Eur. J. Immunol. 1992. 22: 457-462 S6 kiriase in Tccll activation 457

Victor CaIvov., Barbara E. Bierermoo and Terry A. VikmAo

Division of Pediatric Oncology., Dana-Farber Cancer Institute, Hematology-Oncology Division., Brigham and Women’s Hospital, The Children’s HospitalA, Departments of Pediatrics. and Medicineo, Harvard Medical School, Boston, and Department of Cellular and Developmental BiologyA, Harvard University, Cam bridge

Tcell receptor activation of a ribosomal S6 kinase activity*

Stimulation of the Tcell receptor-CD3 complex activates multiple signal transduction pathways, including serinekhreonine and tyrosine protein kinases. Stimulation of the human T cell line Jurkat via the T cell receptor-CD3 complex with anti-CD3 monoclonal antibody or incubation with the tumor promoter phorbol 12-myristate 13-acetate caused increases in S6 kinase and microtubule- associated protein 2 (MAP) kinase activities. An S6 kinase activity that was able to phosphorylate exogenous 40s ribosomal S6 protein was recovered in immunoprecipitates obtained using a 90-kDa ribosomal S6 kinase-specific antiserum and thus represents activation of a member of the 90-kDa ribosomal S6 kinase family. Stimulation of the S6 kinase activity correlated with an increase in a kinase activity able t o phosphorylate exogenous 90-kDa ribosomal S6 kinase (rsk) attributed to a MAP kinase activity. These increases in S6 and MAP kinase activities further correlated with the appearance of a 42-kDa phosphoprotein detected by anti-phosphotyrosine immunoblotting. However, while the tyrosine phosphorylation of the 42-kDa protein and the MAP kinase activity are dependent on protein kinase C activity, residual S6 kinase activity can be detected following protein kinase C depletion and subsequent anti-CD3 stimulation.Thus, T cell activation through theT cell receptor-CD3 complex results in activation of a member of the 90-kDa S6 kinase family which correlates with, but can be independent of, MAP kinase activation.

1 Introduction

The TcR-CD3 complex plays a central role in Tcell activation and function [1, 21. Triggering of the TcR-CD3 complex has been shown to stimulate tyrosine kinase activity [3]. Additionally,TcR activation has been shown to activate phosphoinositide-specific phospholipase C, gener- ating two second messengers, inositol phosphates and diacylglycerol [4]. Elevation of these metabolites leads to an increase in intracellullar calcium concentration and activation of serine/threonine specific PKC [S, 61. Although serinehhreonine as well as tyrosine phosphoryla- tion events appear to be essential for Tcell activation [7], the molecular pathways by which these initial activities result in later functional responses are incompletely defined.

Phosphorylation on multiple serine residues of the 40s ribosomal subunit protein termed S6 is a highly conserved response of animal cells to a variety of external stimuli, including growth factors (epidermal growth factor, platelet-

[I 97701

4: This work was supported by grants from the American Heart Association (B.E.B.), the James S. McDonnell Foundation (B.E.B.) and the NIH (B.E.B.,T.A.V.). Supported by a posdoctoral fellowship of the Spanish Ministry for Education and Science. Present address: Riley Hospital for children, Room 2612, Indianapolis, IN 46202, USA

Correspondence: Victor Calvo, Division of Pcdiatric Oncology, Room D1610B, Dana-Farber Cancer Institute, 44 Binney Street, Boston MA 02115. USA

Abbreviations: MAP: Microtubule-associated protein 2 rsk: YO-kDa ribosomal S6 kinase

derived growth factor) and hormones (progesterone, insu- lin) (reviewed in [S]). Phosphorylation of S6 has been suggested to increase the efficiency of mRNA translation [Y, lo]. Two major classes of S6 kinases have been charac- terized: the 90-kDa family (termed rsk) and the 70-kDa family [S]. cDNA clones corresponding to members of these two groups have recently been isolated [ll-131, and both classes of S6 kinases are activated by phosphorylation on serinekhreonine but not tyrosine residues [S]. While upstream activators and downstream targets of the S6 kinases are incompletely defined, a microtubule-associated protein 2 (MAP) kinase of - 42-kDa appears to be an upstream regulator of the 90-kDa S6 kinase. In vitro, an insulin-stimulated - 42-kDa MAP kinase from preadipo- cytes has been shown to phosphorylate and activate a 90-kDa S6 kinase [14]. Unlike 00-kDa S6 kinase, phospho- rylation on both tyrosine and serine/threonine residues appears to be required for MAP kinase activity [15].

In this report, we show that stimulation of Tcells via the TcR-CD3 complex does not induce an increase in total S6 phosphorylation. However, using a specific antiserum to detect the 90-kDa rsk family of S6 kinases, an increase in S6 kinase activity was observed upon TcR-CD3 triggering that corresponded to activation of the 90-kDa-related S6 kinase(s). We then analyzed MAP kinase activation using a recombinant 90-kDa S6 kinase [16] as an in vitro substrate and observed that stimulation through the TcR activates a MAP kinase able to phosphorylate a 90-kDa S6 kinase. MAP kinase activation correlated with the appearance of a - 42-kDa tyrosine phosphorylated protein as detected by antiphosphotyrosine immunoblotting. Both the tyrosine phosphorylation and MAP kinase activity were shown to be dependent on PKC and abrogated by PKC depletion. However, after PKC depletion. a small but clearly detect- able increase in 90-kDa S6 kinase activity was observed after TcR-CD3 stimulation, suggesting the existence of multiple pathways of S6 kinase activation.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992 0014-29 8O/Y2/O202-0457$3.50 + .25/0

458 V. Calvo, B. E. Bierer and T. A.Vik Eur. J. Immunol. 1992. 22: 457-462

For antiphosphotyrosine immunoblotting, an aliquot cor- responding to 8 x 106 cells was washed once in cold PBS containing 0.4 mM sodium vanadate, 5 mM EDTA, 10 mM NaF and 10 mM sodium pyrophosphate. Cells were recov- ered by centrifugation and lysed on ice in 0.1 ml of lysis buffer B (50 mM Tris-HC1, pH 7.6, 300 mM NaCl, 0.5% Triton X-100, 4 mM PMSF, 1.5 U/ml aprotinin, 10 pg/ml leupeptin, 10 mM iodoacetamide, 1 mM sodium vanadate, 5 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate) for 30 min. After a 15-min centrifugation in a microcentri- fuge at 4 "C, the protein concentration in the supernatants was determined by a colorimetric method [21]. Equivalent protein amounts of each supernatant were resolved by reducing 10% SDS-PAGE. Resolved proteins were trans- ferred to nitrocellulose membranes and developed using the anti-phosphotyrosine mAb 4G10 (provided by Dr. B. Druker, Dana-Farber Cancer Institute, Boston, MA) and anti-mouse IgG alkaline phosphatase-conjugated anti- body as recommended by the manufacturer (Promega, Madison, WI). 4G10 is a murine mAb generated using phosphotyramine as the immunogen and has been pre- viously shown to be specific for phosphotyrosine [22] .When used for immunoblotting, the addition of 1 mM phospho- tyrosine completely eliminates all immunoreactive bands, while phosphoserine or phosphothreonine have no effect P I .

2 Materials and methods

2.1 Cells

The human T leukemia cell line Jurkat (American Type Culture Collection, ATCC, Rockville, MD) was cultured in RPMI 1640 medium supplemented with 6% heat-inacti- vated FCS, 2 mM L-glutamine, 10 mM Hepes, pH 7.2, and 50 p~ 2-ME (termed RPMI-6%FCS), either in the absence or the presence of PMA (50 ng/ml).

2.2 Stimulation of cells, in vitro kinase assays, and antiphosphotyrosine immunoblotting analysis

Aliquots of 10' Jurkat cells were resuspended in 1 ml of RPMI-6Y0FCS and either left untreated or stimulated for the indicated times with either the anti-CD3 mAb OKT3 (ATCC, 1/100 dilution of ascites fluid) or PMA (50 ng/ml) at 37 "C. The cells were divided into two aliquots, one for in vitro kinase assays and one for anti-phosphotyrosine immu- noblotting.

For in vitro kinase assays, an aliquot corresponding to 2 x lo6 cells was spun and lysed by resuspending the cell pellets in 0.3 ml of lysis buffer A (10 mM potassium phosphate, pH 7.05, 0.5% Triton X-100, 5 mM EGTA, 10 mM MgC12, 50 mM P-glycerophosphate, 1 mM sodium vanadate, 2 mM DTT, 40 pg/ml PMSF). After 30 rnin on ice, nuclei were pelleted by a 5-min centrifugation in a microcentrifuge at 4 "C and supernatants were used for kinase assays.

For the detection of total S6 kinase activity, direct S6 phosphorylation assays were performed with 5 p1 of super- natant in a total volume of 20 pl containing 20 mM Tris- HCl, pH 7.25, 10mM MgC12, 5 0 p ~ ATP with 5 yCi = 185 kBq [Y-~~PIATP, and 40s ribosomal subunits as substrate (0.1-0.25 mg/ml) prepared from Xenopus luevis as described [17]. For S6 kinase assays specific,for 90-kDa S6 kinase (referred to as rsk), 150 p1 of supernatant were incubated with 5 p1 of a specific rabbit antiserum (termed 125) raised against recombinant Xenopus 90-kDa S6 kinase produced in bacteria [MI. This antiserum is specific for 90-kDa S6 kinase as shown in studies performed in Xenopus and chicken cells [19, 201. This anti-90-kDa S6 kinase antiserum recognizes both activated and unacti- vated 90-kDa S6 kinase [19,20]. Immunocomplexes were adsorbed to Staphylococcus aureus and washed as previous- ly described [16]. S6 phosphorylation assays were per- formed with the immunocomplexes in the conditions described above.

To assay for MAP kinase activity, 5 yl of supernatant were used in a total reaction volume of 20 pl containing 20 mM Tris-HC1, pH 7.25, 10 mM MgC12, 100 pM ATP with 5 pCi [Y-~~PIATP, and - 1 pg of unactivated Xenopus 90-kDa S6 kinase. The protein was partially purified from insect cells infected with recombinant baculoviruses expressing a Xenopus rsk cDNA [16].

In all cases kinase reactions were carried out at 30°C for 15 rnin and stopped by boiling in electrophoresis sample buffer. Proteins were resolved by SDS-PAGE and analyzed by autoradiography with Kodak X-OMAT film.

2.3 Column chromatography

Aliquots of 3 x 10* Jurkat cells, either unstimulated or following stimulation with OKT3 or PMA for 5 rnin as described above, were lysed in 8 ml of lysis buffer A by homogenizing for 25 strokes in a Dounce homogenizer. Lysates were clarified by a 100000 x g centrifugation for 30 rnin at 4 "C. Supernatants were applied to a 1 ml Mono-Q column (Pharmacia, Piscataway, NJ) equilibrated in buffer containing 20 mM Tris-HC1, pH 7.2, 1 mM EDTA, 50 mM p-glycerophosphate, 100 pM sodium vanadate and 1 mM 2-ME. Bound proteins were eluted with a 40ml NaCl gradient and l-ml fractions were assayed for total S6 kinase, 90-kDa S6 kinase, and MAP kinase activities as described above. Following autoradiography, the recombinant 90- kDa S6 kinase band was excised from the gel and radioac- tivity was quantitated by Cherenkov counting. The pres- ence of phosphotyrosine proteins was analyzed by immu- noblotting as described above.

3 Results and discussion

Jurkat cells were stimulated with the antLCD3 mAb OKT3 or PMA for various times. Cell lysates were assayed in vitro for total S6 kinase activity using as substrate S6-containing 40s ribosomal subunits. No significant increase in total S6 kinase activity was observed (Fig. 1A). When S6 kinase activity was measured in anti-rsk immunoprecipitates a significant increase in S6 kinase activity could be detected as early as 2.5 min after OKT3 stimulation and 0.5 min after incubation with PMA (Fig. 1 B).Thus, stimulation of Tcells through the TcR-CD3 complex activates a 90- kDa-like S6 kinase, while the activity of other S6 kinase(s) seems unaffected.

Eur. J. Immunol. 1992. 22: 457-462

OKT3 PMA t I 1 I

0 0.5 2.5 5 15 30 60 120 0.5 2.5 5 15 30 60 120 (rnin.)

(A1

+ rsk

S6 kinase in Tcell activation 459

1 2 3 4

- 110

- 70

- 45 4- PP42

- 44

- 29

Figure I . Time course of activation of S6 kinase and MAP kinasc and the appearance of a 42-kDa phosphotyrosine protein. Jurkat cells were stimulated with mAb to the TcR-CD3 complex (anti-CD3, OKT3) or PMA for the indicated times. Lysates were analyzed for (A) total S6 kinase activity, (B) S6 kinase activity due to 90-kDa S6 kinase, (C) MAP kinase activity, or (D) induction of tyrosine phosphorylation. For the in vitro kinase assays (A-C), only the portions of the gels containing the corresponding substrates (indicated on the right) are shown. For the antiphosphotyrosine immunoblot (D), the positions of the 42-kDa substrate and molecular weight markers (in kDa) are indicated on the right. (E) Jurkat cell lysates were analyzed for induction of tyrosine phosphorylation as described in section 2.2, except that a 7.5% gel was used. Cells were either left untreated (lane l), or incubated with OKT3 (lane 2), or PMA (lanes 3 and 4) for 5 min. No incubation with anti-phosphotyrosine antibody was performed in lane 4. The positions of molecular weight markers are indicated on the right. The band migrating below the 44-kDa marker which is strongly induced upon incubation with OKT3 or PMA treatment corresponds to the 42-kDa substrate.

In parallel, MAP kinase activity was measured in the lysates using as substrate partially purified Xenopus 90-kDa S6 kinase produced in a baculovirus expression system. PMA treatment and TcR stimulation with anti-CD3 mAb resulted in a marked increase in rsk phosphorylation, revealing the activation of a MAP kinase (Fig. 1C). Analysis of tyrosine phosphorylated proteins by immuno- blotting of SDS-PAGE resolved cell lysates with an anti- phosphotyrosine mAb revealed the rapid appearance of a band corresponding to an M, of - 42-kDa (pp42, Fig. 1 D). This - 42-kDa phosphotyrosine protein detected by immu- noblotting correlated temporally and in intensity with MAP kinase activity detected by rsk phosphorylation (Fig. 1 C).

To correlate further MAP kinase activity with the 42-kDa tyrosine phosphoprotein, lysates from cells stimulated for 5 min with either anti-CD3 mAb or PMA were resolved on an anion exchange chromatography column. Fractions were assayed for the ability to phosphorylate recombinant 90-kDa S6 kinase (Fig. 2 ) . Low levels of activity were detected in fractions from unstimulated cells. In contrast, the MAP kinase activity profile of anti-CD3-stimulated cell lysates showed a peak encompassing fractions 8 to 13 (Fig. 2 A , 2B). An identical elution profile was obtained when PMA-stimulated lysates were analyzed (Fig. 2 A, 2 B). The differences in activity were not due to differences

in the protein content of the column fractions, as monitored by the A280 profiles of the chromatographic separation (Fig. 2C). The asymmetric shape of the MAP kinase activity peak suggests that it may contain more than one of several related enzymes, as described recently in neural cells [23]. cDNA clones corresponding most likely to several - 42-kDa MAP kinases have recently been isolated [23]. Northern blot analysis using these probes has detected several different transcripts in lymphoid tissues [23], raising the possibility that several MAP kinases are involved in signal transduction pathways in lymphocytes. Anti-phos- photyrosine immunoblotting of column fractions showed that a tyrosine-phosphorylated protein co-eluted with the MAP kinase activity in the fractions from stimulated cell extracts, although the high salt concentration of the samples caused distortion of the protein bands (data not shown).

The column fractions were also assayed for S6 kinase activity (data not shown). The major peak of stimulated activity, identified as the 90-kDa S6 kinase by immunopre- cipitation and subsequent in vitro kinase assay, eluted earlier (fractions 5-7) than the MAP kinase activity (fractions 8-13). It was necessary to demonstrate this separation, as this S6 kinase appears to autophosphorylate in vitro (T. Vik, unpublished data). S6 kinase activity that was neither stimulated nor immunoprecipitated by the

460

anti-rsk antiserum eluted later (fractions 23-27) and was probably due to an S6 kinase of the 70-kDa family [24]. These data additionally support the hypothesis that stimu- lation through theTcR-CD3 complex results in activation of a 90-kDa S6 kinase present in Jurkat T cells, while the basal level of 70-kDa S6 kinase activity remains unchanged.

V. Calvo, B. E. Bierer and T. A.Vik Eur. J. Immunol. 1992. 22: 457-462

The observation that direct activation of PKC with the phorbol ester PMA induced the same effects as TcR-CD3 stimulation on S6 kinase and MAP kinase activities

prompted us to investigate the role of PKC in TcR-CD3 activation of S6 and MAP kinases. Jurkat cells were either left untreated or treated chronically with PMA for 44 h to deplete them of active PKC and then stimulated with anti-CD3 mAb or PMA for 5 min. Cell lysates were assayed for 90-kDa S6 kinase and MAP kinase activities and analyzed by anti-phosphotyrosine immunoblotting (Fig. 3). The levels of PKC activity in PMA-pretreated cells were < 10% of those present in untreated cells as deter- mined by a histone H1 in vitro kinase assay (data not shown).

Fraction Number

(B)

OKT3

PMA

8 9 10 11 12 13

Fraction #

0.0 4 6 8 10 12 14 10 18 20 22 24 28 28 30 32 34 36 38

Fraction Number

Figure 2. Column chromatography of Jurkat cell lysates. (A) Graphic representation of the peaks of MAP kinase activity in unstimulated (O) , OKT3 stimulated (H), or PMA stimulated (A) Jurkat cells. Fraction numbers and NaCl gradient are indicated and activity is expressed in terms of pmoles phosphate incorporated into rsk/min/ml. (B) Autoradiogram of phosphorylated rsk over the peak fractions of MAP kinase activity. Fraction numbers and stimuli are as indicated. (C) profile of the chromatographic separation. Only the untreated cell profile is shown, since the ones corresponding to OKT3 and PMA-treated cells were virtually superimposable.

Figure 3. Effect of down regulating PKC activity by chronic PMA treatment of cells. Jurkat cells were grown in the absence or presence of PMA at 50 ng/ml for 44 h. Aliquots were then pelleted immediately or stimulated as described in the text with OKT3 or PMA for 5 min prior to pelleting. Lysates were made and supernatants were immunoprecipitated with anti-rsk antiserum and assayed for in vitro S6 kinase activity (A), assayed for MAP kinase activity (B), or analyzed by immunoblotting with anti- phosphotyrosine mAb (C). The presence or absence of chronic PMA as well as the acute stimuli used are as indicated. The positions of the substrates of the kinase assays (A, B) and the tyrosine phosphorylated 42-kDa protein (C) are indicated on the right.

In untreated cells stimulated with anti-CD3 mAb (lane 2) or PMA (lane 3), 90-kDa S6 kinase (Fig. 3A) and MAP kinase (Fig. 3B) activities were present, along with the 42-kDa tyrosine phosphoprotein (Fig. 3 C). However, in PMA-pretreated cells, neither anti-CD3 (lane 5) nor PMA (lane 6) induced MAP kinase activity (Fig. 3B) or the appearance of the 42-kDa tyrosine phosphoprotein (Fig. 3C). Interestingly, anti-CD3 mAb but not PMA incubation of PMA-pretreated cells induced detectable activation of 90-kDa S6 kinase (Fig. 3 A). Quantitation by multiple scanning densitometries showed mean relative

Chronic PMA - - - + + +

-4- S6

C rsk (B)

(C)

Eur. J. Immunol. 1992. 22: 457-462 S6 kinase in Tcell activation 461

values of 1.00,2.57,2.47,0.87,1.44, and 0.69 for lanes 1-6, respectively in Fig. 3 A. Thus, the presence of active PKC appears to be required for TcR-CD3-mediated activation of MAP kinase within the limits of detection of the in vitro MAP kinase assay. In contrast, some degree of S6 kinase activation appears to be inducible by TcR-CD3 triggering in PKC-depleted cells.

Activated MAP kinase isolated from various cell types requires both serinetthreonine and tyrosine phosphory- lated residues [15]. The results of this PKC depletion experiment suggest that PKC-dependent activation of a tyrosine kinase is required to induce MAP kinase activity in T cells stimulated via theTcR-CD3 complex. This is consist- ent with previous reports in several cell types including lymphoid cells [25, 261. Nevertheless, multiple pathways may lead to 90-kDa S6 kinase activation after TcR-CD3 stimulation, and some of them may be PKC independent as suggested here. For instance, in Swiss 3T3 fibroblasts, platelet-derived growth factor stimulation of 90-kDa S6 kinase activity appears to have a greater dependency on PKC than epidermal growth factor stimulation [27]. Two mouse cDNA clones corresponding to 90-kDa S6 kinases have been described [18], suggesting the existence of several members of the 90-kDa S6 kinase family within a given species. Thus, more than one 90-kDa S6 kinase may exist and become activated in human T cells, and some of these activation processes may be PKC independent.

The current view of Tcell activation via the antigen receptor includes the participation of both serinekhreonine and tyrosine protein kinases. The fact that both types of phosphorylation are required to maintain MAP kinase activity has led to the suggestion that MAP kinase may be able to integrate signals from two different kinase pathways to elicit further downstream events [ 151.This view has been challenged by recent reports showing that: (a) MAP kinase autophosphorylates on tyrosine and threonine residues [28] and (b) a MAP kinase activator is itself a kinase regulated exclusively by serinehhreonine phosphorylation [29]. Nev- ertheless, since several members of a kinase family that includes MAP kinase have recently been characterized at the molecular level 1231, it is conceivable that the different members of this family may be activated by pathways differing in the requirement for two types of phosphoryla- tion and/or participation of PKC.

Several protein kinases and phosphatases have been shown to be relevant in Tcell activation and may therefore regulate MAP kinase activity [3, 4, 30, 311. Fyn is one candidate for the tyrosine kinase(s) involved in MAP kinase activation, because of its physical association with TcR-CD3 complex polypeptides [32]. The tyrosine kinase Ick is also likely to be involved in this pathway, since perturbing the CD4-lck complex has been shown to modulate MAP kinase activation [33]. The data presented here as well as previous observations [26] suggest that the serinekhreonine kinase PKC itself or a PKC-dependent kinase are involved in MAP kinase activation. CD45, a T cell surface glycoprotein containing a tyrosine phospha- tase cytoplasmic domain, is associated with the TcR-CD3 complex [30, 31, 341, is able to deactivate MAP kinase in vitro [15] and is able to regulate MAP kinase activation in Tcells [3S]. Whether CD4S also regulates 90-kDa S6 kinase activation remains to be determined.

The results presented here demonstrate that TcR stimula- tion induces the activation of a MAP kinase that is able to phosphorylate a 90-kDa S6 kinase, but other upstream regulators of 90-kDa S6 kinase activity may exist in T lymphocytes. Besides the detected increase in translation efficiency [9, 101, little is known about the functional consequences of S6 kinase activation. In vitro substrates for S6 kinase appear to include lamin C, a component of the nuclear matrix involved in its disassembly during mitosis [36]. The assessment of the physiological relevance of these and other cellular phosphorylation substrates will open new perspectives on the role of S6 kinase in signal transduction pathways leading to cell division and differentiation.

We thank 7: Roberts, B. Druker and A. Currera for rnAD 4G10 and helpful suggestions, 7: J. Martin5 for technical assistance and S. J. Buraltoff and R. L. Erikson f o r support and critical review of this manuscript.

Received July 18, 1991; in revised form October 7, 1991.

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