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J Biol Chem, Vol. 274, Issue 15, 10140-10144, April 9, 1999
,,
From the Department of Adult Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, Boston, Massachusetts 02115, Division of Experimental Medicine and Hematology,
Deaconess Medical Center, Harvard Institutes of Medicine, Boston,
Massachusetts 02115, § Department of Molecular Biology,
Diabetes Research Laboratory, Massachusetts General Hospital, Boston,
Massachusetts 02114, and ¶ Department of Biochemistry and
Molecular Biology, Howard Hughes Medical Institute, Program in
Molecular Medicine, University of Massachusetts Medical School,
Worcester, Massachusetts 01605
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The stress-activated p38 mitogen-activated
protein kinase (p38 MAPK), a member of the subgroup of mammalian
kinases, appears to play an important role in regulating inflammatory
responses, including cytokine secretion and apoptosis. The upstream
mediators that link extracellular signals with the p38 MAPK signaling
pathway are currently unknown. Here we demonstrate that pp125 focal
adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2,
CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the
activation of p38 MAPK. Furthermore, overexpression of a
dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38
MAPK activation. MKK3 and MKK6 are known potential constituents of p38
MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators
of SAPK/JNK and ERK pathways, respectively. We observe that the
dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1
inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results
demonstrate that treatment of cells with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken
together, these findings indicate that RAFTK represents a
stress-sensitive mediator of the p38 MAPK signaling pathway in response
to certain cytotoxic agents.
The mitogen-activated protein kinases (MAPKs)1 are
induced in response to diverse classes of
inducers in the transduction of signals from the cell membrane to the
nucleus. MAPKs are proline-directed Ser/Thr protein kinases that are
regulated by extracellular signals including growth factors and
cellular stress (1-3). The well characterized MAPK subfamily includes
ERK1 and ERK2, which are activated by growth factors via the conserved
Ras/Raf/MEK pathway (4-7).
c-Jun N-terminal protein kinases (JNKs) or stress-activated protein
kinases (SAPKs) represent a second class of the mammalian MAPKs, which
are primarily activated in response to tumor necrosis factor,
interleukin-1, UV-, and DNA-damaging agents (5, 8-11). A recently
identified novel protein tyrosine kinase, related adhesion focal
tyrosine kinase (RAFTK) (12) (also known as Pyk2, Refs. 13 and 14);
CADTK, Ref. 15) has been shown to be involved upstream to ERKs and JNK
signaling pathways (14, 16). RAFTK is also a close relative to pp125
FAK tyrosine kinase and is activated by various extracellular signals
that increase intracellular calcium concentrations (13). Moreover,
RAFTK can tyrosine phosphorylate and modulate the action of ion
channels and appears to function as an intermediate that links various
calcium signals with both short- and long-term responses in neuronal
cells (13).
An additional class, which presents substantial similarity to the
Saccharomyces cerevisiae HOG1 kinase involved in response to
increased extracellular osmolarity (17), is p38 MAPK. p38 MAPK can also
be activated by changes in osmolarity, lipopolysaccharides, and in
response to DNA-damaging agents (3, 18-20). Other studies have
demonstrated that the Rho GTPases and multiple p21 activated kinases
regulate p38 MAPK (21, 22). Moreover, in contrast to activation of
ERKs, interleukin-1 and tumor necrosis factor are potent activators of
p38 MAPK, suggesting upstream signals via Ras does not play a key role
in p38 MAPK activation. Previous studies have shown that diverse
genotoxic agents activate p38 MAPK and that this response is
mediated by c-Abl protein tyrosine kinase-dependent and
-independent mechanisms (20). Taken together, although certain insights
are available, the precise upstream mechanisms responsible for
activation of p38 MAPK are presently unclear.
The results of the present study demonstrate that in contrast to
ionizing radiation (IR), cis-platinum (CDDP), or ultraviolet radiation (UV), RAFTK is activated in response to certain cytotoxic agents such as methylmethane sulfonate (MMS) or hyperosmolarity. Overexpression of RAFTK leads to the activation of p38 MAPK.
Furthermore, the dominant-negative mutant of MKK3 but not of MKK6
(potential upstream mediators of p38 MAPK signaling pathway) inhibits
RAFTK-induced p38 MAPK activity. These findings indicate that RAFTK
represents a stress-sensitive mediator of the p38 MAPK pathway.
Cell Culture and Reagents--
Human U-937 myeloid leukemia
cells were grown in RPMI 1640 supplemented with 10% heat-inactivated
fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin,
and 2 mM L-glutamine. PC12 cells were grown in
Dulbecco's modifed Eagle's medium containing 10% heat-inactivated
horse serum, 5% heat-inactivated fetal bovine serum and antibiotics.
293T cells were grown in Dulbecco's modifed Eagle's medium containing
10% fetal bovine serum and antibiotics. Cells (1 × 106/100-mm culture dish) were plated 24 h before
treating with 1 mM MMS (Sigma), 100 µM CDDP
(Sigma), 500 mM NaCl, 20 µM BAPTA-AM (Sigma),
or 20 Gy IR at room temperature with a Gammacell 1000 (Atomic Energy of
Canada, Ottawa) under aerobic conditions with a 137Cs
source emitting at a fixed dose rate of 0.76 Gy min Immunoprecipitation and Immunoblot Analysis--
U-937 or PC12
cells were treated with 1 mM MMS, 500 mM NaCl,
100 µM CDDP, 20 Gy IR, 20 µM BAPTA-AM, or
40 J/m2 UV and harvested at different time intervals. The
cells were subjected to immunoprecipitation with anti-RAFTK antibody as
described (23) and analyzed by immunoblotting with anti-P-Tyr. U-937
cells were also treated with 1 mM MMS and harvested at
different time intervals. Total cell lysates were subjected to
immunoblotting with anti-phospho-MKK3/MKK6 antibody (New England
Biolab). The antigen-antibody complexes were visualized by
chemiluminescence (ECL, Amersham).
Transient Transfections, Immunoprecipitations, and Immune Complex
Kinase Assays--
293T cells were transiently transfected with vector
or Flag-RAFTK with HA-p38 MAPK or pEBG-SAPK using a standard calcium
phosphate method as described by Kharbanda et al. (24).
Cells were co-transfected separately with dominant-negative mutants of
Flag-MKK3, Flag-MKK6, MEK1, or SEK1 with HA-p38 MAPK. After 48 h
of transfections, cells were washed with phosphate-buffered saline and
lysed in 1 ml of lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 µg/ml leupeptin and aprotinin)
as described by Kharbanda et al. (25). Total cell lysates
were subjected to immunoprecipitation with anti-HA antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) for 1 h at 4 °C and then for
1 h after addition of protein A-Sepharose. The immune complexes
were washed three times with lysis buffer, once with kinase buffer, and
resuspended in kinase buffer containing [
PC12 cells were transiently transfected with vector or Flag-RAFTK (K-M)
with HA-p38 MAPK using LipofectAMINETM (Life Technologies,
Inc.). After transfection, cells were treated with MMS, and total cell
lysates were subjected to incubation with anti-HA antibody. The
immunoprecipitates were analyzed by immune complex kinase assays using
GST-ATF2 as substrate as described. The reactions were incubated for 15 min at 30 °C and terminated by the addition of SDS sample buffer.
The proteins were analyzed by 10% SDS-PAGE and autoradiography. The
immune complexes were also analyzed by immunoblotting with anti-HA.
Previous studies have demonstrated that certain agents, such as
phorbol esters and sorbitol, activate RAFTK (15, 16). Activation of
RAFTK by tumor necrosis factor or UV, as detected by its
phosphorylation on tyrosine, is controversial and may be cell type
specific (15, 16). RAFTK has also been linked to cell and inducer type
specific activation of JNK/SAPK and ERK1/2 signaling pathways
(14-16).
To determine whether RAFTK is involved in activation of p38 MAPK
pathway, we transiently overexpressed wild type RAFTK along with HA-tag
p38 MAPK in 293T cells and analyzed the anti-HA protein precipitates
for GST-ATF2 phosphorylation. In a parallel experiment, we also
overexpressed wild type RAFTK with pEBG-SAPK, and GST protein
precipitates were analyzed for GST-Jun phosphorylation. The results
demonstrate increased GST-ATF2 phosphorylation (increased p38 MAPK
activity) in cells transfected with wild type RAFTK compared with that
with vector (Fig. 1A). The
activation of p38 MAPK was dependent on expression of the RAFTK protein
(data not shown). Similar results were obtained when wild type RAFTK
transfected cells were analyzed for JNK activity (Fig. 1B).
The RAFTK-induced increase in p38 MAPK or JNK activities occurred in
the absence of changes in p38 or JNK protein levels, respectively (Fig.
1, A and B). To confirm expression of RAFTK, we
assayed the anti-RAFTK immunoprecipitates for reactivity with
anti-RAFTK or anti-P-Tyr antibody. The results demonstrate increased
protein levels and tyrosine phosphorylation of RAFTK in overexpressed
cells (Fig. 1, A and B). Taken together, these
findings indicate that RAFTK acts as an upstream mediator of the p38
MAPK pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1 as
determined by dosimetry. Cells were also treated with 40 J/m2 UV (UV StratalinkerTM, 1800, Stratagene).
-32P]ATP
(6,000 Ci/mmol; NEN Life Science Products) and GST-ATF2 (1-109) (20).
After 48 h of transfection separately, total cell lysates were
also incubated with GST, and the protein precipitates were analyzed by
immune complex kinase assays using GST-Jun (1-102) as substrate (26).
The reactions were incubated for 15 min at 30 °C and terminated by
the addition of SDS sample buffer. The proteins were analyzed by 10%
SDS-PAGE and autoradiography. The immune complexes were also analyzed
by immunoblotting with anti-HA, anti-JNK (Santa Cruz Biotechnology),
anti-RAFTK (12), or anti-P-Tyr (4G10; Upstate Biotechnology, Lake
Placid, NY).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Activation of p38 MAPK and JNK/SAPK by
overexpression of RAFTK. 293T cells were transiently transfected
with vector or with wild type Flag-RAFTK. The cells were also
co-transfected with HA-p38 MAPK (A) or with pEBG-SAPK
(B). After 48 h, total cell lysates were
immunoprecipitated with anti-HA antibody, and in vitro
immune complex kinase reactions were performed using GST-ATF2 fusion
protein as a substrate (A). Total cell lysates were also
incubated with GST and in vitro c-Jun kinase assays were
performed in the protein precipitates using GST-Jun as substrate
(B). The proteins were separated by 10% SDS-PAGE, stained
with Coomassie Blue, and analyzed by autoradiography (top
panels, A and B). Anti-HA immunoprecipitates
or GST protein precipitates were also analyzed by immunoblotting with
anti-p38 MAPK and anti-GST-SAPK, respectively (second
panels, A and B). Anti-RAFTK
immunoprecipitates were also analyzed by immunoblotting with anti-RAFTK
(third panels, A and B) or anti-P-Tyr
(fourth panels, A and B). The fold
increase in p38 MAPK or JNK/SAPK activities is shown (bottom
panels, A and B) as the mean ± S.E.
for four independent experiments.
Because RAFTK is activated in part by phosphorylation on Tyr (13), we
next examined the effect of different potent p38 MAPK activators on the
status of RAFTK phosphorylation in human myelomonocytic leukemia
(U-937) cells and in rat pheochromocytoma (PC12) cells. In contrast to
IR or CDDP, stimulation with MMS or hyperosmolarity lead to enhance
tyrosine phosphorylation of RAFTK in U-937 and PC12 cells (Fig.
2). Exposure of U-937 or PC12 cells to UV
caused, little, if any, increase in the tyrosine phosphorylation of
RAFTK (Fig. 2A and data not shown). Moreover, all of these
stress inducers activate p38 MAPK in both cell types (Fig. 2,
B and D and data not shown). Taken together,
these findings indicated that not all stress signals stimulate
activation of RAFTK.
|
Our findings suggest that RAFTK acts as an upstream mediator of the p38
MAPK pathway. To further confirm a direct role for RAFTK in MMS-induced
activation of p38 MAPK, PC12 cells were transiently transfected with a
dominant-negative mutant of RAFTK (RAFTK K-M). The cells were also
cotransfected with HA-p38 MAPK. PC12 cells were used in this study
because RAFTK is expressed in these cells and the transfection
efficiency in PC12 cells is comparatively better than that in U-937
cells. After transfection, cells were treated with MMS and assayed for
activation of p38 MAPK. As a control, PC12 cells expressing the empty
vector and HA-p38 MAPK were treated with MMS. The results demonstrate
that treatment of PC12 cells expressing RAFTK K-M mutant but not the
empty vector with MMS is associated with a significant inhibition of
p38 MAPK activity (Fig. 3).
Anti-HA-immunoprecipitates were also analyzed by immunoblotting with
anti-HA as a control to determine equal protein expression of p38 MAPK
(Fig. 3). Taken together, these findings indicate that RAFTK acts as an
upstream activator of the p38 MAPK signaling pathway at least in the
cellular response to MMS.
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RAFTK is activated by various extracellular signals that increase
intracellular calcium levels such as carbachol, angiotensin II, or
sorbitol (13, 16). To examine the role of calcium on MMS-induced
activation of RAFTK and induction of p38 MAPK activity, we treated
U-937 cells with MMS in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid, tetra(acetoxymethyl)-ester (BAPTA-AM). BAPTA-AM is a
membrane-permeable calcium chelator and has been shown to inhibit JNK
activation in response to various inducers that raise intracellular
calcium, such as angiotensin II, thapsigargin, or ionophore (27). Other
studies have shown that BAPTA-AM acts also as an intracellular calcium
channel blocker (28). The results demonstrate that MMS-induced tyrosine
phosphorylation of RAFTK was significantly inhibited in cells that were
treated in the presence of BAPTA-AM (Fig.
4A). Moreover, in the presence of BAPTA-AM, p38 MAPK activity induced in response to MMS is also significantly inhibited but not completely blocked (Fig. 4B,
top panel). Furthermore, the inhibition of MMS-induced p38
MAPK activity by BAPTA-AM was without any significant effect on p38
MAPK protein levels (Fig. 4B, bottom panel).
Treatment of U-937 cells with MMS in the presence and absence of
BAPTA-AM. These results suggest that MMS-induced activation of RAFTK is
mediated, at least in part, by the increase in intracellular calcium
levels that is induced by this stimuli. Thus inhibition in MMS-induced
p38 MAPK activity by BAPTA-AM further confirms the role of RAFTK in
mediating p38 MAPK activation in response to MMS. Because MMS-induced
activation of p38 MAPK is not completely blocked in the presence of
BAPTA-AM, it is likely that MMS activates the p38 MAPK by multiple
upstream pathways. Therefore, RAFTK may function as one and not the
only upstream mediator in the MMS response.
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The p38 MAPK signal transduction pathway is activated by diverse
classes of stimuli such as proinflammatory cytokines, environmental stress, and DNA damage (1, 20, 29). These stimuli can also activate
other signal transduction pathways such as ERKs or JNKs (5, 8-10, 30).
Moreover, the role of selective upstream activators of these pathways
such as MKK3 and MKK6 for p38 MAPK, MEK1 and MEK2 for ERK, and MKK4
(SEK1) and MKK7 are necessary for JNK activation (29, 31-34). To
determine whether RAFTK-induced activation of p38 MAPK involves MKK3 or
MKK6, we used catalytically inactive mutants of MKK3 (MKK3 K-A) and
MKK6 (MKK6 K-A) (31), which act as dominant-negative inhibitors. 293T
cells were transiently transfected with wild-type RAFTK, Flag-MKK3 K-A
or Flag-MKK6 K-A with HA-p38 MAPK. After 48 h, anti-HA
immunoprecipitates were analyzed for p38 MAPK activity. The results
demonstrate that RAFTK-induced activation of p38 MAPK is significantly
inhibited by MKK3 K-A but not by MKK6 K-A (Fig.
5A). As a control, 293T cells
were also transfected separately with dominant-negative mutants of SEK1 or MEK1 in the presence of wild-type RAFTK and HA-p38 MAPK and anti-HA
immunoprecipitates were analyzed for p38 MAPK activity. The results
demonstrate that in contrast to MKK3 K-M, dominant-negative mutants of
SEK1 or MEK1 are not associated with significant inhibition in p38 MAPK
activity (Fig. 5B). To assess whether treatment of cells
with MMS induces phosphorylation of MKK3, U-937 cells were treated with
MMS and harvested at different time intervals. Cell lysates were then
analyzed by immunoblotting with antibody raised against the
phosphorylated form of MKK3. The results demonstrate that treatment
with MMS is associated with induction in the phosphorylated form of
MKK3 (Fig. 5C). Taken together, these findings demonstrated that RAFTK acts upstream to MKK3 in the stress response to MMS.
|
The present results demonstrate that diverse types of stress inducers
induce p38 MAPK activity in U-937 and PC12 cells. The findings also
demonstrate that MMS and NaCl induce p38 MAPK activity by
RAFTK-dependent mechanisms. Previous work has shown that
IR- and CDDP-induced activation of p38 MAPK occurs by c-Abl tyrosine kinase-dependent mechanisms. Furthermore, MMS, UV, tumor
necrosis factor, and NaCl induce p38 MAPK by c-Abl-independent
mechanisms. The finding correlates that MMS- and NaCl-induced p38 MAPK
activity requires activation of RAFTK, further supports distinct
signaling events used by various cytotoxic agents. The UV response in
mammalian cells is initiated in an extranuclear compartment (35). The finding that Ha-Ras contributes to the induction of JNK by UV is also
consistent with the hypothesis that UV response is initiated in the
cytoplasm. MMS is a monofunctional alkylating agent that alkylates DNA
and damages membrane proteins (36, 37). Therefore, MMS may also
activate p38 MAPK activity by DNA damage-independent mechanisms. In
this context, taken together with our previous findings (20) in
contrast to DNA-damaging agents, MMS and NaCl induces p38 MAPK activity
by RAFTK-dependent and c-Abl-independent mechanisms.
Therefore, these results demonstrate that activation of the stress
response to diverse agents can be distinguished by
c-Abl-dependent, RAFTK-dependent, and/or other
tyrosine kinase-dependent mechanisms.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Kyriakis, Joseph Avruch, and Leonard Zon for providing various SAPK cDNAs and anti-GST-SAPK antibody. We thank Rebecca Farber for technical assistance.
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FOOTNOTES |
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* This investigation was supported by Public Health Service Grants CA75216 (to S. K.), CA65861 (to R. J. D.) awarded by NCI, and by RO1 HL55445 (to S. A.) from National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Adult
Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2938; Fax: 617-632-2934; E-mail: surender_kharbanda{at}dfci.harvard.edu.
1 MAPK, mitogen-activated protein kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; RAFTK, related adhesion focal tyrosine kinase; CADTK, calcium-dependent tyrosine kinase; IR, ionizing radiation; CDDP, cis-platinum; MMS, methylmethane sulfonate; UV, ultraviolet radiation; anti-P-Tyr, anti-phosphotyrosine; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester; Gy, gray; HA, hemagglutinin; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis ERK, extracellular regulated kinase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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 J. Ohanian, P. Cunliffe, E. Ceppi, A. Alder, E. Heerkens, and V. Ohanian Activation of p38 Mitogen-Activated Protein Kinases by Endothelin and Noradrenaline in Small Arteries, Regulation by Calcium Influx and Tyrosine Kinases, and Their Role in Contraction Arterioscler. Thromb. Vasc. Biol., December 1, 2001; 21(12): 1921 - 1927. [Abstract] [Full Text] [PDF]
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S. S. Chuang, P. R. Kumaresan, and P. A. Mathew 2B4 (CD244)-Mediated Activation of Cytotoxicity and IFN-{gamma} Release in Human NK Cells Involves Distinct Pathways J. Immunol., December 1, 2001; 167(11): 6210 - 6216. [Abstract] [Full Text] [PDF]
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 M. Del Corno, Q.-H. Liu, D. Schols, E. de Clercq, S. Gessani, B. D. Freedman, and R. G. Collman HIV-1 gp120 and chemokine activation of Pyk2 and mitogen-activated protein kinases in primary macrophages mediated by calcium-dependent, pertussis toxin-insensitive chemokine receptor signaling Blood, November 15, 2001; 98(10): 2909 - 2916. [Abstract] [Full Text] [PDF]
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Y. Rikitake, S. Kawashima, T. Takahashi, T. Ueyama, S. Ishido, N. Inoue, K.-I. Hirata, and M. Yokoyama Regulation of tyrosine phosphorylation of PYK2 in vascular endothelial cells by lysophosphatidylcholine Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H266 - H274. [Abstract] [Full Text] [PDF]
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P. Siljander, R. W. Farndale, M. A. H. Feijge, P. Comfurius, S. Kos, E. M. Bevers, and J. W. M. Heemskerk Platelet Adhesion Enhances the Glycoprotein VI-Dependent Procoagulant Response : Involvement of p38 MAP Kinase and Calpain Arterioscler. Thromb. Vasc. Biol., April 1, 2001; 21(4): 618 - 627. [Abstract] [Full Text] [PDF]
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 U. Kintscher, S. Wakino, S. Kim, E. Fleck, W. A. Hsueh, and R. E. Law Angiotensin II Induces Migration and Pyk2/Paxillin Phosphorylation of Human Monocytes Hypertension, February 1, 2001; 37(2): 587 - 593. [Abstract] [Full Text] [PDF]
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 D. Tian, V. Litvak, and S. Lev Cerebral Ischemia and Seizures Induce Tyrosine Phosphorylation of PYK2 in Neurons and Microglial Cells J. Neurosci., September 1, 2000; 20(17): 6478 - 6487. [Abstract] [Full Text] [PDF]
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