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The Journal of Immunology, 1998, 160: 7-11.
Copyright © 1998 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: p21-Activated Kinase (PAK) Is Required for Fas-Induced JNK Activation in Jurkat Cells1 ,2

Thomas Rudel3, Frank T. Zenke, Tsung-Hsien Chuang and Gary M. Bokoch4

The Scripps Research Institute, Departments of Immunology and Cell Biology, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The process of apoptosis is a critical component of normal immune system development and homeostasis, and in many cells this involves signaling through the c-Jun amino terminal kinase (JNK) pathway. In Jurkat T cells, Fas-induced JNK activity is dependent upon activation of the caspase cascades known to be central components of the apoptotic program. We show in Jurkat cell lines expressing a dominant negative PAK construct that PAK signaling is necessary for JNK activation in response to Fas receptor cross-linking. Inhibition of JNK activation induced by Fas does not impair cell death as assessed by DNA fragmentation. However, expression of the catalytically active C terminus of PAK2, which is generated through caspase action during Fas-mediated apoptosis, induces Jurkat cell apoptosis. We conclude that PAK activity resulting from caspase-mediated cleavage is a necessary component of JNK activation induced by Fas receptor signaling and that PAK2 can contribute to the induction of cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis, or regulated cell death, is a fundamental process in the development of multicellular organisms, playing important roles in the control of normal morphogenesis and development of the immune, endocrine, and nervous systems. Apoptosis acts to preserve peripheral T and B cell homeostasis and participates in the removal of immature thymocytes during thymic development and in the elimination of T and B cells following stimulation by autoantigens (1, 2, 3, 4, 5). Fas (CD95), a member of the TNF receptor/nerve growth factor receptor family, is highly expressed in activated lymphocytes, and the ligand for Fas becomes expressed on activated T cells (5, 6). Fas receptors can rapidly initiate the cell death pathway, serving to down-regulate immune reactions, to establish immune-privileged sites, and to mediate target cell killing by CTL (1, 4, 5). Because of the critical role of Fas in immune and developmental processes, abnormalities in its apoptotic signaling can lead to developmental, autoimmune, lymphoproliferative, and inflammatory disorders (5, 7, 8).

All apoptotic cells undergo a similar sequence of characteristic morphologic and biochemical events (9, 10, 11, 12). Current evidence implicates a cascade of ICE5/CED-3 family cysteine proteases (termed caspases; 13) as a common and critical component of the cell death pathway (12, 13, 14, 15). A sequential activation cascade, which is not yet well defined, occurs subsequent to FADD-like IL-1ß-converting enzyme/MORTI-associated CED-3 homolog 1/Caspase 8 activation (16, 17, 18). The DEVD-sensitive CPP32/Caspase 3 is activated from a proto-form through the action of upstream YVAD-sensitive protease(s) (18) and has been strongly implicated as an important downstream mediator of cell death. Inhibition of CPP32 activity by either tetrapeptide inhibitors or CrmA attenuates apoptosis in vitro in a wide array of cell types, and the generation of a CPP32 null mouse has established a critical role for CPP32 in neuronal apoptosis (19, 20). The downstream targets for proteolytic cleavage by CPP32 and related DEVD-sensitive proteases are few, and their role in subsequent apoptotic events remains unclear.

Stimuli that induce cellular stress, including ultraviolet light, inflammatory cytokines, chemotherapeutic drugs, withdrawal of growth factors, etc., often induce apoptosis (21, 22). These agents are also known to effectively induce the activation of JNK (c-Jun amino terminal kinase) and the related p38 kinase (23, 24). Increases in JNK activity have been noted in cells undergoing apoptosis (25, 26, 27, 28, 29, 30), and JNK activity is stimulated by ceramide, a proposed mediator of apoptotic signaling (31). In some cells, defects in the ability to activate JNK or the blockade of JNK signaling pathways can inhibit apoptosis (25, 32, 33). It has been suggested that a balance of signals between the JNK and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathways serves as an important determinant of whether cells survive or undergo apoptosis (33).

The small GTPases Rac and Cdc42 are important upstream regulators of the protein kinase cascades that control JNK activity (34, 35, 36), and we have recently established that expression of constitutively active forms of Cdc42 can induce apoptosis through a mechanism requiring signaling through JNK (37). One of the immediate downstream mediators of Rac and Cdc42 in this pathway are the p21-activated kinases (PAKs)(36, 38). PAKs are serine/threonine kinases that bind Rac or Cdc42 only when these GTPases are in the GTP-bound active form (39, 40). PAKs contain a catalytic C-terminal domain that is highly homologous to the kinase domain of Ste20 in Saccharomyces cerevisiae, and a family of mammalian Ste20-related kinases appears to exist (41). We have recently described that PAK2 is proteolytically cleaved and activated through the action of a DEVD-sensitive protease during Fas-induced apoptosis (42). In the current paper, we show that expression of the constitutively activated PAK2 C terminus induces Jurkat T cell apoptosis and that PAK activity is required for activation of the JNK pathway by Fas receptor in Jurkat T cells. These data indicate that PAK2 may be an important component of cell death signaling.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Jurkat E6 T cells were routinely cultivated in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, streptomycin, and penicillin at 37°C under a 5% CO2 atmosphere. Ste-20 VI antisera was from Kinetek Biotechnology Corporation, Vancouver, B.C., Canada. pBJ5 vector was from L. Holsinger, Stanford University, and pCMV5-MEKK+ from M. Karin, University of California at San Diego. The Jurkat cell lines (TD8, TA12) expressing dominant negative PAK1(H83L, H86L, and K299R) under the control of the IPTG-inducible Lac repressor system were prepared and handled as described previously (42). Transient transfections were performed as described previously (37).

JNK Assay

An amount equal to 3 x 106 cells was lysed in 500 µl of cell extraction buffer (CEB = 25 mM HEPES, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 0.5 mM PMSF, 0.5 µg/ml leupeptin, 5 µg/ml aprotinin), and lysates clarified by centrifugation at 14,000 x g for 10 min. Ten micrograms of glutathione S-transferase (GST)-cJun (aa 1–79) coupled to glutathione Sepharose beads was added, and JNK kinase reactions were performed as described previously (36, 37). The samples were resolved on a 13% polyacrylamide gel and analyzed using a phosphoimager (Molecular Dynamics, Sunnyvale, CA).

Apoptosis

Apoptosis was induced by adding Fas-IgM Ab (Immunotech Inc., Westbrook, ME) at 150 ng/ml to Jurkat T cells in complete medium for the times indicated. Jurkat T cells were suspended in propidium iodide buffer (PI buffer = 0.1% Na-citrate, 0.1% Triton-X-100, 20 µg/ml propidium iodide) and analyzed by FACS as previously described (43). DNA fragmentation was confirmed by TUNEL assay (42).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of JNK by Fas in Jurkat T cells is dependent upon caspase activity

The stress-activated protein kinase (SAPK/JNK) pathway has been shown to be activated as a result of stress-related, proapoptotic stimuli in a variety of cell types (21, 24, 26). We established that the JNK pathway is induced upon ligation of the Fas receptor in Jurkat T cells. Cells were stimulated by cross-linking the Fas receptor with anti-Fas IgM mAb, and JNK kinase activity was determined (Fig. 1Go, A and B). About 1 h after Fas ligation, a strong JNK activity was observed that peaked at 2 h and declined again after 3 h. The same samples analyzed for JNK activity were also examined for apoptosis by PI staining, and exhibited a significant increase in apoptotic cells after 1 h (Fig. 1GoB). Thus Fas ligation induced a strong activation of JNK which correlated with the onset of apoptosis.



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  		FIGURE 1. Stimulation of JNK activity by Fas cross-linking in Jurkat cells correlates with PAK2 cleavage and is blocked by caspase inhibitors. A, The activity of JNK was evaluated at the indicated times after Fas receptor cross-linking with anti-Fas IgM, as described in Materials and Methods. Activity was dramatically increased by 60 min. Stimulation by PMA is shown as a control. B, The results of A are quantitated and plotted along with the time course of apoptotic cell death, determined as described in Materials and Methods. JNK activation exactly (solid squares) paralleled cell death (solid triangles) and PAK2 cleavage in all experiments (more than three). The corresponding course of PAK2 cleavage is shown in the inset as an immunoblot using the C-terminal-specific Ste20 antiserum to detect the PAK2 C terminal cleavage product (CP). The legend "CPM (x 103)" on the y-axis indicates cpm incorporated into the cJun substrate as determined by Phosphorimager analysis of the kinase assay. C, Fas-stimulated JNK activation was blocked by YVAD-cmk and DEVD-ald (each at 300 µM) in Jurkat cells. JNK activity was determined at 2 h after Fas ligation, while inhibitors were added just before Fas Ab. For each peptide inhibitor, the block of JNK activation correlated to the potential of that inhibitor to block PAK2 cleavage (41).

 
Since the Fas receptor has been shown to be directly coupled to a cascade of ICE-related proteases (caspases), we examined whether JNK activation initiated by cross-linking of the Fas receptor in Jurkat cells was dependent upon caspase activity. We observed that Fas-stimulated JNK activation in intact Jurkat cells was sensitive to inhibition by peptide-derived caspase inhibitors, with inhibition by the YVAD-cmk peptide inhibitor being more effective than inhibition by DEVD-ald at the same peptide concentration (Fig. 1GoC), likely due to the poor membrane permeability of the latter. These data indicate that a substantial portion of JNK activation involves a pathway initiated downstream of and subsequent to activation of the caspase cascade.

PAK activity is required for Fas-induced JNK activation

We previously reported that only PAK2 appeared to be activated by Fas in the Jurkat cell system, and this activation occurred via proteolytic removal of the regulatory N terminus of PAK2 by DEVD-sensitive caspase(s) (42). As shown in Figure 1GoB (inset), JNK activation induced by Fas cross-linking correlated temporally with PAK2 cleavage. We investigated whether PAK2 might be involved upstream in the Fas-induced signaling cascade leading to JNK by constructing Jurkat cell lines (TD8, TA12) stably expressing the PAK1 (H83L,H86L,K299R) dominant negative mutant kinase under an IPTG-inducible promoter. This PAK mutant contains a K299R mutation that inactivates the catalytic activity of the kinase domain; mutation of this site has been demonstrated to induce dominant negative activity when introduced into various signaling kinases (44). The presence of the histidine 83 and 86 mutations in the small GTPase binding site prevents GTPase binding (45) and enables us to avoid potentially complicating effects due to titration of Rac and/or Cdc42 GTPases. Since mammalian PAKs (PAK1, 2, 3) are all over 90% identical in their C-terminal kinase domains, it is likely that the PAK1 (H83L,H86L,K299R) mutant will inhibit downstream signaling of all three PAK isoforms. We therefore examined the effect of expression of this protein on Fas-mediated JNK activation.

PAK1 (H83L,H86L,K299R) was induced about threefold in the stable Jurkat line TD8 in the presence of IPTG as determined by immunoblotting, although background expression levels were also detectable in the absence of IPTG (Fig. 2GoA). Fas ligation in Jurkat TD8 cells resulted only in weak activation of JNK even in the absence of IPTG, where dominant negative PAK was expressed at a lower level (Fig. 2Go, B and C). Induction of further PAK1 (H83L,H86L,K299R) expression with IPTG reduced the JNK activation even below that of the unstimulated Jurkat TD8 control (Fig. 2Go, B and C). JNK expression levels remained similar under each condition. Essentially the same result was obtained using the independently derived TA12 clone expressing PAK1 (H83L,H86L,K299R). In contrast, JNK activation was substantially increased in Fas-treated control Jurkat T cells. Furthermore, the phorbol ester PMA, which activates JNK via a protein kinase C-mediated pathway, effectively induced JNK activation in both control cells and Jurkat TD8 cells, suggesting that Fas activates the JNK pathway specifically via a PAK-dependent pathway.



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  		FIGURE 2. Dominant negative PAK inhibits Fas-stimulated JNK activation in Jurkat cells. A, The expression of dominant negative PAK1 (H83L,H86L,K299R) in stable Jurkat cell lines was assessed by immunoblotting with myc epitope Ab in the presence or absence of IPTG induction. Induced expression in the TD8 line was about threefold, while expression in the TA12 line was at least fivefold. B, JNK activity was determined using a solid phase assay with GST-cJun as substrate in the presence or absence of Fas cross-linking or PMA, and in the presence or absence of IPTG, as indicated. C, Quantitation by phosphoimager analysis of the data shown in B.

 
The PAK2 kinase domain induces apoptosis in Jurkat T cells

We observed that even though JNK activity was completely inhibited after Fas ligation in the IPTG-induced TD8 or TA12 cell lines, there was little or no effect on the rate of apoptotic cell death, nor upon the number of dead cells, as evaluated by PI staining of nuclei and flow cytometry, as well as by TUNEL assay (42). These data indicate that JNK activity is not absolutely required for Fas-induced killing of Jurkat T cells and that the Fas receptor is able to couple to alternative cell death pathway(s) not involving JNK signaling.

Transient expression of the catalytically active PAK2 C terminus in Jurkat cells effectively induced cell death by apoptosis (Fig. 3Go). In contrast, expression of a catalytically inactive mutant of the PAK2 kinase domain (K278R) did not cause apoptosis in the same experiment (Fig. 3Go). As with the active PAK2 C terminus, expression of a constitutively active MEKK, known to induce JNK activation, also produced apoptosis. These results establish that proteolytic activation of PAK2 via removal of the regulatory N terminus is sufficient to induce apoptosis and that this effect is dependent upon PAK catalytic activity.



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  		FIGURE 3. Expression of the catalytically active PAK2 C terminus induces apoptotic death. Jurkat T cells were transiently transfected with the indicated cDNAs, then selected and assayed for apoptotic cell death at the indicated times post-transfection, as described in Materials and Methods. The cDNAs used for transfection were pBJ5 vector control (open bars), pBJ5-PAK2 catalytic domain (closed bars), pBJ5-PAK2K278R catalytic domain (diagonally striped bars), which is rendered catalytically inactive, and pCMV5-MEKK+ (stippled bars), a dominant positive mutant of MEKK. Results shown are the mean +/- SD of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of JNK initiated by Fas cross-linking in Jurkat T cells was blocked by the caspase inhibitors YVAD-cmk and DEVD-ald (Fig. 1GoC), verifying recent reports in several cell types of similar findings (27, 28, 30). Proteolytic activation of PAK2 kinase activity correlated with the onset of JNK activity and with Fas-induced apoptosis (Fig. 1GoB). Jurkat cell lines stably expressing an inducible PAK1 (H83L,H86L,K299R) mutant were almost completely deficient in the ability of the Fas receptor to stimulate JNK activity. This was not due to defects in downstream components of the pathway, because activation induced by phorbol esters was unaffected (Fig. 2Go). These data strongly suggest that PAK2 activation by DEVD-sensitive protease(s)-mediated proteolysis plays a required role in the caspase-initiated JNK activity observed in Jurkat cells and, although not investigated here, likely caspase-induced p38 activation as well.

It has recently been reported that MEKK1, another kinase acting upstream of the JNK pathway, is cleaved and activated during anoikis, a form of apoptosis initiated by loss of cell-substratum contacts (46). Caspase-mediated cleavage and activation of upstream regulatory kinases may be a common theme to ensure the onset and maintenance of JNK activation to induce a proper death response. It remains to be tested whether PAK2 and MEKK1 are cleaved differentially depending upon the apoptotic stimulus and cell type.

In some cells a newly identified adapter protein, termed Daax, couples the Fas receptor to cell death via JNK activation (47). However, in contrast to our model of caspase-dependent, PAK2-regulated JNK activation in Jurkat T cells, this signaling mechanism seems not to be dependent on caspase activity. Such a mechanism may account for the residual YVAD- and DEVD-insensitive JNK activity we observed (see Fig. 1GoC). The intriguing possibility that DAAX may regulate JNK activation via a Rac/Cdc42-mediated PAK activation remains to be examined.

What is the role of PAK2 in cell death responses? Under conditions where JNK activity was blocked, Fas still killed Jurkat cells effectively. These data do not rule out an important role for JNK in regulating apoptosis in response to other known apoptotic stimuli (ceramides, growth factor withdrawal, etc.). We have observed that stimulation of the JNK kinase cascade, by Cdc42 and downstream kinases that lead to JNK and p38 stimulation, can induce regulated cell death (37). Transient expression of the constitutively active PAK2 C terminus induced Jurkat cell apoptosis, and this effect required a catalytically active PAK2 protein (Fig. 3Go). We have not yet been able to determine whether this effect of PAK2 is mediated by JNK activation, however. The current data, as well as that presented in our previous work (42), suggest a model of PAK2 action during the apoptotic response (Fig. 4Go). Since PAK2 is particularly abundant in neuronal tissue and hemopoietic cell lines (39, 40), it may be an important component of JNK-mediated cell death signals in such cells.



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  		FIGURE 4. Model for cell regulation by caspase-activated PAK2. Activation of CPP32 and/or related DEVD-sensitive proteases in response to apoptotic stimuli causes the proteolytic cleavage of PAK2. This leads to the formation of two major PAK2 fragments, cleavage taking place after Asp 212. This site is situated just before the highly conserved catalytic domain of PAK2 and related kinases. Removal of the N-terminal regulatory domain relieves inhibitory constraints on the PAK2 C-terminal fragment (the 34-kDa cleavage product), allowing it to become constitutively active in the absence of Rac or Cdc42. This active kinase can now phosphorylate various substrates, including components leading to stimulation of the SAPK/JNK/p38-signaling cascade and cytoskeletal regulatory substrates, such as myosin. The release of the intact PAK2 N terminus (the 28-kDa cleavage product) may also enhance the affinity of the PXXP SH3-binding motif(s) present in this fragment for regulatory targets, including components that allow PAK to induce cytoskeletal rearrangements (45). Together, the two PAK2 fragments regulate apoptotic signaling cascades and/or the membrane/morphologic changes that are so important for phagocytic disposal of cells in a noninflammatory fashion.

 


   Acknowledgments
 
The authors acknowledge Dr. Yan Wang for excellent technical assistance.


   Footnotes
 
1 T.R. was supported by Deutsche Forschungsgemeinschaft, and T-H.C was supported by an Arthritis Foundation Fellowship award. G.M.B. acknowledges grant support for these studies from the National Institutes of Health and the University of California Breast Cancer Research Program . Back

2 This is publication No. 11093-IMM from The Scripps Research Institute. Back

3 Current address: Max Planck Institut für Infektionsbiologie, Abt. Molekulare Biologie, Berlin, Germany Back

4 Address correspondence and reprint requests to Dr. Gary M. Bokoch, The Scripps Research Institute, Department of Immunology-IMM14, 10550 N. Torrey Pines Road,La Jolla, CA 92037. E-mail address: Back

5 Abbreviations used in this paper: ICE, IL-1ß-converting enzyme; CPP32, 32-kDa cysteine protease; JNK, c-Jun N-terminal kinase; PAK, p21-activated kinase; IPTG, isopropyl ß-D-thiogalactoside; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling; SAPK, stress-activated protein kinase; PI, propidium iodide; MEKK, mitogen-activated protein kinase kinase kinase; caspase, cysteine requiring aspartase protease activity; CED-3, caenorhabditis elegans death gene 3. Back

Received for publication September 15, 1997. Accepted for publication October 20, 1997.


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Top
 Abstract
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 Materials and Methods
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 Discussion
 References
 

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