, with no additions (control), 100 nglml PBu2 (PDBu) or PBu2 plus 100 VM PKC pseudosubstrate peptide inhibitor (Psi). Phosphorylation of GS peptide was quantitated as indicated in Sect. 2.4. Each data point is the average of duplicate determinations. Results are represented as pmol of phosphate incorporated into peptide GS per time of assay and per number of cells used in the assay. Data are representative of three experi- ments.

IL-2-controlled pathway for p2lraS activation must be able to function in the absence of a concomitant PKC controlled route; which supports that PKC is not involved in IL-2 regulation of p2lraS. The failure of PKC activation alone to result in p21ras activation is not unique to 32D cells; a similar absence of PKC control of p21ras has been reported in mast cells and fibroblasts [lo, 131. Whether this difference in PKC function is a consequence of different PKC isoenzyme expression remains to be established. Nevertheless, the present data support the emerging conclusion that, although p21ras activation is a common response to trigger-

Eur. J. Immunol. 1992. 22: 817-821 IL-2 regulation of p21ra5 821

10 Satoh, T., Nakafuku, M., Miyajima, A. and Kaziro,Y, Proc. Natl. Acad. Sci. USA 1991. 88: 3314.

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ing of many growth factor receptors, the intracellular pathways that regulate p2lraS may differ in different cell populations or cell lineages. One challenge for future p21raS studies will be to identify the points at which p2lraS control mechanism diverge.

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