(Received for publication, August 2, 1995; and in revised form, October 31, 1995)
From the
Previous work has shown that treatment of cells with the
antimetabolite 1--D-arabinofuranosylcytosine (ara-C) is
associated with induction of the c-jun gene. The present
studies demonstrate that ara-C activates the c-Abl non-receptor
tyrosine kinase. We also demonstrate that activity of the
stress-activated protein kinase (SAP kinase/JNK) is increased in
ara-C-treated cells. Using cells deficient in c-Abl
(Abl
) and after introduction of the c-abl gene, we show that ara-C-induced c-Abl activity is necessary for
the stimulation of SAP kinase. Other studies using cells transfected
with a SEK1 dominant negative demonstrate that ara-C-induced SAP kinase
activity is SEK1-dependent. Furthermore, we show that overexpression of
truncated c-Abl results in activation of the SEK1/SAP kinase cascade.
1--D-Arabinofuranosylcytosine (ara-C) (
)is the most effective agent used in the treatment of acute
myelogenous leukemia(1) . This agent misincorporates into
cellular DNA (2, 3) and inhibits replication by
site-specific termination of DNA
strands(4, 5, 6) . Although the precise
mechanisms responsible for the lethal effects of this agent remain
unclear, recent studies have supported the activation of nuclear
signaling cascades in ara-C-treated cells. Exposure of human myeloid
leukemia cells to ara-C is associated with induction of c-jun and other early response genes(7, 8) . The
induction of c-jun transcription is positively autoregulated
by its product c-Jun in cells treated with phorbol esters(9) .
Treatment with ara-C is also associated with post-translational
modification of c-Jun and enhancement of Jun/AP-1
activity(10) . Moreover, binding of activated c-Jun to the AP-1
site in the c-jun gene promoter confers ara-C inducibility of
this gene(10) . Two serines (Ser-63 and Ser-73) in the
transactivation domain of c-Jun that are phosphorylated in response to
phorbol ester and UV light have been identified as substrates for the
mitogen-activated and stress-activated protein (SAP)
kinases(11, 12, 13) . Other studies have
demonstrated that the SAP kinase/extracellular signal-regulated kinase
kinase 1 (SEK1) is responsible for activation of SAP
kinase(14, 15) .
The product of the c-abl gene is a non-receptor tyrosine kinase(16) . c-Abl is
localized to the nucleus and cytoplasm(17, 18) and
shares structural features with Src family tyrosine kinases. In
addition, c-Abl contains C-terminal actin binding and DNA binding
domains(17, 18) . The finding that c-Abl associates
with the retinoblastoma (Rb) protein has supported a role for c-Abl in
regulating the cell cycle(19) . Other work has demonstrated
that overexpression of c-Abl is associated with the arrest of growth in
the G phase(20, 21) . Overexpression of a
dominant negative c-Abl results in deregulation of withdrawal from or
reentry into the cell cycle(21) . These findings have suggested
that c-Abl negatively regulates growth. Other studies have demonstrated
that c-Abl is phosphorylated on multiple sites by p34
and that such modification inhibits DNA
binding(17, 22) . c-Abl phosphorylates the C-terminal
domain of RNA polymerase II (23, 24) and stimulates
transcription(19) . Despite these insights into a potential
role for c-Abl, the precise function of this tyrosine kinase remains
unclear.
The present studies demonstrate that c-Abl is activated by ara-C treatment. We also demonstrate that c-Abl is required for ara-C-induced SAP kinase activity and that c-Abl activates SAP kinase through SEK1.
Previous studies have demonstrated that ara-C induces a
stress response that includes activation of Jun/AP-1 and c-jun transcription(7) . In order to determine whether c-Abl is
involved in the cellular response to ara-C, we treated NIH3T3 cells
with this agent and prepared anti-Abl immunoprecipitates from nuclear
lysates. In vitro kinase assays were performed with the Crk
protein as substrate. c-Abl binds to the N-terminal SH3 domain of Crk
and phosphorylates Tyr-221(26, 31) . Analysis of the
anti-Abl immunoprecipitates with a GST-Crk(120-225) fusion
protein demonstrated increased (3-4-fold) Crk
phosphorylation as a consequence of ara-C treatment (Fig. 1A). The finding that there was little if any
phosphorylation of a GST-Crk(120-212) fusion protein, which lacks
the critical Tyr-221 for c-Abl phosphorylation, supported detection of
c-Abl activity (data not shown). The ara-C-induced tyrosine kinase
activity was also studied with a peptide (EAIYAAPRAKKK) recently
identified as a specific substrate for c-Abl (29) . Anti-Abl
immunoprecipitates from ara-C-treated cells phosphorylated this peptide
at a level approximately 3-fold higher than that obtained with similar
immunoprecipitates from untreated cells (Fig. 1B).
These results and the finding that immunoprecipitates with PIRS fail to
demonstrate ara-C-induced peptide phosphorylation (Fig. 1B) support activation of c-Abl by ara-C.
Figure 1: Activation of c-Abl by ara-C in NIH3T3 cells. A, NIH3T3 cells were treated with 10 µM ara-C for 30 min. Nuclei were isolated and the nuclear proteins subjected to immunoprecipitation with anti-Abl. In vitro immune complex kinase assays were performed using a GST-Crk(120-225) fusion protein as substrate. GST-Crk(120-212) fusion protein (which lacks the critical Tyr-221) was used as a negative control (data not shown). B, NIH3T3 cells were treated with 10 µM ara-C and harvested at the indicated times. Nuclear proteins were then subjected to immunoprecipitation with anti-Abl antibody. Immunoprecipitations were also performed with PIRS from cells exposed to 10 µM ara-C and harvested at 30 min. In vitro immune complex kinase assays were performed using the c-Abl substrate EAIYAAPFAKKK. The data (percent control phosphorylation) represent the mean ± S.E. of two separate experiments.
Previous work has shown that SAP kinase is activated in cells
treated with tumor necrosis factor , anisomycin, ionizing
radiation, and UV light(11, 12, 32) . To
determine whether ara-C induces SAP kinase, we analyzed anti-SAP kinase
precipitates for phosphorylation of the transactivation domain of
c-Jun. Using this approach, ara-C treatment was associated with
increased phosphorylation of a GST-Jun(2-100) fusion protein (Fig. 2A). In contrast, cells deficient in c-Abl
(Abl
) failed to respond to ara-C with
stimulation of SAP kinase activity (Fig. 2A). In order
to confirm the involvement of c-Abl in ara-C-induced SAP kinase
activity, we used Abl
cells that had been
infected with a c-Abl containing retrovirus (designated
Abl
). Immunoblot analysis of the Abl
cells demonstrated expression of c-Abl(33) . While ara-C
failed to induce c-Abl activity in Abl
cells,
the Abl
cells responded to ara-C with stimulation of
c-Abl activity (Fig. 2B). Moreover, the Abl
cells responded to ara-C exposure with increases in SAP kinase
activity (Fig. 2C). These findings suggested that c-Abl
is necessary for activation of SAP kinase in cells treated with ara-C.
Figure 2:
Activation of SAP kinase activity by
ara-C. A, NIH3T3 and Abl cells were
treated with 10 µM ara-C and harvested at 2 h. Total
lysates were immunoprecipitated with anti-SAP kinase (anti-SAPK) antibody, and in vitro immune complex
kinase reactions containing GST-Jun(2-100) fusion protein were
analyzed by 10% SDS-PAGE and autoradiography. B,
Abl
and c-Abl
cells were
treated with 10 µM ara-C for 30 min. Nuclear proteins were
subjected to immunoprecipitation with anti-Abl. In vitro immune complex kinase assays were performed using
GST-Crk(120-225) fusion protein. C, total cell lysates
from control and ara-C-treated c-Abl
cells were
subjected to immunoprecipitation with anti-SAP kinase, and in vitro immune complex kinase reactions were performed with
GST-Jun(2-100) fusion protein as
substrate.
SAP kinase is activated by SEK1(14, 15) . In order
to determine whether ara-C-induced SAP kinase activity is
SEK1-dependent, we prepared NIH3T3 cells that stably express a dominant
negative SEK1 in which the critical phosphorylation sites are mutated
as Ser Ala and Thr
Leu (SEK1 AL mutant) (provided by
James Woodgett)(14) . Treatment of the NIH3T3 SEK1 AL
transfectants with ara-C was associated with stimulation of c-Abl
activity as determined by GST-Crk(120-225) phosphorylation (Fig. 3A, lanes 1 and 2). In
contrast, there was little if any phosphorylation of the
GST-Crk(120-212) fusion protein (Fig. 3A, lane 3). While these results supported activation of c-Abl,
the NIH3T3 SEK1 AL cells failed to respond to ara-C with activation of
SAP kinase (Fig. 3B). Similar results were obtained
with other clones stably expressing the SEK1 AL dominant negative
protein (data not shown). Taken together, these findings demonstrate
that ara-C-induced SAP kinase activity is c-Abl- and SEK1-dependent.
Figure 3:
Activation of SAP kinase by ara-C is
blocked in cells overexpressing SEK1 AL dominant negative mutant.
NIH3T3 SEK1 AL cells were treated with 10 µM ara-C for 30
min (A) or 2 h (B). A. Nuclear proteins were analyzed
for c-Abl activity using GST-Crk(120-225) fusion protein as
substrate (lanes 1 and 2). GST-Crk(121-212)
fusion protein was used as a negative control (lane 3). B, total proteins were analyzed for SAP kinase (SAPK)
activity. As a positive control, Abl cells were
treated with 10 µM ara-C for 2 h, and total protein was
analyzed for phosphorylation of
GST-Jun(2-100).
In order to confirm and extend our findings in ara-C-treated NIH3T3
cells, we asked whether other cell types respond similarly to this
agent. Indeed, treatment of 293 kidney cells with ara-C was associated
with activation of c-Abl (Fig. 4A). These cells also
responded to ara-C with increases in SAP kinase activity (Fig. 4B). To further analyze the relationship between
c-Abl and SEK1/SAP kinase, we transfected 293 cells with pEBG-SEK1 and
pGNG Abl (SH3-deleted abl gene) and assayed
glutathione-agarose protein complexes for in vitro phosphorylation of GST-Jun. Using these experimental conditions,
there was no detectable phosphorylation of GST-Jun (Fig. 4C). Similar results were obtained following
transfection of pEBG-MEK1 and pGNG Abl (Fig. 4C). While
transfection of SAP kinase was associated with detectable GST-Jun
phosphorylation, there was little if any effect on the intensity of
this signal by cotransfection with pGNG Abl or pEBG-SEK1 (Fig. 4C). However, transfection of SAP kinase with
both pGNG and pEBG-SEK1 resulted in pronounced GST-Jun phosphorylation
as evidenced by an increase in signal and a decrease in electrophoretic
mobility (Fig. 4C). Moreover, the finding that
transfection of the pEBG SEK1 KR dominant negative completely
blocks Abl stimulation of SAP kinase activity provided further support
for c-Abl involvement in activation of SEK1/SAP kinase. The results
also support the inability of MEK1 to substitute for SEK1 in
stimulation of SAP kinase by pGNG Abl cotransfection (Fig. 4C). Other studies were performed with pGNG
Abl-transfected NIH3T3 cells that stably express the SH3-deleted and
activated Abl mutant (designated
XB)(27) . Transfection of
XB cells with pEBG SAP kinase and different molar ratios of SEK1
K
R resulted in complete abrogation of SAP kinase activity (Fig. 4D). Taken together, these results in 293 and
NIH3T3 cells demonstrate that activation of c-Abl is upstream to the
SEK1/SAP kinase cascade. The finding that SEK1 and SAP kinase are
detectable in the nucleus (
)also suggests that this cascade
may be activated independently of cytoplasmic proteins.
Figure 4:
Role of Abl in activating SAP kinase in
293 cells. A, 293 cells were treated with 10 µM ara-C and harvested at 30 min. Nuclei were isolated and the
nuclear proteins subjected to immunoprecipitation with anti-Abl. In
vitro immune complex kinase assays were performed using a
GST-Crk(120-225) fusion protein as substrate. B, 293
cells were treated with 10 µM ara-C for 2 h. Total lysates
were immunoprecipitated with anti-SAP kinase, and in vitro immune complex kinase assays were performed using
GST-Jun(2-100) as substrate. C, 293 cells were
transiently transfected with the indicated vectors and after 16 h, cell
lysates were incubated with glutathione-agarose for 30 min. In
vitro immune complex kinase assays were performed in the resulting
protein complexes by using GST-Jun as a substrate. Proteins were
separated by SDS-PAGE and analyzed by Coomassie Blue staining (left
panel) and autoradiography (right panel). WT,
wild type; SAPK, SAP kinase. D, pEBG-SAPK (2
µg/plate) was transiently transfected into NIH3T3 XB cells
together with the pEBG SEK1 K
R dominant negative. The molar ratio
of the SAPK vector to the dominant negative plasmid is indicated, with
the total DNA concentration kept constant by supplementation with pEBG
vector. Total cell lysates were incubated with glutathione-agarose for
30 min at 4 °C. In vitro immune complex kinase assays were
performed on the resulting protein complexes using GST-Jun as
substrate. Proteins were separated by SDS-PAGE and analyzed by
autoradiography.
Ara-C acts as an efficient but not absolute DNA chain terminator(4) . The conformational and hydrogen bonding differences of the arabinose sugar moiety (34) are consistent with decreased reactivity of the 3` terminus following ara-C incorporation and thereby slowing or termination of DNA chain elongation. The incorporation of ara-C into DNA results in inhibition of replication forks and the accumulation of DNA fragments(35) . While the event(s) responsible for activation of c-Abl remains unclear, DNA fragmentation may represent an initial signal. In this context, treatment with certain other agents that damage DNA, such as ionizing radiation, is also associated with c-Abl activation(33) . These findings and the present studies suggest that c-Abl is involved in SEK1-dependent activation of SAP kinase in response to DNA damage.