(Received for publication, October 10, 1995)
From the
The c-Jun N-terminal kinases (JNK) are activated by various
stimuli, including UV light, interleukin-1, tumor necrosis factor-
(TNF-
), and CD28 costimulation. Induction of JNK by TNF-
, a
strong apoptosis inducer, implies a possible role of JNK in the
regulation of programmed cell death. Present studies show that lethal
doses of
radiation (GR) induced JNK activities at the early phase
of apoptosis in Jurkat T-cells. We demonstrate that JNK1 was activated
by either the T-cell activation signals, anti-CD28 monoclonal antibody
plus phorbol 12-myristate 13-acetate (PMA), or the apoptosis-inducing
treatment, GR; however, the induction patterns were different. In
contrast to the rapid and transient JNK1 activation caused by CD28
signaling plus PMA, GR induced a delayed and persistent JNK1
activation. This implies a distinct regulatory mechanism and specific
function of JNK1 in irradiated cells. The nuclear and cytosolic JNK1
activities were simultaneously increased in the irradiated cells
without an evident change in the protein levels. The abilities of GR to
induce JNK1 activation and DNA fragmentation were correlated.
Peripheral blood lymphocytes were more sensitive to GR than Jurkat
cells in JNK1 induction. The responsiveness of JNK1 to GR suggests the
involvement of JNK1 in the initiation of the apoptosis process.
Apoptosis is the unique morphological pattern of cell death characterized by chromatin condensation, membrane blebbing, and cell fragmentation. The most prominent event in the early stages of apoptosis is internucleosomal DNA cleavage by undefined endonuclease activities. This programmed cell death is widely observed in different cells of various organisms, from nematodes to mammals. It is generally accepted that apoptosis plays important roles in developmental processes, maintenance of homeostasis, and elimination of cells that have suffered serious damage (reviewed in (1) and (2) ).
radiation (GR) (
)is one of many
stimuli that induce cellular damage and apoptosis(2) . GR can
cause single-stranded and double-stranded breaks in the genomic DNA (3) . Hydroxyl radicals generated by radiolytic attack on
H
O in the cellular aqueous environment can cause oxidative
damages to macromolecules(4) . Although the direct damaging
effects of GR have been well studied, the biochemical and genetic
mechanisms that initiate the active programmed cell death in
radiation-damaged cells remain largely unknown.
The p46/p54 serine/threonine kinases, c-Jun N-terminal kinases (JNK1 and JNK2), are emerging members of the MAP kinase-related family(5) . Similar to MAP kinase, JNK activation requires phosphorylation at 2 residues, Thr-183 and Tyr-185, by MAP kinase kinase 4/JNK kinase(5) , a dual specificity kinase, which is structurally related to MAPK/ERK kinases (MEKs). MAP kinase kinase 4/JNK kinase itself is phosphorylated and activated by the upstream kinase MAPK/ERK kinase kinase 1 (MEKK1) (5) . The JNK kinase cascade can be induced by various mitogenic factors including growth factors, oncogenic Ras, phorbol esters, and T-cell activation signaling(5, 6) . JNK activity is also induced by stimuli such as UV light, protein synthesis inhibitors, osmotic shock, and proinflammatory cytokines(5, 7) . This kinase cascade was shown to be the common pathway shared by cell proliferation and stress-response signaling. The exact mechanism of how JNK kinase cascade integrates with other signaling pathways to achieve specific response to different stimuli remains to be elucidated. There are three cellular proteins currently known to be phosphorylated by JNK, which are the transcription factors c-Jun(8) , ATF-2(9) , and Elk-1(10) . Transcription activities driven by the responsive elements binding these transcription factors are strongly enhanced after JNK activation (5, 9, 10) .
Prominent JNK activation was observed in cells treated with
TNF- (7) , a potent inducer of apoptosis(2) .
Moreover, the tumor suppressor p53, which causes apoptosis on some
occasions(1) , was suggested as an in vivo substrate
of JNK1(11) . Based on these findings and the general
involvement of JNK in responses to various stresses, we proposed that
JNK may be activated by GR. In these studies, we demonstrated that JNK1
was activated in cells exposed to lethal doses of GR. The
radiation-induced JNK1 activation showed a unique kinetics in
comparison with that induced by other stimuli.
Figure 1: Lethal dose of GR induces DNA fragmentation and c-Jun N-terminal phosphorylation activity. Jurkat cells were exposed to 100 Gy of GR, and cells were collected at various time points. A, cellular DNA was extracted and analyzed by electrophoresis on 1.8% agarose gel to detect DNA laddering. B, c-Jun N-terminal phosphorylation activities were precipitated by GST-Jun-(1-79) GSH-Sepharose beads (8) and determined by solid-phase kinase assay. GST-Jun-P, phosphorylated GST-Jun.
To study
the possible JNK activation induced by GR, we used GST-Jun-(1-79)
bound to GSH-Sepharose beads to precipitate JNK or JNK-like activities
from Jurkat cell lysate. The precipitated complexes were washed and
subjected to solid-phase kinase assay. As shown in Fig. 1B, GST-Jun phosphorylation activity dramatically
increased between 1 and 2 h after irradiation and peaked at 3 h; then
it slightly decreased but remained constant through the 6-h time point.
The kinase activity was still higher than the basal level 12 h after
irradiation. We did not, however, detect a significant increase of
GST-Jun phosphorylation activity before the 1-h time point (data not
shown). In comparison with the immediate and transient JNK activation
induced by other stimuli such as TNF- (7) and UV
light(14) , the induction of JNK activity by GR was late and
sustained.
Figure 2:
Anti-JNK1 (Ab101) specifically
recognizes human JNK1 protein. A, Jurkat cells lysate (lane 1) and purified GST-JNK1 fusion protein (lane
2, fusion protein bound on GSH beads; lane 3, eluted
fusion protein) resolved by SDS-PAGE were transferred to nitrocellulose
filter. The filter was subjected to immunoblot with rabbit anti-JNK1
(Ab101). B, kinase activities precipitated by Ab101 from
untreated and UV light (100 J/m), anisomycin (1 µg/ml),
and GR (100 Gy) treated cell lysates (lanes 1-4,
respectively) were examined by in-gel kinase assay using
GST-Jun-(1-331) as the substrate.
Because the kinetics of c-Jun N-terminal phosphorylation activities induced by GR is very different in comparison with those induced by other stimuli, it is possible that GR may induce several kinases, in addition to JNK, that will phosphorylate the N terminus of c-Jun. The kinetics pattern detected by solid-phase kinase assay could be the additive effect of different kinase activities. To determine the JNK1 activation kinetics, we used the anti-JNK1 Ab for immunocomplex kinase assay. As shown in Fig. 3A, the precipitated JNK1 activity from irradiated cells has the same kinetics as that determined by solid-phase kinase assay. This shows that GR induced a delayed and sustained JNK1 activation. To exclude the possibility that this unique kinetics is the specific property of certain Jurkat cell clones, we used anti-CD28 mAb plus PMA, which are the potent stimuli for T-cell activation(12) , to induce JNK activation. As shown in Fig. 3B, after addition of anti-CD28 plus PMA, JNK1 activity rapidly increased within 15 min and reached the peak at the 30-min time point. However, the JNK1 activity induced by anti-CD28 mAb plus PMA diminished significantly 2 h after stimulation. These results show that, although both T-cell activation signals and GR induce JNK1 activation in Jurkat cells, the induction kinetics are quite different. While anti-CD28 mAb plus PMA induced an immediate and transient JNK1 activation, GR induced a relatively delayed and much more prolonged JNK1 activation.
Figure 3: GR and T-cell activation signals induce different kinetics of JNK1 activation. Jurkat cells were treated with: A, 100 Gy of GR; or B, anti-CD28 mAb (1:1,000 dilution) plus PMA (50 ng/ml). Cell lysates were collected at different time points after stimulation. JNK1 activity was immunoprecipitated from cell lysates and determined by using GST-Jun-(1-331) as a substrate. GST-Jun-P, phosphorylated GST-Jun.
Figure 4: Simultaneous activation of JNK1 in the nuclear and cytosolic fraction by GR without apparent translocation. Nuclear and cytosolic fractions of irradiated Jurkat cells were collected at various time points. Intracellular JNK1 protein distribution was determined by immunoblotting (A), and JNK1 activity was examined by immunocomplex kinase assay (B). GST-Jun-P, phosphorylated GST-Jun.
Figure 5:
Peripheral lymphocytes are more
sensitive to GR than Jurkat cells in JNK1 induction. Jurkat cells (A) and peripheral lymphocytes (B) were
-irradiated using various dosages. Cell lysates were collected 3 h
after irradiation. JNK1 activity was determined by immunocomplex kinase
assay. PB, peripheral blood; GST-Jun-P,
phosphorylated GST-Jun.
Because (i) overexpression of MAPK/ERK kinase kinase (MEKK),
the JNK-activating kinase, has a lethal effect on fibroblasts (16) and (ii) TNF- strongly induces JNK
activity(7) , we suspected that the JNK kinase cascade may be
involved in the induction of cell death. In these studies we show that
JNK1 activity is strongly activated during the early phase of apoptosis
caused by GR. Because of the correlation between JNK1 activation and
apoptosis induced by GR, we propose that JNK1 activation may be
involved in the initiation of programmed cell death in response to
radiation damages.
Immediate and transient kinetics of activation is universal in JNK responses to various stimuli(14) . However, the JNK1 induction by GR in Jurkat cells was delayed and persistent. Since the T-cell activation signals, anti-CD28 mAb plus PMA, induced a rapid and transient JNK1 activation in the same cell line, the unique pattern of JNK1 induction should be a specific cellular response to GR rather than a unique property of Jurkat cells. The induction of JNK1 in both T-cell activation and apoptosis indicates that JNK1 is the common kinase shared by these two distinct phenomena. However, the opposite outcomes imply that the presence or absence of other co-activators at different times may be important. In addition, prolonged JNK1 induction in irradiated cells may cause the persistent activation of some cellular factors (e.g. c-Jun or p53) and results in detrimental effects to the cells. Therefore, the different timing and/or duration of JNK1 induction may lead to opposite outcomes, T-cell proliferation or apoptosis.
JNK (JNK1/JNK2) and p38-Mpk protein kinases are coordinately regulated, although to a different extent, by proinflammatory cytokines, UV light, and other environmental stresses(7, 14) . Since the substrate specificities of these kinases are different(14) , they may have distinct functions in response to the stimuli. It will be important to determine whether GR induces all of these stress-responsive kinases. It is possible that different combinations of the kinase members, with various kinetics, may mediate diverse cellular signaling and dictate final outcomes, proliferation or cell death.
The delayed kinetics of JNK1 induction in irradiated cells also implies the existence of a distinct activation or regulation mechanism. In the known JNK activation mechanism, a nuclear translocation of JNK after activation at the proximity of the plasma membrane is required for nuclear function of the kinase(5) . However, we show that nuclear JNK1 is constitutively present and can be activated without evident, nuclear translocation in irradiated cells. The delay of JNK1 activation may be due to the time needed for accumulation of cellular damage to certain threshold levels. Furthermore, the existence of unrepairable damage could be the reason for persistent JNK1 activation, probably through the continued activation of upstream kinases. The other possible explanation for the prolonged JNK1 induction after ionizing radiation is the absence of dual specificity phosphatase activities, which were shown to dephosphorylate ERK and JNK causing down-regulation of the kinase activities(17, 18) . The imbalance between kinase and phosphatase activities may have detrimental effects and lead to cell death. Finally, identification of JNK1 as a potential signaling molecule in mediating apoptosis will open a new avenue for unraveling the signal transduction mechanisms of apoptosis.