Stimulation of Jun N-Terminal Kinase (JNK) by Gonadotropin-Releasing Hormone in Pituitary {alpha}T3–1 Cell Line Is Mediated by Protein Kinase C, c-Src, and CDC42

Nurel L. Levi, Tamar Hanoch, Outhiriaradjou Benard, Meirav Rozenblat, Dagan Harris, Nachum Reiss, Zvi Naor and Rony Seger

Department of Biological Regulation (N.L.L., T.H., O.B., M.R., R.S.) The Weizmann Institute of Science Rehovot, 76100 Israel
Department of Biochemistry (M.R., D.H., N.R., Z.N.) George S. Wise Faculty of Life Sciences Tel Aviv University Ramat Aviv 69978, Israel


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in {alpha}T3–1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in {alpha}T3–1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Intracellular transmission of extracellular signals is mediated in large by several groups of sequentially activated protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK) cascades. In growth factor signaling, the key elucidated MAPK cascade is the extracellular signal-regulated kinase (ERK, also known as p42/44 MAPK) cascade (reviewed in Ref.1), which is initiated by the small GTP-binding protein (sGP) Ras. Upon stimulation, Ras assumes its active, GTP-bound form, and recruits the protein kinase Raf-1 to the plasma membrane where the kinase can be activated (2, 3). At this stage, the signal dissociates from the plasma membrane via a sequential activation of protein kinases, which are MEK, ERK, p90RSK, and under some conditions, also glycogen-synthase kinase 3. However, the ERK cascade is not the only link between membranal receptors and their intracellular targets, and in the past years several other ERK-like cascades have been identified (1). One of the best studied of these cascades is the Jun N-terminal kinase [JNK; also known as stress-activated protein kinase ( SAPK) (4, 5)] cascade that utilizes a sequential activation of PAK1/MLK, MEKK1, SEK1/MKK7, and JNK1/2 to activate transcription factors such as c-Jun, ATF2, and Elk1 (reviewed in Refs. 6 and 7). Additional MAPK cascades are the p38RK [HOG, CSBP (8, 9, 10)] and ERK5 [BMK (11)]. The extent of activation of these different cascades varies according to stimuli, and the ratio between the signals in each cascade may determine signaling specificity. For example, JNK appears to be highly activated by stress, whereas the ERKs are mainly activated by mitogens. Interestingly, MKK4 [SEK1 (12, 13)] activates both JNK and p38RK, thus indicating the existence of some cross-talk between the cascades in mammalian cells.

GnRH, which is a hypothalamic decapeptide, serves as a key regulator of the reproductive system. It induces the synthesis and release of the pituitary gonadotropin LH and FSH. When GnRH binds to its seven-transmembrane receptor, it induces interaction of the receptor with the heterotrimeric Gq protein that leads to activation of phospholipase C and formation of inositol 1,4,5- trisphosphate and diacylglycerol, leading to elevation of Ca2+ and activation of protein kinase C (PKC). Phospholipase D, phospholipase A2, and the formation of bioactive lipoxygenase products are also activated by GnRH, although the mechanisms involved are not yet known (reviewed in Refs. 14 and 15). Recently, the stimulation of the ERK-signaling cascade by GnRH has been demonstrated by several laboratories (16, 17, 18, 19). ERK activation in response to GnRH is unique as it was shown to be fully mediated by PKC and Ca2+, which seem to act in a Ras-independent manner (19). Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein partially inhibited this pathway, which indicates the involvement of a PTK in the GnRH-induced ERK activation.

In this study, stimulation of the JNK pathway by GnRH was examined. Our results show that GnRH-induced JNK activation was greater but occurred more slowly than that of ERK. The signaling pathway that mediates the GnRH stimulation of JNK appears to involve PKC, c-Src, CDC42/Rac1, and probably also MEKK1, and to act independently of the ERK cascade. Thus, the {alpha}T3–1 cells appear to utilize a unique pathway that links a G protein-coupled receptor to the activation of the JNK-signaling cascade through PKC, c-Src, and CDC42.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Stimulation of JNK Activity and Jun Expression by GnRH
Recently, we have shown that the activity of ERK1 and ERK2 is stimulated by GnRH in pituitary {alpha}T3–1 cells (19). To examine whether GnRH can also activate the JNK pathway, serum-starved {alpha}T3–1 cells were stimulated with a GnRH analog (GnRH-a, 10-7 M), and JNK activity was monitored by a solid phase assay (20). As shown in Fig. 1Go, JNK activity was significantly (25- to 55-fold) stimulated by GnRH-a, to an extent similar to that caused by the potent stimulating agents, peroxovanadate (VOOH, 15 min) and the PKC activator tumor-promoting agent (TPA, 30 min). This magnitude of stimulation was higher than that observed for ERK1/2 (5- to 15-fold) in the same cells. However, the time course of JNK activation by GnRH-a was slower than the rapid activation of the ERKs. JNK activation was detected 5 to 10 min after the initiation of the GnRH-a treatment, it peaked by 30 min, and it decreased over the next 90 min (Fig. 1BGo). Thus, although both ERK and JNK might be key mediators of GnRH signaling, they may regulate different cellular processes.



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Figure 1. GnRH-a Stimulates JNK Activity in {alpha}T3–1 Cells

A, Subconfluent {alpha}T3–1 cells were treated with GnRH-a (10-7 M), VOOH (vanadate 100 µM and H2O2 200 µM), and TPA (200 nM) for the indicated times. The stimulation was terminated by washing three times with ice-cold PBS, and JNK activity was measured as described in Materials and Methods. B, ERK1 and ERK2 activation was determined using antiactive-MAPK antibody. The activation of ERK 1 and 2 and JNK activity was quantified by an imaging denstitometer (Bio-Rad). The JNK results are average of three distinct experiments. The solid line represents JNK activity and the dashed line ERK activity. C, Stimulation of c-Jun expression by GnRH-a. Subconfluent {alpha}T3–1 cells were treated with GnRH-a (10-7 M, 30 min) or left untreated and harvested. RNA samples of these cells were subjected to a Northern blot analysis using c-Jun probe as described.

 
Since activation of the JNK cascade is known to induce c-Jun expression in several cell systems (7), we examined whether c-Jun expression is also induced by GnRH in {alpha}T3–1 cells. Northern blot analysis, using c-Jun as a probe, revealed that GnRH-a induced the expression of c-Jun mRNA in these pituitary cells (Fig. 1CGo), thus confirming the findings of Cesnjaj et al. (21). Taken together, these results indicate that the JNK cascade does indeed play a role in GnRH signaling in {alpha}T3–1 cells.

Effects of PKC and PTK Inhibitors on JNK Activation
Activation of ERKs by GnRH-a requires PKC and to some extent also protein tyrosine phosphorylation (16, 17, 18, 19). Therefore, the role of PKC and PTKs in GnRH-induced JNK activation was examined. Exposure of {alpha}T3–1 cells to TPA or to VOOH (general activator of tyrosine kinases) caused a pronounced stimulation of JNK activity (Fig. 1AGo), which suggested that PKC and PTK might stimulate JNK activity in this cell line. Whether PKC is indeed involved in GnRH signaling was determined using PKC depletion and inhibition. Thus, {alpha}T3–1 cells were pretreated with TPA (1 µM, 16 h) which depletes most PKC isoforms, or with the selective PKC inhibitor GF109203X. In both cases there was a significant (80–85%) inhibition of the GnRH-induced JNK activity (Fig. 2AGo). These results suggest that, as in the case of ERK (19), activation of JNK by GnRH-a in these cells is mainly mediated by PKC.



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Figure 2. Effect of PKC and PTK Inhibitors on GnRH-a and TPA Stimulation of JNK Activity

A, Inhibition of PKC: {alpha}T3–1 cells were either pretreated with 1 µM TPA (TPA dep.) for 16 h or with 3 µM GF109203X for 15 min before stimulation or left untreated. GnRH-a (10-7 M) was added for 0, 30, 60, or 120 min, and JNK activity was measured by a solid phase assay using GST-Jun as a substrate as described in Materials and Methods. B, Inhibition of PTKs: {alpha}T3–1 cells were pretreated with 200 µM genistein for 15 min or left untreated as control. GnRH-a (10-7 M) was then added for 0, 15, 30, and 60 min, and JNK activity was measured as above. C, Inhibition of TPA-stimulated JNK activity by genistein: {alpha}T3–1 cells were pretreated with 200 µM genistein for 15 min or left untreated as control. TPA (200 nM) was then added for 0, 15, 30, and 60 min, and JNK activity was measured as above. D, Quantification of the experiments in A, B, and C by an imaging densitometer. •, GnRH-a; {blacksquare}, GnRH-a+ TPA depletion; {blacktriangleup}, GnRH-a+GF109203X; x, GnRH-a+genistein; {blacktriangledown}, TPA; {diamondsuit}, TPA + genistein. All results are from a representative experiment that was reproduced three times.

 
To determine whether PTKs are involved in the GnRH-induced activation of JNK, we pretreated the {alpha}T3–1 cells with the PTK inhibitor, genistein. Genistein completely abolished the GnRH-stimulated JNK activity for up to 1 h after stimulation (Fig. 2BGo) but only inhibited ERK activation by ~30% (19), indicating the existence of a genistein-sensitive PTK that mediates the activation of JNK by GnRH-a. To determine whether this PTK is located upstream or downstream to PKC, genistein-treated {alpha}T3–1 cells were stimulated with TPA. As shown in Fig. 2Go, C and D (that quantitate the results of panels A–C), stimulation of JNK activity by TPA was most prominent after 60 min (~30-fold), and this stimulation was completely abolished by pretreatment with genistein. TPA bypasses the GnRH receptor-Gq complex and activates PKC directly; these results indicate that PKC is located upstream to the PTK(s) in the pathway that mediates the activation of JNK by GnRH. Sequential activation of PKC and PTKs that leads to JNK activation in G-protein coupled receptors signaling seems to be unique to GnRH-stimulated {alpha}T3–1 cells, as will be discussed below.

c-Src Is Involved in the GnRH-JNK Signaling Pathway
The involvement of a putative PTK that was implicated by the use of genistein initiated an effort to identify the PTK that is involved in JNK activation by GnRH. c-Src or other member(s) of the Src family of PTKs are genistein-sensitive protein kinases that have been previously reported to participate in G-protein signaling (22, 23, 24). To examine whether a c-Src family member is the PTK that mediates the effect of GnRH on JNK, we first asked whether these kinases are expressed in the {alpha}T3–1 cells. Western blot analysis revealed that the {alpha}T3–1 cells contain a substantial amount of c-Src, a small amount of Fyn, and, as expected, no Lyn (Fig. 3AGo). Activation of c-Src by GnRH-a was probed by immunoprecipitating c-Src from GnRH-a-stimulated cells using specific antibodies and measuring its activity using enolase as a substrate (25). GnRH-a caused an ~3.2-fold increase in c-Src activity, which peaked at 5 min after stimulation and was maintained for an additional 15 min (Fig. 3Go, B and C). Although the fold activation seems to be small, it is comparable to the activation reported in other cellular systems (22) and might be due to a high basal activity of this PTK. c-Src was activated also by TPA (2.6-fold; Fig. 3Go, B and C), and the activation of c-Src by GnRH was sensitive both to PKC and to PTK inhibitors, thus indicating that the activation of c-Src in response to GnRH-a is downstream of PKC.



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Figure 3. Content and Activation of Src by GnRH-a and TPA in {alpha}T3–1 Cells

A, Western blot analysis of Src family members in {alpha}T3–1 cells. Total cell extracts were separated by SDS-PAGE and subjected to Western blotting using anti-Src, anti-Fyn, and Anti-Lyn anti-peptide antibodies (Santa Cruz, according to the manufacturer instruction and using alkaline phosphatase-coupled secondary antibody). To assure the specificity of the antibodies, the antigenic peptide was used (100 µg/ml) for competition. B, c-Src activation by GnRH-a and TPA: {alpha}T3–1 cells were treated with GnRH-a (10-7 M) for the indicated times, or pretreated with GF109203X (3 µM, 15 min) and with genistein (200 µM, 15 min) and then treated with GnRH-a (top panel). Alternatively, the cells were stimulated with 200 nM TPA (lower panel) Stimulation was terminated by washing with ice-cold PBS followed by c-Src immunoprecipitation. c-Src activity toward acid-denatured enolase was determined as described in Materials and Methods. C, Quantification of the results in panel B. The reactions were analyzed by an SDS-PAGE, and the amount of phosphate incorporated into enolase was determined by imaging densitometer. These results are an average of two experiments. •, GnRH-a activation of Src; {blacksquare}, GnRH-a + GF109203X; {blacktriangleup}, GnRH-a + genistein. Inset, TPA stimulation.

 
The involvement of c-Src in GnRH-induced JNK activation was then examined by cotransfecting Csk, which serves as a Src-dominant interfering kinase (22), together with hemagglutinin (HA)-JNK2 into the {alpha}T3–1 cells. GnRH-stimulated JNK activity was markedly inhibited by the overexpression of Csk, whereas the basal activity of JNK was not affected (Fig. 4AGo), indicating that most, if not all, the GnRH signal toward JNK is indeed mediated by c-Src. To further verify this point we also cotransfected the constitutively active form of c-Src, Y527F-Src, together with HA-JNK2 into the same cells. Y527F-Src by itself activated JNK up to 4-fold above basal activity, and this activity was not enhanced by the addition of GnRH-a to the cells (Fig. 4Go, B and C), further indicating that most of the GnRH signal is funneled through c-Src. Moreover, the activation of JNK by Y527F-Src was sensitive to genistein (Fig. 4Go, B and C), indicating that c-Src by itself is a target for genistein and therefore might be the genistein- sensitive PTK in the GnRH-JNK pathway. As expected, GF109203X did not influence the Y527F-Src activation of JNK (up to 60 min treatment with the inhibitor). Therefore, GnRH signal toward JNK appears to be mediated mainly via the genistein-sensitive c-Src (or a family member), operating downstream of PKC.



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Figure 4. The Effect of Y527F-Src and Csk on GnRH-a Stimulation of JNK Activity

A, {alpha}T3–1 cells were cotransfected with either the Src-dominant interfering kinase Csk or with vector control together with HA-JNK2. Two days after transfection the cells were serum starved for 16 h and then either treated with GnRH-a (10-7 M) or left untreated. JNK activity toward GST-Jun was determined as described above (GST-Jun). The amount of immunoprecipitated HA-JNK was determined by Western blot analysis with anti-JNK antibody (Sigma). B, {alpha}T3–1 cells were cotransfected with either the constitutively active Y527F-Src (Src) or vector control (left lane) together with HA-JNK2. Two days after transfection the cells were serum starved for 16 h and then either treated with GnRH-a (right lane), treated with GF109203X (GF, 3 µM, 15 min), genistein (Gen, 200 µM, 15 min), or left untreated (two left lanes). JNK activity toward GST-Jun was determined as described above. Similar results were obtained when the GF109203X was added for 1 h. The amount of immunoprecipitated HA-JNK was determined by Western blot analysis with anti-JNK antibody (HA-JNK). C, The amount of phosphate in GST-Jun was determined by an imaging densitometer. These results are an average of three experiments.

 
JNK Stimulation by GnRH Is Mediated by CDC42/Rac1 but Not Ras
The JNK signaling cascade is known to act downstream of several sGPs, such as Rac1, CDC42, and Ras (26, 27). Therefore, we examined which, if any, of the sGPs is involved in the GnRH-stimulated, genistein-sensitive, JNK activation. Thus, the dominant interfering mutants of three sGPs (T17N-Rac2, T17N-CDC42, and S17N-H-Ras) were cotransfected into {alpha}T3–1 cells together with HA-JNK2. After cell treatment with the desired stimuli and inhibitors, lysis of the cells and immunoprecipitation with anti-HA antibodies, JNK2 activity was measured using GST-Jun as a substrate. All three inactive forms of the sGP had an inhibitory effect on the GnRH-stimulated JNK2 activity (Fig. 5Go, A and B). T17N-CDC42 inhibited GnRH-stimulated JNK activity by as much as 62 ± 5%, the T17N-Rac1 by 36 ± 4%, and the S17N-H-Ras by only 17 ± 4%.



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Figure 5. The Involvement of CDC42 and Rac1, But Not Ras, in GnRH-a and TPA Stimulation of JNK Activity

A, {alpha}T3–1 cells were cotransfected with HA-JNK together with the dominant negative (DN-sGP) and constitutively active (CA-sGP) forms of CDC42, Rac1 and H-Ras, or with a vector control (Control and GnRH lanes). Two days after transfection the cells were starved for 16 h followed by a treatment with GnRH-a (10-7 M, 30 min, two left lanes), with GF109203X (GF, 3 µM, 15 min), with genistein (Gen, 200 µM, 15 min) or left untreated (two central lanes). The cells were then harvested and JNK activity was determined as described in Materials and Methods. It should be noted that the results of the different sGPs are from separate experiments and therefore the intensity of the 32p incorporated GST-Jun should only be compared within each gel strip. B, The amount of phosphate incorporated into GST-Jun in cells transfected with DN-sGP was quantified by an imaging densitometer. The average of three experiments is presented. C, The influence of GF109203X (3 µM, 0.25, 1, and 2 h) and genistein (200 µM, 0.25, 1, and 2 h) over prolonged time on JNK activation by the constitutively active CDC42, Rac1, and Ras was determined as described above. •, GF109203X; {blacktriangleup}, genistein; {blacksquare}, JNK activation by GnRH-a (10-7 M, 30 min) under these conditions. The fold activation in time 0 represents fold activation by the constitutively activated sGPs, and the broken line represents a time course of GF109203X and genistein influence on these activations. The results in this figure are from one representative experiment that was reproduced four times. D, {alpha}T3–1 cells were cotransfected with HA-JNK together with the dominant negative forms of CDC42 (right lanes) or vector control (left lanes). Two days after transfection the cells were starved (16 h) and then treated with TPA (200 nM, 30 min) or left untreated. The cells were then harvested, and JNK activity was determined as above. Comparable amounts of JNK were detected in the immunoprecipitants of all the above experiments (data not shown).

 
Since the inhibition of GnRH stimulation of JNK activity by the dominant interfering forms of the sGPs was incomplete, some of the GnRH-induced, genistein-sensitive signaling may have been mediated by an sGP-independent pathway. To test this possibility, constitutively active forms of the same sGPs (Q61L-CDC42, Q61L-Rac1, and Q61L-H-Ras) were cotransfected into {alpha}T3–1 cells together with HA-JNK2, and the stimulation of JNK activity was determined as above. The constitutively active CDC42 stimulated JNK activity greatly [8.8 ± 1.3 fold (n = 3), which is 65% of GnRH stimulated JNK activity under these conditions]. The constitutively active form of Rac1 had a moderate effect (3.8 ± 0.5 fold), and the constitutively active H-Ras had only a marginal effect (1.9 ± 0.7 fold; Fig. 5CGo). The stimulation of HA-JNK2 by Q61L-CDC42 or by Q61L-Rac1 was not affected by the PKC inhibitor GF109203X nor by the PTK inhibitor genistein (Fig. 5CGo). In addition, TPA stimulation of JNK activity was significantly inhibited by the T17N-CDC42 (Fig. 5DGo), which supports CDC42 and also, to a lesser extent, Rac1 acting downstream of PKC and c-Src. The effects of the PKC and PTK inhibitors on cells cotransfected with the constitutively active form of H-Ras could not be determined because of the low stimulation with this construct.

The above results indicate that most of the genistein-sensitive, GnRH-induced JNK activation is mediated by CDC42. Although in many cases CDC42 and Rac1 seem to use similar guanine nucleotide exchange factors, the effect of Rac1 on the GnRH-JNK pathway seems to be limited. Similar phenomenon, where CDC42 transmits downstream signals without the involvement of Rac1, was recently reported for serum-induced proliferation (28). Ha-Ras does not seem to be significantly involved in GnRH-induced signaling that contributes to the JNK cascade.

GnRH Stimulation of JNK Is Mediated by MEKK1 But Not by MEK1
Several reports have recently suggested that the stress-activated JNK cascade consists of JNK, JNKK (SEK1, MKK7), MEKK1 (6), and either PAK1 (29) or MLK (30). To determine whether the genistein-sensitive GnRH signal toward JNK is mediated via this cascade, {alpha}T3–1 cells were cotransfected with HA tagged-JNK2 and the truncated, constitutively active form of MEKK1 (31) and processed as described for the sGPs above. The constitutively active MEKK1 elicited a strong activation (~25 fold) of JNK2, which was somewhat lower than the activation caused by GnRH-a (data not shown) but could not be further activated by treatment of the transfected cells with GnRH-a (Fig. 6Go). The lack of synergistic activation with MEKK1 and GnRH indicates (although does not prove) that MEKK1 might be involved in the GnRH-JNK signaling pathway. Indeed, the MEKK1 effect was not blocked by GF109203X or genistein when added for 15 (Fig. 6Go), 60, or 120 min (data not shown), thus indicating that PKC and the genistein-sensitive PTK(s) are probably situated upstream of MEKK1, (although they may lie on a distinct pathway in case MEKK1 is not the activatory component as suggested above). On the other hand, coexpression of HA-JNK2 with the constitutively active form of MEK1 [{Delta}N-EE-MEK (32, 33)] which is involved in the distinct ERK signaling cascade, did not affect JNK activity under conditions in which MEKK1 caused its strong activations (data not shown). This later observation is in agreement with our previous findings on the effects of {Delta}N-EE-MEK in COS7 cells (32). Thus, in {alpha}T3–1 and COS7 cells, but unlike in U937 cells (34), the JNK signaling cascade appears to be completely distinct from the MEK-ERK cascade.



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Figure 6. Effect of Constitutively Active MEKK on JNK Activity

{alpha}T3–1 cells were cotransfected with either N-terminally deleted, constitutively active MEKK1 (MEKK), or vector control together with HA-JNK2 as described in Materials and Methods. Two days after transfections the cells were serum starved for 16 h followed by a treatment with GF109203X (GF, 3 µM, 15 min), genistein (Gen, 200 µM, 15 min), GnRH-a (10-7 M, 30 min), or control PBS as indicated. GF109203X and genistein had no effect even when added for up to 2 h. Comparable amounts of JNK were detected in the immunoprecipitants of all lanes (data not shown).

 
Taken together, our results support a model wherein GnRH-stimulated JNK activation is mediated by a pathway involving PKC, c-Src, CDC42/Rac1, and possibly MEKK1, which might lead to the expression of Jun detected upon GnRH stimulation of the {alpha}T3–1 cells. A divergence of the GnRH-stimulated ERK and JNK pathways at the level of PKC stimulation of PTK activity was suggested by the differential sensitivity of the two MAPKs to genistein (Fig. 7Go).



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Figure 7. A Schematic Representation of GnRH-Induced ERK and JNK Signaling Pathways.

For details see text.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of GnRH to its membranal receptor elicits a series of signaling pathways (14), including activation of the ERK cascade (16, 17, 18, 19). However, the ERK cascade is not the only route by which GnRH communicates with the nucleus. Here the JNK pathway is shown to be significantly activated in response to GnRH-a, to a much greater extent than the ERK cascade. The time course of this GnRH-induced JNK activation in response to GnRH-a is slower than that of ERK (Fig. 1BGo) and is similar to time courses observed for the induction of JNK by other heterotrimeric G protein-coupled receptors (35) and tyrosine kinase receptors (36). However, this slower time course does not occur in all cells. For example, in nerve growth factor-treated PC12 cells, the short-term JNK activation is rapid (10–30 min) and transient (37). Thus, like the ERK pathway (38), the duration of JNK activity may determine the outcome and specificity of the signal. The delayed response of JNK observed here (Fig. 1Go) may indicate that JNK is involved in a later stage of transcription regulation and suggests that the signaling machinery that is involved in the activation of JNK is different from that leading to ERK activation. This upstream machinery was elucidated in this study, and our results suggest that GnRH binding to its receptors triggers the activation of PKC, which occurs most probably via the heterotrimeric Gq protein and PLC. The signal is then transmitted to a Src-family PTK and further to the sGPs CDC42 and possibly Rac1. The sGPs activate, in turn, a sGP-activated kinase whose identity is still controversial and which might be PAK1 (29), MLK (30), or another unknown protein kinase. Finally, the JNK cascade, including JNKKK (probably MEKK1), JNKK, and JNK (reviewed in Ref.6), is activated, causing the stimulation of several transcription factors including, most likely, c-Jun (6) and the expression of c-Jun protein (Fig. 7Go).

It is well established by now that PKC mediates many of the downstream effects of GnRH, including the activation of the ERK cascade (14). In {alpha}T3–1 cells, JNK activation, like that of ERK (19), is activated by TPA and markedly inhibited (80–85%) by the PKC-specific inhibitor GF109203X or by TPA depletion of PKC (Fig. 2Go). This degree of activation would imply that most of the GnRH signal toward JNK is mediated by PKC, although the small amount of residual activity upon PKC inhibition could suggest an additional signaling machinery. This involvement of PKC in JNK activation by GnRH seems to be unique to our system. In other cell lines TPA does not stimulate JNK activity (35, 39, 40) or may even cause a decrease in the basal activity of JNK (32). A clue regarding the identity of the putative components of GnRH signaling was first achieved by the effect of PTK inhibitors on JNK activity. Thus, the PTK inhibitor, genistein, completely blocked GnRH-stimulated JNK activation (Fig. 2Go) whereas it inhibited only ~30% of ERK activation by GnRH (19). Moreover, TPA-induced JNK activation was also blocked by genistein (Fig. 2Go), indicating that the PTK is located downstream to PKC. Since TPA bypasses the GnRH receptor-Gq complex and activates PKC directly, our results indicate that the PKC-PTK step is located in the pathway leading to GnRH activation of JNK. This is reminiscent of the involvement of PTKs in PKC signaling toward ERK in other systems (19, 41, 42). The difference in the extent of inhibition by genistein suggests that two (or more) distinct PTKs may be involved in the activation of ERK and JNK by GnRH. Therefore, the activation of PTKs by PKC might be the point of divergence between the JNK and ERK cascades in {alpha}T3–1 cells (Fig. 7Go).

Recently, Src family protein kinases have been implicated in some G protein-coupled receptor signaling pathways. Lyn is involved in the activation of MEK in DT40 cells (23), and c-Src is involved in Angiotensin II signaling in COS cells (43). Our results showing that c-Src is involved in the stimulation of JNK activity (Figs. 3Go and 4Go) in a GF109203X-independent, but genistein-dependent, manner implies that c-Src (or a member of its family) is the PTK that mediates PKC-dependent signaling toward JNK. Since JNK activation by GnRH-a was completely blocked by a Src-dominant interfering kinase (Csk), and JNK could not be further activated by GnRH in cells transfected with the constitutively active form of c-Src, this PTK seems to act as a central component that funnels all the PKC-mediated GnRH signals toward JNK. The mechanism by which PKC induces c-Src activation in the GnRH-JNK system is not clear. PKC has already been shown to directly activate c-Src by phosphorylation of its serines 12 and 48 (44), which are required for the enhanced response to ß-adrenergic agonists in cells overexpressing c-Src. The importance of the c-Src phosphorylation on serine 12 was supported also by Liebenhoff et al. (45), who suggested that this phosphorylation by PKC induces cytoskeletal association necessary for c-Src activation. Furthermore, G protein-coupled receptors may mediate their signal through a cascade of PTK that involves activation of c-Src by Pyk2 (22) or c-Src and FAK (46). Therefore, in the GnRH-JNK pathway studied here, PKC may activate an upstream PTK, which in turn activates c-Src. However, since some reports have implied that PKC may be located downstream of c-Src in the signaling pathway leading to proliferation (47, 48), the relationship between PKC and c-Src is not yet fully understood, and some other signaling components may be involved in the activation of c-Src by PKC.

The activation of JNK involves in most cases the sGPs, CDC42 and Rac1, that might operate downstream to Ras (26, 27). Both dominant-negative and constitutively active forms of these G proteins were used to determine whether they participate in the genistein-sensitive GnRH-stimulated pathway leading to JNK activation. Here we show that CDC42 and, to some extent, also Rac1 are involved in this activation, whereas the involvement of Ras is unlikely (Fig. 5Go). The activation of Rac1 and CDC42 by stress-activated signals was previously shown to be Ras-dependent (27). Therefore, the activation of JNK by GnRH in {alpha}T3–1 cells seems to differ and involves a Ras-independent activation of Rac1 and CDC42 by c-Src. Although the mechanism of activation is not yet known, it might involve a distinct set of adaptor molecules and nucleotide exchange factors.

In summary, we demonstrated here the activation of the JNK cascade by a unique signaling pathway that involves PKC, c-Src family PTK, CDC42/Rac1, and probably MEKK1, which leads to JNK activation and most likely to the c-Jun induction in response to GnRH. Similar to the ERK cascade, the activation of JNK by GnRH is PKC dependent, and the point of divergence between the two cascades seems to be the activation of PTK downstream of PKC. Furthermore, the JNK pathway in these cells seems to be different from most other signaling pathways that link G protein-coupled receptors to the activation of JNK in that it includes the activation of CDC42/Rac1 by PKC and c-Src in a Ras-independent mechanism.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Buffers
Homogenization buffer (buffer H) consisted of 50 mM ß-glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM benzamidine, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and pepstatin (2 µg/ml). HEPES lysis buffer consisted of 20 mM HEPES (pH 7.6), 10 mM EGTA, 40 mM ß-glycerophosphate, 1% Nonidet P-40, 2.5 mM MgCl2, 1 mM dithiothreitol, 2 mM sodium orthovanadate, aprotinin (20 µg/ml), leupeptin (20 µg/ml), and 1 mM phenylmethylsulfonyl fluoride. The JNK buffer consisted of 20 mM HEPES (pH 7.6), 20 mM MgCl2, 40 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, and 2 mM dithiotritol. The PTK buffer consisted of 50 mM HEPES (pH 7.6), 150 mM NaCl, 5 mM MnCl2, 5 mM MgCl2, 0.1 mM sodium orthovanadate, and 1 mM dithiothreitol.

Stimulants, Inhibitors, Antibodies, and Miscellaneous Reagents
[D-Trp6]-GnRH, a stable GnRH analog, genistein (PTK inhibitor), enolase, and protein A-Sepharose were obtained from Sigma Chemical Co. (St. Louis, Mo). GF109203X (a PKC inhibitor) and TPA were purchased from Calbiochem (La Jolla, CA). GST-Jun (1–97) was prepared as previously described (20). Mouse monoclonal antihemagglutinin (HA)-antibodies were produced by the Antibody Unit of The Weizmann Institute of Science. Rabbit polyclonal anti-c-Src antibodies, N-16 and SRC 2, and anti-Fyn and anti-Lyn antibodies and their control peptides were purchased from Santa Cruz (San Diego, CA). Mouse monoclonal anti-active MAPK antibodies were from Sigma (Rehovot, Israel).

Plasmids
The CDC42 constructs (Q61L and T17N-CDC42) were a gift from Dr. G. Bokoch (The Scripps Research Institute, La Jolla CA). The Csk construct was a gift from Dr. S. Courtneidge (Sugen, Inc. Redwood City, CA). MEKK1, GST-c-Jun JNK2, and Rac constructs were provided by Dr. Y. Ben-Neriah (Hebrew University, Jerusalem, Israel). {Delta}N-EE-MEK was cloned in pCDNA1 (Invitrogen, San Diego, CA) as previously described (32).

Solid Phase Assay for JNK Activity
Pituitary {alpha}T3–1 cells were grown to 80% confluency serum starved for 16 h, and the examined stimulants were added for various time intervals. The cells were then washed (twice with PBS and once with buffer H), scraped into 250 µl of buffer H and sonicated (50 W, 2 x7 sec), all at 4 C. After centrifugation (20,000 x g, 15 min, 4 C), aliquots of the resulting supernatant were assayed by the Coomassie protein assay (Pierce, Rockford, IL) for protein. JNK activity was detected according to Hibi et al. (20). Briefly, aliquots (100–150 µg protein) of the cell extracts were incubated (2 h, 4 C) with GST-Jun to allow the JNKs to bind to the substrate. After extensive washing, the JNK activity was measured by phosphorylation of the GST-Jun, which was mediated by the bound kinase in the presence of 20 mM MgCl2, 20 µM [{gamma}-32P]-ATP (300 cpm/pmol) for 20 min at 30 C. The reactions were terminated by the addition of sample buffer, and the samples were subjected to SDS-PAGE analysis. The gels were dried, autoradiographed on Kodak X-100 films, and the phosphorylation of GST-Jun was quantitated by densitometry (Bio-Rad 690 densitometer, Bio-Rad, Richmond, CA).

Transfection of {alpha}T3–1 Cells
Subconfluent {alpha}T3–1 cells were cotransfected with 5 µg each of HA-JNK2-SR{alpha} and one of the following plasmids (MEKK-pCMV5, Q61L-Rac1-pCDNA3, T17N-Rac1-pCDNA3, Q61L-CDC42-pCDNA3, T17N-CDC42-pCDNA3, Q61L-H-Ras-pCMV5, S17N-H-Ras-pCMV5, Y527F-Src-RK5, and Csk-pRK5) using the calcium phosphate technique (49). The total amount of plasmid was adjusted to 10 µg with vector DNA in the control experiments. The transfection efficiency was 10–30%, as determined by transfection with a plasmid that contained ß-Gal and appropriate staining. Two days after transfection, the cells were serum starved for 16 h and incubated for the desired time intervals with GnRH-a in the presence or absence of various inhibitors. The cells were then lysed with 250 µl of HEPES lysis buffer at 4 C, vortexed, and kept on ice for 5 min. After centrifugation (20,000 x g, 20 min), the supernatant was assayed for protein content as above.

Immunoprecipitation with Anti-HA Antibodies
Lysates from transfected cells (400–500 µg protein in HEPES lysis buffer) were incubated (1 h, 4 C) with anti HA-antibodies (3 µg Ab/reaction). The immunocomplexes were precipitated with Protein A-Sepharose, and the resulting precipitates were washed twice with PBS containing 1% Nonidet P-40 and 2 mM sodium vanadate, once with 100 mM Tris (pH 7.5) containing 0.5 M LiCl and once with JNK buffer. The immunoprecipitates were then resuspended in 30 µl of JNK buffer, and the JNK activity was measured (30 min, 30 C with constant mixing) using GST-c-Jun as a substrate. Phosphorylation of GST-c-Jun was monitored by autoradiography.

Immunoprecipitation with Anti c-Src Antibodies
Cell lysates (400–500 µg protein in HEPES lysis buffer) were incubated (1 h at 4 C) with anti-c-Src-antibodies (5 µg/reaction) and then precipitated with Protein A-Sepharose. The immunocomplexes were washed twice with PBS containing 1% Nonidet P-40 and 2 mM sodium vanadate, once with 100 mM Tris (pH 7.5) containing 0.5 M LiCl, and once with PTK buffer. The washed immunoprecipitates were resuspended in PTK buffer and the c-Src activity was determined by using acid-denatured enolase (3 mM) as substrate (final volume of 40 µl) in the presence of 20 µM [{gamma}32P]-ATP (8,000 cpm/pmol). The enzymatic reactions were terminated by the addition of sample buffer. The samples were then subjected to SDS-PAGE, autoradiography, and densitometry analysis.

RNA Extraction and Northern Blot Analysis
Total RNA was isolated from cells by extraction in guanidium thiocyanate containing 8% ß-mercaptoethanol by the LiCl method. For Northern blot analysis, total RNA (15 µg) was fractionated on 1.2% denaturing agarose gel and transferred to GeneScreen membrane (Dupont, NEN, Boston, MA). After baking and prehybridization, the membranes were hybridized (16 h) with the specifc cDNA probes labeled to high specific activity using a random primer labeling kit (Boehringer Mannheim, Indianapolis, IN). The filters were washed at high stringency and autoradiographed.


    ACKNOWLEDGMENTS
 
We would like to thank Dr. B. Schick for helping in the preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Rony Seger, Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel.

This work was supported by grants from the Israeli Ministry of Health, the Forschheimer Fund, and Keren Naftali (to R.S.) and a postdoctoral fellowship award from the Israel Cancer Research Fund (to N.L.L). R.S. is an incumbent of the Samuel and Isabela Friedman Career Development Chair.

Received for publication December 15, 1997. Revision received February 4, 1998. Accepted for publication February 19, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Seger R, Krebs EG 1995 The MAPK signaling cascade. FASEB J 9:726–735[Abstract/Free Full Text]
  2. Zhang XF, Settleman J, Kyriakis JM, Takeuchi SE, Elledge SJ, Marshall MS, Bruder JT, Rapp UR, Avruch J 1993 Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature 364:308–313[CrossRef][Medline]
  3. Leevers SJ, Paterson HF, Marshall CJ 1994 Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369:411–414[CrossRef][Medline]
  4. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR 1994 The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156–160[CrossRef][Medline]
  5. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, Davis RJ 1994 JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025–1027[Medline]
  6. Davis RJ 1994 MAPKs: new JNK expands the group. Trends Biochem Sci 19:470–473[CrossRef][Medline]
  7. Karin M 1995 The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem 270:16483–16486[Free Full Text]
  8. Han J, Lee JD, Bibbs L, Ulevitch RJ 1994 A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265:808–811[Medline]
  9. Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal M, Heys JR, Landvatter SW 1994 A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372:739–746[CrossRef][Medline]
  10. Rouse J, Cohen P, Trigon S, Morgane M, Alonso-Llamazares A, Zamanillo D, Hunt T, Nebreda AR 1994 A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78:1027–1037[Medline]
  11. Zhou G, Bao ZQ, Dixon JE 1995 Components of a new human protein kinase signal transduction pathway. J Biol Chem 270:12665–12669[Abstract/Free Full Text]
  12. Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ 1995 Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267:682–685[Medline]
  13. Yan M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, Templeton DJ 1994 Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature 372:798–800[Medline]
  14. Naor Z, Shacham S, Harris D, Seger R, Reiss N 1995 Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: cross-talk of calcium, protein kinase C (PKC) and arachidonic acid. Cell Mol Neurobiol 15:527–544.[Medline]
  15. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499[Medline]
  16. Mitchell R, Sim PJ, Leslie T, Johnson MS, Thomson FJ 1994 Activation of MAP kinase associated with the priming effect of LHRH. J Endocrinol 140:8–9
  17. Roberson MS, Misra PA, Laurance ME, Stork PJ, Maurer RA 1995 A role for mitogen-activated protein kinase in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone. Mol Cell Biol 15:3531–3539[Abstract]
  18. Tsai PS, Werner S, Weiner RI 1995 Basic fibroblast growth factor is a neurotropic factor in GT1 gonadotropin-releasing hormone neuronal cell lines. Endocrinology 136:3831–3838[Abstract]
  19. Reiss N, Llevi LN, Shacham S, Harris D, Seger R, Naor Z 1997 Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary of alphaT3–1 cell line: differential roles of calcium and protein kinase C. Endocrinology 138:1673–1682[Abstract/Free Full Text]
  20. Hibi M, Lin A, Smeal T, Minden A, Karin M 1993 Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7:2135–2148[Abstract]
  21. Cesnjaj M, Catt KJ, Stojilkovic SS 1994 Coordinate actions of calcium and protein kinase-C in the expression of primary response genes in pituitary gonadotrophs. Endocrinology 135:692–701[Abstract]
  22. Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J 1996 A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383:547–550[CrossRef][Medline]
  23. Wan Y, Kurosaki T, Huang XY 1996 Tyrosine kinases in activation of the MAP kinase cascade by G-protein-coupled receptors. Nature 380:541–544[CrossRef][Medline]
  24. Wan Y, Bence K, Hata A, Kurosaki T, Veillette A, Huang XY 1997 Genetic evidence for a tyrosine kinase cascade preceding the mitogen-activated protein kinase cascade in vertebrate G protein signaling. J Biol Chem 272:17209–17215[Abstract/Free Full Text]
  25. Feder D, Bishop JM 1990 Purification and enzymatic characterization of pp60c-src from human platelets. J Biol Chem 265:8205–8211[Abstract/Free Full Text]
  26. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS 1995 The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the J NK/SAPK signaling pathway. Cell 81:1137–1146[Medline]
  27. Minden A, Lin A, Claret FX, Abo A, Karin M 1995 Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81:1147–1157[Medline]
  28. Molnar A, Theodoras AM, Zon LI, Kyriakis JM 1997 Cdc42Hs, but not Rac1, inhibits serum-stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK. J Biol Chem 272:13229–13235[Abstract/Free Full Text]
  29. Zhang S, Han J, Sells MA, Chernoff J, Knaus UG, Ulevitch RJ, Bokoch GM 1995 Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J Biol Chem 270:23934–23936[Abstract/Free Full Text]
  30. Teramoto H, Coso AM, Miyata H, Igishi T, Miki T, Gutkind JS 1996 Signaling from the small GTP-binding protein Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway: a role for mixed lineage kinase 3/protein-tyrosine kinase 1. A novel member of the mixed linage kinase family. J Biol Chem 271:27225–27228[Abstract/Free Full Text]
  31. Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, Johnson GL 1993 A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science 260:315–317[Medline]
  32. Jaaro H, Rubinfeld H, Hanoch T, Seger R 1997 Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc Natl Acad Sci USA 94:3742–3747[Abstract/Free Full Text]
  33. Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Wounde-Vande GF, Ahn NG 1994 Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966–970[Medline]
  34. Franklin CC, Kraft AS 1995 Constitutively active MAP kinase kinase (MEK1) stimulates SAP kinase and c-Jun transcriptional activity in U937 human leukemic cells. Oncogene 11:2365–2374[Medline]
  35. Coso OA, Chiariello M, Kalinec G, Kyriakis JM, Woodgett J, Gutkind JS 1995 Transforming G protein-coupled receptors potently activate JNK (SAPK). Evidence for a divergence from the tyrosine kinase signaling pathway. J Biol Chem 270:5620–5624[Abstract/Free Full Text]
  36. Karunagaran D, Tzahar E, Beerli RR, Chen X, Graus PD, Ratzkin BJ, Seger R, Hynes NE, Yarden Y 1996 ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 15:254–264[Abstract]
  37. Pinkas-Kramarski R, Soussan L, Waterman H, Levkowitz G, Alroy I, Klapper L, Lavi S, Seger R, Ratzkin BJ, Sela M, Yarden Y 1996 Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions. EMBO J 15:2452–2467[Abstract]
  38. Marshall CJ 1995 Specificity of receptor tyrosine kinase signaling: transient vs. sustained extracellular signal-regulated kinase activation. Cell 80:179–185[Medline]
  39. Zohn IE, Yu H, Li X, Cox AD, Earp HS 1995 Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase. Mol Cell Biol 15:6160–6168[Abstract]
  40. VanDam H, Wilhelm D, Herr I, Steffen A, Herrlich P, Angel P 1995 ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J 14:1798–1811[Abstract]
  41. Seger R, Biener Y, Feinstein R, Hanoch T, Gazit A, Zick Y 1995 Differential activation of mitogen-activated protein kinase and S6 kinase signaling pathways by 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin. Evidence for involvement of a TPA-stimulated protein-tyrosine kinase. J Biol Chem 270:28325–28330[Abstract/Free Full Text]
  42. Lev S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, Schlessinger J 1995 Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature 376:737–745[CrossRef][Medline]
  43. Luttrell LM, Hawes BE, van-Biesen T, Luttrell DK, Lansing TJ, Lefkowitz RJ 1996 Role of c-Src tyrosine kinase in G protein-coupled receptor and Gß{gamma} subunit-mediated activation of mitogen-activated protein kinase. J Biol Chem 271:19443–19450[Abstract/Free Full Text]
  44. Moyers JS, Bouton AH, Parsons SJ 1993 The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src. Mol Cell Biol 13:2391–2400[Abstract]
  45. Liebenhoff U, Greinacher A, Presek P 1994 The protein tyrosine kinase pp60c-src is activated upon platelet stimulation. Cell Mol Biol 40:645–652
  46. Schlaepfer DD, Hunter T 1997 Focal adhesion kinase overexpression enhances ras-dependent integrin signaling to ERK2/mitogen-activated protein kinase through interactions with and activation of c-Src. J Biol Chem 272:13189–13195[Abstract/Free Full Text]
  47. Gschwendt M, Kielbassa K, Kittstein W, Marks F 1994 Tyrosine phosphorylation and stimulation of protein kinase C delta from porcine spleen by src in vitro. Dependence on the activated state of protein kinase C delta. FEBS Lett 347:85–89[CrossRef][Medline]
  48. Zang Q, Frankel P, Foster DA 1995 Selective activation of protein kinase C isoforms by v-Src. Cell Growth Differ 6:1367–1373[Abstract]
  49. Wigler M, Pellicer A, Silverstein S, Axel R 1978 Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725–731[Medline]