(Received for publication, May 30, 1995)
From the
The stress-activated protein (SAP) kinases are induced by tumor
necrosis factor, oncoproteins, and UV light. The present studies
demonstrate that ionizing radiation (IR) activates p54 SAP kinase.
IR-induced activation of SAP kinase is associated with binding to the
SH2/SH3-containing adaptor protein Grb2. This interaction is mediated
by the SH3 domains of Grb2 and the proline-rich sequence PPPKIP in the
carboxyl-terminal region of SAP kinase. We also demonstrate that SAP
kinase and the p85-subunit of phosphatidylinositol (PI) 3-kinase
form a complex in irradiated cells. The results indicate that this
complex involves binding of the p85
subunit of PI 3-kinase to the
SH2 domain of Grb2. The functional role of linking SAP kinase to PI
3-kinase is further supported by the finding that wortmannin, an
inhibitor of PI 3-kinase, stimulates SAP kinase activity. These results
suggest that the cellular response to IR may include regulation of SAP
kinase by a PI 3-kinase-dependent signaling pathway.
The c-jun early response gene is induced by diverse
mitogenic stimuli. Although the role of c-Jun in mitogenesis remains
unclear, studies have shown that this protein is involved in the
G/G
transition and progression through late
G
/S(1, 2) . Other studies have indicated
that c-Jun is involved in induction of cellular
differentiation(3) . Moreover, the finding that transcription
of c-jun is rapidly induced in cells exposed to UV light (4) and other DNA-damaging agents (5, 6) has
supported a role for c-Jun in the response to genotoxic stress.
The
cellular response to ionizing radiation (IR) ()includes
activation of DNA repair, cell cycle arrest, and cell
death(7) . Recent studies have shown that exposure of
eukaryotic cells to IR is also associated with activation of c-jun transcription. These findings have provided support for the
transduction of early nuclear signals to longer term changes in gene
expression in the response to radiation(3) . Other studies have
demonstrated that the pp44/42 mitogen-activated protein (MAP) kinases
are activated in irradiated cells and that this event is temporally
related to the activation of pp90
and early
response gene expression(8) . The involvement of MAP kinase in
the regulation of c-Jun (9) has suggested that this pathway may
contribute to IR-induced c-jun expression.
The
stress-activated protein (SAP) kinases, also known as c-Jun
amino-terminal kinases, are a MAP kinase-related family of p54/p46
serine/threonine kinases that phosphorylate
c-Jun(10, 11, 12) . Two serine residues
(Ser and Ser
) in the amino-terminal
transactivation domain of c-Jun that are phosphorylated in response to
mitogens, Ras, and phorbol esters have been identified as substrates
for SAP kinase(4, 11, 12) . SAP kinases are
activated by tumor necrosis factor, sphingomyelinase, and UV
light(10, 11, 12) . The demonstration that
Ha-Ras is also involved in the activation of SAP kinase has supported
the involvement of this pathway in protection against UV-induced damage
to cellular components other than DNA(11) . Recent studies have
demonstrated that SAP kinase is activated by a pathway involving MAP
kinase and SAP kinase-activating kinase 1(13, 14) .
The present work has examined involvement of the SAP kinases in IR-induced signaling. The results demonstrate that IR exposure is associated with the activation of SAP kinase and the formation of a Grb2-mediated complex involving SAP kinase and phosphatidylinositol (PI) 3-kinase.
Human U-937 myeloid leukemia cells were exposed to 20 grays IR and harvested at 1 h. Lysates were subjected to affinity chromatography with a GST-Jun fusion protein. Adsorbates from IR-treated cells exhibited an increase in Jun kinase activity compared with that of control cells (Fig.1A). IR-induced stimulation of Jun kinase activity was comparable with that obtained in tumor necrosis factor- (Fig.1A) or sphingomyelinase-treated cells (data not shown). In order to determine whether the IR-induced Jun kinase activity is due to stimulation of SAP kinase, we assayed anti-p54 SAP kinase immunoprecipitates for phosphorylation of GST-Jun. The finding that IR activates SAP kinase to phosphorylate GST-Jun (Fig.1B) and not GST (data not shown) supports the involvement of this kinase in IR-induced signaling.
Figure 1:
IR induces activation of SAP kinase. A, lysates from control (U-937) and IR-treated cells were
incubated with glutathione-Sepharose beads containing 5 µg of
GST-Jun amino acids(2-100) for 2 h. The beads were washed and
then incubated in kinase buffer containing
[-
P]ATP for 15 min at 30 °C. Reactions
were terminated by addition of 6
SDS-PAGE sample buffer. The
phosphorylated proteins were resolved by 10% SDS-PAGE, dried, and
analyzed by autoradiography. B, lysates from control and
IR-treated cells were subjected to immunoprecipitation with anti-p54
antibody. Immune complex kinase assays were performed by the addition
of GST-Jun fusion protein and [
-
P]ATP and
incubation for 15 min at 30 °C. The proteins were analyzed by 10%
SDS-PAGE and autoradiography. TNF, tumor necrosis
factor.
Analysis of anti-p54 immunoprecipitates suggested that other proteins associate with p54 after IR exposure. For example, the finding that a 26-kDa protein coprecipitates with SAP kinase raised the possibility of an interaction with Grb2. Grb2, also known as Ash, Sem-5, or Drk (17, 18, 19, 20) , is an SH2- and SH3-containing adaptor protein that links activated protein-tyrosine kinase receptors to the Ras activator protein Sos. Immunoblot analysis of anti-p54 immunoprecipitates with anti-Grb2 revealed increased reactivity with a 26-kDa protein following IR exposure (Fig.2A, left panel). To provide additional support for an association between Grb2 and SAP kinase, anti-Grb2 immunoprecipitates were analyzed for phosphorylation of GST-Jun. The results demonstrate that Grb2 associates with a Jun kinase activity that is induced by IR treatment (Fig.2A, middle panel). The interaction between Grb2 and SAP kinase was further examined by incubating lysates from control and IR-treated cells with a GST-Grb2 (full-length) fusion protein. Analysis of the adsorbates to GST-Grb2 supported binding of a kinase that phosphorylates GST-Jun (Fig.2A, right panel). Analysis of the adsorbates by immunoblotting with anti-p54 also revealed increased reactivity with a 54-kDa protein in the irradiated cells (Fig.2B). Lysates were similarly incubated with GST fusion proteins prepared from the SH2 and SH3 (carboxyl- and amino-terminal) domains of Grb2. Adsorbates obtained with the GST-Grb2 SH2 fusion protein exhibited no detectable anti-p54 reactivity (Fig.2B). In contrast, adsorbates from GST-Grb2 N-SH3 or GST-Grb2 C-SH3 revealed binding of SAP kinase when assaying lysates from irradiated but not control cells (Fig.2B). Lysates from irradiated cells were also incubated with GST-Lyn SH3 and GST-Fyn SH3 fusion proteins. The results demonstrate little if any binding of SAP kinase to these SH3-containing proteins (data not shown). These findings indicated that the IR-induced association between Grb2 and SAP kinase is specifically mediated through the SH3 domains of Grb2.
Figure 2:
Association of p54 SAP kinase and Grb2. A, lysates from control and IR-treated cells were
immunoprecipitated with anti-p54 antibody (left panel) or
anti-Grb2 antibody (middle panel) or subjected to affinity
chromatography with GST-Grb2 (full-length) (right panel). The
immune complexes were analyzed by immunoblotting with anti-Grb2.
Anti-Grb2 immune complexes and GST-Grb2 protein complexes were
incubated in kinase buffer containing
[-
P]ATP and 5 µg of GST-Jun fusion
protein for 15 min at 30 °C. The proteins were resolved by 10%
SDS-PAGE, dried, and analyzed by autoradiography. B, lysates
from control and IR-treated cells were incubated with GST-Grb2
(full-length), GST-Grb2 SH2, GST-Grb2 C-SH3, GST-Grb2 N-SH3, or
GST-Grb2 N-SH3 in the absence(-) or presence (+) of
competitor peptide (AEAPPPKIPDKQ). The bound proteins were resolved by
7.5% SDS-PAGE and immunoblotted with anti-p54 antibody. C,
lysates from control and IR-treated cells were incubated with GST-Grb2
N-SH3. Lysates from IR-treated cells were also immunoprecipitated with
anti-pp90
or anti-Erk1/2 antibodies. The
adsorbed or immunoprecipitated proteins were resolved by 7.5% SDS-PAGE
and analyzed by immunoblotting with anti-pp90
or
anti-MAP kinase antibodies.
Alignment of the Grb2, Crk, Nck, and Abl SH3-binding
sites has identified two proline residues spaced by two nonconserved
amino acids. The consensus PXXP has also been observed for
certain other SH3-binding sites(21, 22, 23) .
These findings suggested that a proline-rich candidate sequence
(PPPKIP) in SAP kinase may be responsible for the association with Grb2
SH3 domains. A synthetic peptide corresponding to amino acids
330-341 (AEAPPPKIPDKQ) of SAP kinase was used in competition
assays. Preincubation of GST-Grb2 N-SH3 with the peptide inhibited
binding of SAP kinase by 50% in lysates of irradiated cells (Fig.2B). In contrast, there was no detectable
inhibition of SAP kinase binding when using an unrelated peptide
(LQHPYINVWYDP) as competitor (data not shown). Previous studies have
demonstrated that IR also induces MAP (Erk1/2) and pp90 kinase activity(8) . To assess specificity of SAP
kinase binding to SH3 domains of Grb2, experiments were performed in
which the IR-induced lysates were incubated with GST-Grb2 N-SH3 or
GST-Grb2 C-SH3 and the adsorbed proteins assayed by immunoblotting with
either anti-MAP or anti-pp90
antibodies. The
results demonstrate that, unlike SAP kinase, there is no detectable
association of MAP and pp90
with the Grb2 SH3
domains (Fig.2C and data not shown). These findings
indicate that the IR-induced binding of Grb2 and SAP kinase is mediated
through the SH3 domains of Grb2 and the PPPKIP site in SAP kinase.
The significance of Grb2 binding to SAP kinase in irradiated cells
is presumably to link SAP kinase to an upstream or downstream effector
in the IR response. We therefore studied anti-p54 immunoprecipitates to
define other proteins that might associate with a Grb2-SAP kinase
complex. Staining demonstrated increased binding of a protein at
approximately 85 kDa (data not shown). Because the PI 3-kinase consists
of 85 and 110 kDa subunits, we asked whether p85 of PI 3-kinase
associates with SAP kinase. Analysis of anti-p54 immunoprecipitates
with anti-PI 3-kinase (p85
) demonstrated increased reactivity in
IR-treated as compared with control cells (Fig.3A).
Although these findings suggest that SAP kinase and PI 3-kinase
associate in irradiated cells, the results do not distinguish between
direct binding of these molecules or an indirect association through
other proteins. One possible explanation for an association between SAP
kinase and PI 3-kinase is through Grb2. In this regard, recent studies
have demonstrated that PI 3-kinase associates with Grb2 during growth
factor stimulation(24) . To address this issue, we assayed Grb2
immunoprecipitates for the presence of PI 3-kinase p85
. The
results demonstrate binding of p85
to Grb2 in control and
irradiated cells (Fig.3B, left panel).
Analysis of anti-p85
immunoprecipitates confirmed binding to Grb2 (Fig.3B, right panel), although there was no
detectable anti-Grb2 reactivity when assaying precipitates with
preimmune rabbit serum or anti-c-Abl (data not shown). In order to
define the domain of Grb2 involved in binding to PI 3-kinase, lysates
from control and IR-treated U-937 cells were subjected to affinity
chromatography with GST-Grb2 (full-length), GST-Grb2 SH2, GST-Grb2
C-SH3, or GST-Grb2 N-SH3 domains. The adsorbed proteins were then
immunoblotted with anti-p85
. The results demonstrate that
association of p85
with the SH2 domain of Grb2 is increased by IR
exposure (Fig.3C). Moreover, the finding that tyrosine
phosphorylation of p85
is increased in irradiated cells provides
an explanation for the IR-stimulated interaction of this protein with
the Grb2 SH2 domain (Fig.3D).
Figure 3:
Association of the p85 subunit of PI
3-kinase with p54 SAP kinase and Grb2. A, lysates from control
and IR-treated cells were subjected to immunoprecipitation with
anti-p54 antibody. Proteins were resolved by 7.5% SDS-PAGE and analyzed
by immunoblotting with anti-p85
antibody. B, lysates from
control and IR-treated cells were immunoprecipitated with anti-p85
and anti-Grb2 antibodies. The proteins were resolved by SDS-PAGE and
immunoblotted with anti-Grb2 and anti-p85
. C, lysates
from control and IR-treated cells were incubated with GST-Grb2 SH2
protein. Lysates from IR-treated cells were also incubated with GST and
GST-Grb2 (full-length). The bound proteins were resolved by 7.5%
SDS-PAGE and subjected to immunoblotting with anti-p85
antibody. D, lysates from control and IR-treated cells were subjected to
immunoprecipitation with anti-Tyr(P) (anti-P-Tyr) antibody.
The bound proteins were resolved by 7.5% SDS-PAGE and analyzed by
immunoblotting with anti-P85
.
Whereas these
findings are in support of a Grb2-mediated association between SAP
kinase (through the Grb2 SH3 domains) and PI 3-kinase p85 (through
the Grb2 SH2), we also asked whether there is a potential functional
relationship between these two proteins. In addressing this
possibility, we pretreated cells with wortmannin, a potent PI 3-kinase
inhibitor(25, 26) , to determine whether PI 3-kinase
is coupled to IR-induced SAP kinase signaling. Wortmannin treatment was
associated with stimulation of constitutive levels of SAP kinase
activity (Fig.4A). Moreover, activation of SAP kinase
in cells treated with both wortmannin and IR was similar to that
obtained with wortmannin alone (Fig.4A). In contrast,
rapamycin (inhibitor of pp70
)(27) , H-7
(a nonspecific protein kinase C inhibitor)(28) , and HA1004 (an
inhibitor of cyclic nucleotide-dependent protein kinase) (29) had little effect on SAP kinase activity (Fig.4B and data not shown). These results suggest a
potential role for PI 3-kinase in the regulation of SAP kinase.
Figure 4:
Activation of SAP kinase by wortmannin. A, cells were exposed to IR, harvested at 1 h, pretreated with
250 nM wortmannin for 30 min (WORT) or with
wortmannin and then IR (WORT/IR). Lysates were
immunoprecipitated with anti-p54 antibody, and the resulting immune
complexes were incubated in kinase buffer containing 10 µg of
GST-Jun and [-
P]ATP for 15 min at 30
°C. The reactions were stopped by the addition of SDS-PAGE sample
buffer. Phosphorylated proteins were separated by 10% SDS-PAGE, dried,
and analyzed by autoradiography. B, cells were treated with 1
ng/ml rapamycin (RAPA), 250 nM wortmannin (WORT), 50 µM H-7, or 50 µM HA1004
(data not shown) for 30 min. Lysates were subjected to
immunoprecipitation with anti-p54 antibody. GST-Jun phosphorylation
assays were performed as described above.
PI
3-kinase possesses lipid and serine kinase
activities(30, 31) . The 85-kDa subunit of PI 3-kinase
binds to tyrosine-phosphorylated proteins through its SH2 domains, and
the 110-kDa subunit exhibits catalytic activity. Although IR treatment
is associated with increased tyrosine phosphorylation of p85 (Fig.3D), the precise mechanism by which p85
interacts with the SH2 domain of Grb2 requires further study. The
activation of PI 3-kinase results in the formation of PI-3,4-P
and PI-3,4,5-P
. The finding that the
Ca
- and phorbol ester-insensitive
isoform of
protein kinase C is activated by PI-3,4,5-P
has suggested
that this kinase may be a target for PI 3-kinase
signaling(32) . The serine kinase activity of PI 3-kinase
contributes to the phosphorylation of p85 subunit(31) . Other
work has indicated that PI 3-kinase phosphorylates the insulin receptor
substrate 1 on serine(33) . Wortmannin inhibits both the lipid
and serine phosphorylation activities of PI 3-kinase(33) .
Thus, the effects of wortmannin on PI 3-kinase signaling pathways may
be complex. The present demonstration that wortmannin treatment is
associated with activation of SAP kinase suggests that PI 3-kinase may
be involved in the regulation of SAP kinase. The findings that PI
3-kinase and SAP kinase form a complex in irradiated cells also
supports a possible functional interaction between these two molecules.
However, although SAP kinase-activating kinase 1 has been shown to
directly stimulate SAP kinase activity(13, 14) ,
further studies are needed to more precisely define a potential
interaction between PI 3-kinase and SAP kinase.