Rapid tyrosine phosphorylation of an 85,000 Mr protein after phorbol ester stimulation of EL4 thymoma cells

Cellular Signalling Vol. 7. No. I, pp. 17-30. 1995. Copyright © 1995 Elmvier Science Ltd Printed in Great Bntain. All rights re~rved 0898-6568/95 $9.50 + 0.00

Pergamon 0898-6568(94)00068-9

R A P I D T Y R O S I N E P H O S P H O R Y L A T I O N O F A N 85,000 M r P R O T E I N A F T E R PHORBOL ESTER STIMULATION OF EL4 THYMOMA CELLS ALISON F. RICHARDSON and JULIANNE J. SANDO* Department of Pharmacology and Cancer Center, University of Virginia, Charlottesville, VA, 22908. U.S.A. (Received 1 August 1994; and accepted 15 August 1994) Abstract--Early signalling events between protein kinase C (PKC) activation and lyrnphokine transcription were compared between phorbol ester-sensitive and -resistant EL4 cell lines which do or do not respond with interleukin 2 (IL2) production, respectively. The earliest event detected in the sensitive cell line was a dramatic increase in the tyrosine phosphorylation of an 85,000 Mr protein (p85; 30 s), followed by mobility shifts of raf-l, mitogen-activated protein kinase kinase (MEK), mitogen-activated protein (MAP) kinase, lck and ZAP-70 (within 5 min). In contrast, p85 was not detected in the resistant cell line and lck and raf-I mobility shifts exhibited delayed kinetics. Both vanadate and okadaic acid blocked the phorbol ester-stimulated p85 tyrosine phosphorylation in the sensitive cell line, suggesting that a phosphatase activity downstream of PKC activation may be required for p85 tyrosine phosphorylation. Characterization of p85 and its regulation should help elucidate some of the earliest events in this PKC pathway. Key words: Protein kinase C; tyrosine phosphorylation, T cells, tyrosine kinases, serine kinases, signal transduction.

INTRODUCTION

gene family of tyrosine kinases [9, 10]. Lck is non-covalently associated with the CD4 and CD8 membrane proteins which function as T cell receptor co-receptors during T cell activation [ 11 ], and a proportion of fyn molecules is associated with the T cell receptor [12]. Supportive evidence for the involvement of lck and fyn in T cell signalling includes the finding that T cells which are deficient in lck or which overexpress a kinasedeficient form offyn exhibit defective T cell activation [7, 13]. The ZAP-70 tyrosine kinase is most closely related to the B cell kinase syk [14]. Its role in T cell signal transduction was demonstrated by a marked increase in cellular tyrosine phosphorylation after ZAP-70 co-expression with either lck or fyn in cos cells [14]. After T cell stimulation, ZAP-70 is induced to bind to the tyrosine phosphorylation activation motif sequences of the T cell receptor ~ and e chains, where it undergoes tyrosine phosphorylation itself [15, 16]. T cell activation can be mimicked by the combination of tumour promoting phorbol esters and

T cell activation results in changes in gene expression leading to interleukin 2 (IL2) production (reviewed in [1]); however, the signalling pathways e m p l o y e d are incompletely understood. A n t i p h o s p h o t y r o s i n e i m m u n o b l o t t i n g and inhibitor studies have confirmed that one of the earliest events in this process is the activation of tyrosine phosphorylation [2-4]. None of the eight integral membrane proteins of the T cell receptor complex possesses an intrinsic kinase activity, suggesting that the T cell receptor-couples to a non-receptor tyrosine kinase (reviewed in [5]). Three tyrosine kinases, lck, fyn, and ZAP-70, are highly enriched in T cells and they have been implicated in transduction of signals from the T cell receptor [6-8]. Lck and fyn are members of the src protoonco-

*Correspondence to: Julianne J. Sando, Box 448, Department of Pharmacology, University of Virginia. 1300, Jefferson Park Avenue, Charlottesville, VA 22908, U.S.A.

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A.F. RICHARDSONand J. J. SANDO

calcium ionophores, suggesting roles for both protein kinase C (PKC) and calcium [17]. PKC is a family of closely related, lipid-dependent, serine/threonine kinases, most of which can be stimulated directly by phorbol ester treatment [for reviews see 18, 19]. The precise role of PKC and calcium in the activation of T cell tyrosine kinases remains unresolved; however, the observation that phorbol ester treatment induces Ick phosphorylation in the human Jurkat T cell line is suggestive of a role for PKC upstream of lck [20]. Phorbol ester stimulation of Jurkat cells also results in activation of the serine kinase, mitogen-activated protein (MAP) kinase [20]. M A P kinase has been implicated as a component of many mitogenic signalling pathways [21-24], and is itself part of a cytoplasmic kinase cascade acting as a substrate for the tyrosine/threonine kinase, MEK (MAP kinase kinase) [25, 26], which in turn acts as a substrate for the serine/threonine kinase, raf-1 [27, 28]. The evidence that PKC can phosphorylate raf-1 [29-31] argues for a role of PKC upstream of this phosphorylation cascade. The demonstration that MAP kinase can translocate to the nucleus [32] and that it can phosphorylate and positively regulate the c-Jun transcription factor [33], provides a mechanism whereby a kinase cascade from PKC may result in the regulation of gene transcription. A convenient model for studies of PKC-mediated T cell activation is provided by an EL4 mouse thymoma cell line which requires only phorbol ester treatment to stimulate IL2 production [34]. Comparison with an EL4 variant which is resistant to phorbol ester-stimulated IL2 production [35] has enabled investigations into the essential components for IL2 gene transcription. Other work has revealed deficient induction of cJun and Fra transcription factors [36] and deficient activation of MEK [37] in phorbol estertreated resistant EL4 cells. Thus a defect upstream of MEK is likely to be present in resistant EL4 cells. In addition, recent work has demonstrated that tyrosine kinase inhibitors (genistein and herbimycin) block phorbol ester-stimulated IL2 m R N A production in sensitive EL4 cells [Sando and Holman. manuscript submitted], suggesting

an important role for tyrosine phosphorylation. In the work described in this paper, early T cell signalling events have been compared between phorbol ester sensitive and resistant EL4 cells. These studies have revealed a temporal sequence of phorbol ester-induced events in sensitive EL4 cells, several of which are either deficient or delayed in the phorbol ester-resistant EL4 cell line. M A T E R I A L S AND METHODS Reagents Anti-phosphotyrosine (P-Tyr), phosphatidylinositol 3-kinase (PI3K) and lck antibodies were supplied by Upstate Biotechnology Incorporated (Lake Placid, NY); The MAP kinase antibody was a generous gift from Dr Michael Weber (University of Virginia, VA). All other antibodies were supplied by Santa Cruz Biotechnology Ltd (Santa Cruz, CA). Phorbol dibutyrate (PDB), aprotinin and sodium orthovanadate were purchased from Sigma (St Louis, MO) and enhanced chemiluminescence (ECL) reagents were obtained from Amersham (Arlington Heights, IL). Phorbol ester-sensitive and -resistant EL4 cells were purchased from ATCC (Rockville, MD). and foetal calf serum was supplied by Gibco BRL (Gaithersburg, MD). Phorbol ester treatment and electrophoresis EL4 cells were grown in RPM1 1640 medium supplemented with 5% heat inactivated foetal calf serum and 2 mM glutamine to a density of approximately 2 x 106/ml. Cells (107) were treated with 150 nM PDB or vehicle (0.015% ethanol) for the times indicated. Cells were collected by centrifugation (1 rain; 1000 g) and washed once with ice-cold phosphate-buffered saline supplemented with 100 p.M sodium vanadate. The cell pellet was resuspended in boiling 2× Laemmli sample buffer (150 p.l) [381 and 10" cells were analysed by polyacrylamide gel electrophoresis. Western blotting Proteins were transferred to nitrocellulose (Schleicher and Schuell, Keene, NH) using a wet transfer apparatus (Hoeffer, San Francisco, CA), and equal loading of protein was confirmed by ponceau red staining of the nitrocellulose. Filters were blocked by incubation (1 h) with either 1% bovine serum albumin in Tris-buffered saline-Tween (10 mM Tris pH 7.4, 150 mM NaCI and 0.1% Tween 20), for P-Tyr and MAP kinase blots or with 5% non-fat dried milk in Tris

Early phorbol ester-stimulatedevents in EL4 cells buffered saline-Tween. Membranes were exposed for 1 h to primary antibody diluted as follows: 1 lag/ml PTyr, 1:200 dilution lck, 1:500 dilution PI3K, 1:1000 dilution MAP kinase, and 2 ~tg/ml for all other antibodies. Membranes were washed with Tris buffered salineTween and horseradish peroxidase-linked secondary antibody was added for a further 60 min. The membranes were washed and proteins were visualized by addition of enhanced chemiluminescence reagents exactly as described by the manufacturers. RESULTS

Tyrosine phosphorylation The observations that increased tyrosine phosphorylation is detected only a few seconds after T cell activation [2] and that tyrosine kinase inhibitors block phorbol ester-stimulated IL2 production in EL4 cells [Sando and Holman, manuscript submitted] prompted a comparison of tyrosine phosphorylation events between sensitive EL4 cells, which exhibit phorbol ester-induced IL2 transcription, and an EL4 variant which is resistant to phorbol ester stimulation. Lysates from control cells and cells stimulated with 150 nM PDB for 5 min were analysed using antiphosphotyrosine (clone 4G10) immunoblotting and the presence of a number of tyrosine phosphoproteins was demonstrated (Fig. 1). Proteins of 34,000, 35,000 and 44,000 M r were detected in both cell lines; however, the 56,000 and 85,000 M r phosphoproteins were detected in sensitive cells only. After stimulation with phorbol ester, both cell lines demonstrated the appearance of a 45,000 Mr band. An increase in the intensity of the phosphotyrosine band at 85,000 M r (p85) was detected after stimulation of sensitive EL4 cells. Interestingly, pretreatment of the cells with vanadate, an inhibitor of tyrosine phosphatases [reviewed in 39], prevented the phorbol esterinduced increase of the p85 phosphotyrosine signal.

Tyrosine kinase levels The detection of p85 only in sensitive EL4 cells suggested that resistant EL4 cells might be defective in the tyrosine kinase responsible for this phosphorylation. Therefore immunoblotting

19

with antibodies specific for the candidate tyrosine kinases, lck, fyn, and ZAP-70, was performed (Fig. 2a, b, c). All three kinases could be detected in both cell lines, although relatively lower levels of fyn were detected in resistant EL4 cells (Fig. 2b). Phorbol ester treatment induced the appearance of a higher molecular weight species of lck in sensitive EL4 cells (Fig. 2c) suggestive of phosphorylation, but the shifted band was less pronounced in the resistant cell line. Although pretreatment with vanadate did not alter the phorbol ester-induced lck mobility shifts, vanadate did significantly decrease ZAP-70 detection in both sensitive and resistant cells (Fig 2a, lanes 5 and 6).

Levels of MAP kinase, MEK and raf-1 Since resistant EL4 cells fail to induce c-Jun in response to phorbol ester treatment [36], they were compared to sensitive cells for expression of the MAP kinase cascade which has been implicated in c-Jun activation [33]. Figure 3 illustrates the immunoblotting of EL4 cell lysates with antibodies specific for raf-1, MEK and MAP kinase. Similar amounts of these kinases were found in both cell lines. Phorbol ester treatment induced the appearance of a higher molecular weight form of MAP kinase in sensitive and resistant cells. MAP kinase has been demonstrated previously to migrate at an apparently higher molecular weight after activation by phosphorylation [e.g. 40]. Phorbol ester treatment also caused a small shift of MEK and a decrease in the level of the lower band detected. Vanadate pretreatment did not affect the mobility shifts of MAP kinase or MEK, however the MEK signal was much weaker after vanadate treatment in both cell lines. Raf-1 exhibited a greater increase in its apparent molecular weight after phorbol ester treatment of sensitive EL4 cells as compared with the resistant cell line (Fig. 3c).

Temporal effect of phorbol ester Since phorbol ester stimulation of sensitive EL4 cells induced tyrosine phosphorylation of

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A.F. RICHARDSONand J. J. SANDO

p85 and gel mobility shifts of lck, MAP kinase, MEK and raf-1, the possibility that these events could be ordered temporally after phorbol ester stimulation was investigated. Lysates from cells stimulated with PDB for various times up to 20 min were analysed by immunobiotting with antibodies specific for the proteins of interest. Lysates from resistant cells treated under identical conditions were also analysed. Figure 4 shows western blots probed with antibodies to phosphotyrosine and the three T cell tyrosine kinases, lck, fyn, and ZAP-70. An increase in p85 tyrosine phosphorylation was observed 30 s after phorbol ester addition to sensitive cells (Fig. 4a). 58,000 (p58) and 62,000 M~ (p62) proteins were detected after 5 min of stimulation and a 63,000 Mr protein (p63) was detected by 15 min (Fig. 4a). None of these tyrosine phosphorylation events was detected in the phorbol ester-resistant EL4 cell line. Lck immunoblotting also revealed the presence of 58,000, 62,000 and 63,000 Mr proteins in phorbol ester-stimulated sensitive EL4 cell lysates (Fig. 4d). The 58,000 and 62,000 Mr species were detected 5 rain after phorbol ester treatment of sensitive EL4 cells and the 63,000 M r band was apparent after 15 rain of phorbol ester stimulation. Lck mobility shifts (58,000 and 62,000 M~) in resistant EL4 cells were detected 7 min after phorbol ester treatment but the 63,000 Mr form of lck was not detected over the 20 rain time course of stimulation. The appearance of a higher molecular weight doublet of ZAP-70 was just detectable 5 min after phorbol ester addition to sensitive EL4 cells (Fig. 4b). A similar shift was not readily detected after phorbol ester treatment of the resistant EL4 cells line. Fyn did not undergo phorbol ester-induced mobility shifts in either cell line (Fig. 4c) during the time course of stimulation. MAP kinase exhibited a mobility shift 3 min after phorbol ester treatment of sensitive and resistant EL4 cells (Fig. 5a and b), and a small MEK mobility shift was just detectable at this time in both cell lines. Although a raf-I mobility shift was observed 3 rain after treatment with phorbol ester in sensitive cells, this was not detected until 7 min of stimulation in the resistant EL4 cell line (Fig. 5c).

Effect of genistein, herbimycin, okadaic acid and vanadate The inhibition by vanadate of phorbol esterstimulated p85 tyrosine phosphorylation prompted us to examine the effects of other kinase and phosphatase inhibitors on p85 tyrosine phosphorylation. Figure 6 shows the effects of the tyrosine kinase inhibitors, genistein and herbimycin [41, 42], as well as the phosphatase inhibitors, vanadate and okadaic acid [39, 43] on the basal and phorbol ester-induced levels of p85. The genistein (30/ag/ml) and herbimycin (0.3 ~tg/ml)concentrations used caused maximal inhibition of IL2 production in sensitive EL4 cells [Sando J. and Holman G., manuscript submitted] and 15 laM okadaic acid was previously shown to activate MEK activity in EL4 cells [37]. Neither genistein nor herbimycin had any detectable effect on basal or phorbol ester-stimulated levels of the 85,000 Mr phosphotyrosine band. In contrast, vanadate and okadaic acid blocked the phorbol ester-induced increase in the p85 signal and caused a decrease in the basal level of p85 detected. In resistant, as well as sensitive EL4 cells, a band just below p85 was induced after 20 min of phorbol ester treatment. In addition, both vanadate and herbimycin pretreatment induced the appearance of this band (most obvious in the exposure shown for resistant cells). Vanadate pretreatment also caused an increase in the phosphotyrosine signal of bands of 56,000-63,000 Mr in resistant cells.

Identity of p85 The possible identity of p85 was investigated by western analysis with antibodies specific for vav [44], HS1 [45], cortactin [46], p85 MAP kinase [47], interferon-stimulated gene factor 3 (ISGF3) [48] and cPLA2 [49]. These studies revealed the absence of cortactin in sensitive and resistant EL4 cells and showed that p85 did not co-migrate with any of the proteins tested (data not shown). In addition, p85 did not co-migrate with PCK-~5 (data not shown), which was recently demonstrated to undergo tyrosine phosphorylation in murine keratinocytes expressing an oncogenic

Early phorbol ester-stimulated events in ELA cells

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A.F. RICHARDSON and J. J. SANDO

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A.F. RICHARDSON and J. J. SANDO

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A.F. RICHARDSON and J. J. SANDO

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Fig. 6. Effects of genistein, herbimycin, vanadate and okadaic acid on the phorbol ester-induced increase in the 85,000 Mr tyrosine phosphoprotein band. EL4 cells were either untreated or incubated for 30 min with: 30 pg/ml genistein (Gen), 300 ng/ml herbimycin (Herb), 1 mM vanadate (Van) or 15 ktM okadaic acid (OKA). Following the incubations, either cell lysates were prepared or cells were treated with 150 nM PDB for the times indicated in the figure. Lysates were analysed by electrophoresis on 10% polyacrylamide gels followed by blotting with anti-phosphotyrosine antibodies. Results shown are representative of two (genistein, herbimycin and okadaic acid) or three (vanadate) separate experiments. (A), sensitive cells; (b), resistant cells. The resistant blot has been overexposed relative to the sensitive.

Early phorbol ester-stimulatedevents in EL4 cells ras Ra gene [50], or with PKC-0 and PKC-rl (data not shown), p85 did co-migrate with the 85,000 Mr subunit of PI3K [51]. PI3K was detected in both cell lines and relatively higher levels were observed in the resistant cell line. To determine whether p85 was PI3K, immunoprecipitation studies with antibodies specific for PI3K and phosphotyrosine were performed. The PI3K antibody immunoprecipitated the 85,000 M~ subunit of PI3K but this protein was not recognized by the phosphotyrosine antibody (data not shown). The phosphotyrosine antibody, however, was only able to immunoprecipitate a small proportion (< 10%) of p85, and no reactivity of this protein with the PI3K antibody was observed. DISCUSSION The results presented in this study reveal a temporal sequence of phorbol ester-stimulated signalling events in phorbol ester-sensitive EL4 cells and a number of deficiencies in an EL4 variant which is resistant to phorbol ester stimulation. The earliest event detected after stimulation of the sensitive cells was an increase in amount of tyrosine phosphorylated p85 (within 30 s). This was followed by the induction of polyacrylamide gel mobility shifts of raf-1, p44 MAP kinase and MEK (3 min) and electrophoretic shifts of lck and ZAP-70 (5 min) suggestive of phosphorylation [20, 52]. Resistant cells exhibited similar MAP kinase and MEK mobility shifts; however, several other events were either deficient (p85), or delayed, as demonstrated for mobility shifts of lck (7 min) and raf-1 (7 min), suggesting defective phosphorylation events. Other resistant cell defects include the lack of induction of c-Jun and Fra, although c-Fos, Fos-B, Jun-B and Jun-D were induced as in the sensitive cells [36]. In addition to p85, a number of other tyrosine phosphorylated proteins were detected in sensitive and resistant cells (Fig. 1). The 44,000 and 45,000 Mr tyrosine phosphoproteins most likely represent p44 MAP kinase, which exhibits phorbol esterinduced mobility shifts due to phosphorylation (Fig. 3a) [40]. The observation that phorbol ester treatment induced a MAP kinase mobility shift in

27

both sensitive and resistant cells contrasts with a previous investigation [37] which demonstrated that phorbol ester treatment induced activation and a mobility shift of p38 MAP kinase in sensitive EL4 cells only. This difference suggests that not all forms of MAP kinase present in resistant EL4 cells exhibit defective phosphorylation. Indeed, a large number of MAP kinase species has been reported to date and immunoblotting with an antibody specific for p42, p44, p54 and p90 MAP kinases demonstrated the presence of all four of these kinases in both sensitive and resistant EL4 cells (data not shown). Phorbol ester-induced lck mobility shifts have been reported previously [20] and these are clearly induced during the time course of phorbol ester treatment in both sensitive and resistant cells (Fig. 4d). It appears that the higher molecular weight forms of lck are tyrosine phosphorylated only in sensitive EL4 cells since phosphotyrosine immunoblotting revealed an identical set of bands at 55,000--65,000 M r in sensitive EL4 cells alone (Fig. 4a). Thus, the 56,000 Mr tyrosine phosphoprotein apparent in sensitive EL4 cells (Fig. 1) probably represents the 56,000 Mr lck tyrosine kinase. This difference between the two cell lines could not be explained by the absence of the lck tyrosine kinase c-src kinase (csk) [53] in resistant cells, since western analysis revealed similar amounts of csk in both cell lines (data not shown). However, the possibility of an enhanced tyrosine phosphatase activity in resistant cells is suggested by the appearance of phosphotyrosine bands at 55,000-60,000 Mr after vanadate treatment of these cells. The identity of the other bands detected after phosphotyrosine immunoblotting of sensitive and resistant EL4 cell lysates remains unknown. MEK, which requires phosphorylation for activation [25], exhibited a small mobility shift after phorbol ester treatment of both sensitive and resistant EL4 cells. An earlier investigation showed that a MEK protein isolated from a phorbol estertreated resistant EL4 cell line was inactive [37]. At least two forms of MEK are known to exist [54] and it is possible that the MEK detected in this study represents a different and phorboi ester-

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A.F. RICHARDSONand J. J. SANDO

activatable form. Another possibility is that the cell lines themselves may differ; a number of EL4 cell lines have been described [55]. MEK activity has been shown to be stimulated by the raf-1 serine/threonine kinase [27, 28] and raf-1 itself is both phosphorylated and activated by PKC [29-31 ]. Raf-1 phosphorylation is associated with a mobility shift [29] and this effect is readily observed after phorbol ester treatment of sensitive EL4 cells. A similar shift in resistant cells required a longer incubation with phorbol ester, possibly suggesting a delayed activation of an upstream kinase. Alternatively, raf-1 may also be a substrate for a downstream kinase and only this phosphorylation is detected in the resistant cells. A good candidate for this role is MAP kinase which has been shown to phosphorylate raf [56] and at least one form of MAP kinase, p44, appears to be activated in the resistant cell line prior to raf-1 phosphorylation (Fig. 3a). The most dramatic difference between sensitive and resistant EL4 cells reported here is the presence of a tyrosine phosphorylated 85,000 Mr protein in sensitive EL4 cells only. Whether p85 is absent in the resistant EL4 cell line or is merely not tyrosine phosphorylated is not yet clear. The rapid phorbol ester-stimulated increase in the amount of tyrosine-phosphorylated p85 suggests that changes in the phosphorylation level rather than in the amount of protein occur. Indeed, the ability of vanadate, a tyrosine phosphatase inhibitor [39], and okadaic acid, a serine/threonine phosphatase inhibitor [43], to block the phorbol ester-induced increase in the 85,000 M r band argues for control at the level of phosphorylation. A potential candidate for the site of action of these phosphatase inhibitors is the membrane-associated tyrosine phosphatase CD45, which is required for the dephosphorylation of lck resulting in an increased activity [57]. CD45 undergoes serine phosphorylation in a cytokine-stimulated murine T cell line and, although the serine phosphorylation does not increase CD45 activity, it has been suggested that it may allow CD45 to assume a conformation to allow it to interact with a new set of substrates within the cell [58]. Thus, if okadaic acid prevents dephosphorylation of

CD45 it may alter the association of CD45 with a kinase responsible for p85 tyrosine phosphorylation. The possibility that defective p85 tyrosine phosphorylation in resistant cells is due to a lack of CD45 was discounted after fluorescence activated cell sorting revealed similar levels of CD45 in both cell lines (data not shown). Interestingly, a phosphotyrosine band just below p85 is induced after 20 min of phorbol ester treatment in both sensitive and resistant cells and this is readily observed after herbimycin and vanadate pretreatment of resistant cells. The identity of this band is unknown. Since an increase in the 85,000 M r phosphotyrosine band is the earliest event detected after phorbol ester stimulation of sensitive EL4 cells, characterization of this protein and its regulation should help elucidate some of the earliest events in this PKC pathway. Initial results described here have compared p85 to a number of known signalling proteins of approximately 85,000 Mr by western blotting and immunoprecipitation. The results suggest that p85 is unlikely to represent the 85,000 Mr subunit of PI3K, and does not represent vav, HS 1, cortactin, cPLA 2, PKC-8, PKC-0, PKCrl, p90 MAP kinase, or ISGF3. The recently described 85,000 Mr nuclear protein found in sensitive EL4 cells is also unlikely to represent p85 since the nuclear protein did not undergo tyrosine phosphorylation [59]. Thus, purification and cloning of p85 will be necessary in order to test for a possible role in IL2 production or other downstream events.

Acknowledgements--We thank Drs J. Clark, T. Parsons and M. Weber for the generous gifts of cPLA 2, csk and MAP kinase antibodies, respectively. This work was supported by Health and Human Services grant DK 40031. REFERENCES 1. Smith K. A. (1988) Science 240, 1169-1176. 2. June C. H., Fletcher M. C., Ledbetter J. A. and Samelson L. E. (1990) J. lrnmunol. 144, 1591-1599. 3. Trevillyan J. M., Lu Y., Atlura D., Phillips C. A. and Bjorndahl J. M. (1990) J. Immunol. 145. 3223-3230.

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