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J Biol Chem, Vol. 275, Issue 10, 7395-7402, March 10, 2000
,From the Kennedy Institute of Rheumatology, London W6 8LH, United Kingdom
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ABSTRACT |
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Pyridinyl imidazole inhibitors, particularly
SB203580, have been widely used to elucidate the roles of p38
mitogen-activated protein (MAP) kinase (p38/HOG/SAPKII) in a wide array
of biological systems. Studies by this group and others have shown that
SB203580 can have antiproliferative activity on cytokine-activated
lymphocytes. However, we recently reported that the antiproliferative
effects of SB203580 were unrelated to p38 MAP kinase activity. This
present study now shows that SB203580 can inhibit the key cell cycle
event of retinoblastoma protein phosphorylation in
interleukin-2-stimulated T cells. Studies on the proximal regulator of
this event, the phosphatidylinositol 3-kinase/protein kinase B
(PKB)(Akt/Rac) kinase pathway, showed that SB203580 blocked the
phosphorylation and activation of PKB by inhibiting the PKB kinase,
phosphoinositide-dependent protein kinase 1. The concentrations
of SB203580 required to block PKB phosphorylation
(IC50 3-5 µM) are only approximately
10-fold higher than those required to inhibit p38 MAP kinase
(IC50 0.3-0.5 µM). These data define a new
activity for this drug and would suggest that extreme caution should be
taken when interpreting data where SB203580 has been used at
concentrations above 1-2 µM.
Interleukin-2 (IL-2)1 is
a potent T cell growth factor that mediates its effects via a high
affinity heterotrimeric receptor comprising The above data suggest the existence of a novel target of SB203580 that
is critical for mitogenic signaling. To help understand the mechanism
of action and possible targets of SB203580, the effect of this
inhibitor on cell cycle-regulated proteins was examined. We found that
the IL-2-induced hyperphosphorylation of Rb was greatly reduced in
SB203580-treated cells, whereas the drug had no effect on
cytokine-induced p27kip degradation or Myc expression.
Subsequent studies investigated the effects of SB203580 on PI
3-kinase/protein kinase B (PKB), as these kinases have been implicated
in the regulation of Rb phosphorylation (10). We observed that although
PI 3-kinase activity was unaffected, the phosphorylation of PKB on
Thr308 and Ser473 was inhibited, resulting in
an inhibition of IL-2-induced kinase activity. The concentrations of
SB203580 required were commensurate with antiproliferative effects of
the drug and provide evidence that 3-phosphoinositide-dependent
protein kinase 1 (PDK1; the Thr308 kinase) is inhibited by
SB203580. It has recently been reported that PDK1 also acts as the
Ser473 kinase (11). These results could provide a mechanism
for the antiproliferative effect of SB203580. Furthermore these data
will have important implications for the interpretation of results from
numerous published studies in which SB203580 has been used at
concentrations in excess of 2 µM.
Materials
IL-2 was a generous gift from Dr. P. Lomedico (Roche Molecular
Biochemicals). Antiphospho-specific Ser473 and
Thr308 PKB were from New England Biolabs (Hitchin, Herts,
UK), whereas sheep anti-PKB and other reagents for in vitro
PKB kinase assay were sold as a kit by Upstate Biotechnology, Inc.
(Lake Placid, NY). Anti-Rb was from Pharmingen (San Diego, CA), and the
monoclonal anti-cyclin D3 and anti-p27kip1 were from
Pierce. Rabbit anti-p70S6 kinase was from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA) and was used for both immunoprecipitation and
Western blotting. Rabbit anti-c-Myc antibody was from Santa Cruz (Santa
Cruz, CA). SB203580 was purchased from Calbiochem-Novabiochem, and U5
anti-p85 Methods
Cells and Proliferation Assay
The IL-2-dependent murine T cell line, CT6, was
grown and maintained as described previously (12). These cells were
rested by washing three times in RPMI and culturing overnight in RPMI, 5% fetal calf serum in the absence of growth factor, antibiotics, or
Kinase Assays
PKB Kinase Assay--
Cells were lysed in Buffer A (see below)
for Western blotting and PKB kinase assays. Kinase assays were
performed according to the manufacturer's instructions. Briefly, 4 µg of sheep anti-PKB PI 3-Kinase Assay--
Cells were lysed in PI 3-kinase lysis
buffer (40 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EGTA supplemented with 1 mM DTT, 1 mM Na3VO4, and 10 µg/ml each of
aprotinin, pepstatin, leupeptin) at 10 × 106
cells/ml, and the post-nuclear lysate was precleared with 25 µl of
protein G-Sepharose for 1 h then preincubated with 5 µg of
monoclonal anti-p85 p70S6 Kinase Assay--
Cells were lysed in 0.5 ml of p70S6
kinase lysis buffer (10 mM potassium phosphate, pH 7.05, 0.5% Triton X-100, 1 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 µg/ml aprotinin), and the postnuclear lysate was
precleared with 20 µl of protein A-agarose for 30 min. The precleared
supernatant was then preincubated with 5 µl of rabbit antiserum to
p70S6 kinase for 1 h and additionally with 25 µl of protein
A-agarose with mixing for a further 1 h, all at 4 °C. The final
immune complex was washed twice in 0.5 ml of lysis buffer and twice in
0.5 ml of kinase assay buffer (50 mM MOPS, pH 7.2, 1 mM DTT, 30 mM ATP, 5 mM
MgCl2, 10 mM
p-nitrophenylphosphate). To washed immune complex pellet was
added 45 µl of assay mixture (made up of 35 µl of kinase assay
buffer, 5 µl of 125 mM substrate peptide (KKRNRTLTK), 5 µl of 50 µM protein kinase A inhibitor, 5 µCi of
[ Recombinant PDK1/PKB Assays--
Lipid vesicles were made by
drying down a mixture of phosphatidylcholine and phosphatidylserine
in vacuo and reconstituting with lipid buffer (0.2 M NaCl, 20 mM HEPES, 2 mM EGTA) to
a final 5 times working stock (500 µM
phosphatidylcholine, 500 µM phosphatidylserine, and 100 µM phosphatidylinositol 3,4,5-trisphosphate
(PIP3) and sonicated before use. EE-tagged recombinant PDK1
and PKB Western Blotting--
After separation by SDS-polyacrylamide gel
electrophoresis, proteins were transferred onto nitrocellulose or
polyvinylidene difluoride membranes that were then blocked with 5%
(w/v) milk powder in Tris-buffered saline/Tween 20 (0.1%) for 1 h
at room temperature. The blocked membranes were then incubated in
1:1000 dilution of first-stage antibody (made up in Tris-buffered
saline Tween, 2% bovine serum albumin supplemented with 0.1% azide)
overnight, then washed and incubated with 1:1000 horseradish
peroxidase-conjugated secondary antibody (made up in Tris-buffered
saline Tween, 5% (w/v) milk powder) for 1 h at room temperature
before developing with ECL.
SB203580 Inhibits IL-2-induced Rb Hyperphosphorylation--
As we
have shown previously (4, 5), the IL-2-induced proliferation of primary
human T cells, murine CT6 T cells, or BAF F7 B cells is prevented by
the p38 MAP kinase inhibitor with an IC50 of 3-5
µM (Fig. 1). However, as
our recent studies (5) showed that IL-2-induced proliferation and the
inhibitory effects of SB203580 on this event were independent of p38
MAP kinase or even p54 MAP kinase activity in both T cells and B cells,
we endeavored to identify other possible targets involved in mediating
the anti-proliferative effects. To do this, we investigated events
associated with cell cycle progression. SB203580 had no effect on Myc
expression, except for a small reduction at 30 µM only
(Fig. 2a). Furthermore,
nuclear staining of SB203580-treated CT6 cells with propidium iodide
showed no evidence of apoptosis after stimulation with IL-2 for 20 h (data not shown). The expression of hyperphosphorylated Rb and degradation of p27kip1 were also measured as markers of
S-phase entry. The addition of IL-2 to resting CT6 cells caused the
hyperphosphorylation of Rb as detected by Western blotting (Fig.
2b). The presence of SB203580 in the antiproliferative
(0-30 µM) range resulted in a dose-dependent
reduction in the hyperphosphorylated form (Fig. 2b). There
also appeared to be some reduction in the total levels of Rb protein.
Similar inhibitory effects on Rb hyperphosphorylation and protein
levels were obtained with wortmannin and LY294002, both inhibitors of
PI 3-kinase. The decrease in Rb protein is likely to be due to the
IL-1-converting enzyme (ICE)-mediated proteolysis of the
hypophosphorylated form, which has been previously reported (16-18).
These results would also agree with previous studies on the role of PI
3-kinase in IL-2-induced Rb hyperphosphorylation and protein by Brennan
et al. (10) and would support previous indications that PI
3-kinase is a proximal regulator of Rb (10, 19, 20). The effects of
SB203580 on Rb hyperphosphorylation were confirmed in similar studies
on activated primary human T cells (Fig. 2d).
We also investigated a second cell cycle-regulated protein,
p27kip1. The addition of IL-2 to resting CT6 cells induces
the degradation of p27kip1(Fig. 2c). This
degradation was unaffected by SB203580, which if anything, further
reduced levels of the protein. Wortmannin and LY294002 similarly had no
inhibitory effect on p27kip1 degradation. Again, these
studies on p27kip1 degradation were repeated in activated
primary human T cells, with no significant inhibition observed with
SB203580 or wortmannin, although LY294002 had some inhibitory effect
(Fig. 2e).
SB203580 Inhibits the Phosphorylation and Activation of
PKB--
The characteristic, S-phase hyperphosphorylation of Rb
induced by IL-2 has been reported to be mediated by the PI 3-kinase pathway via the distal effector PKB (10). Furthermore, both the
mitogenic and survival functions of the PI 3-kinase pathway have, in
several reports, been attributed to PKB (10, 15, 19-22). We were
therefore interested in investigating the possibility that SB203580
mediates its effects on Rb by inhibiting these kinases, especially as
wortmannin and LY294002 displayed similar effects. The activation of
PKB requires the PI 3-kinase-generated second messenger
PIP3 as well as phosphorylation on Thr308 and
Ser473 mediated by the
PIP3-dependent kinases, PDK1 and PDK2,
respectively (23, 24). We investigated the effect of SB203580 on PKB
activation by looking at IL-2-induced phosphorylation of residue
Ser473 of PKB in whole cell lysates using a
phospho-specific antibody. In both CT6 and activated human T cells,
SB203580 inhibited the phosphorylation of Ser473 in a
dose-dependent manner (Fig.
3, a and b).
Similar studies on the IL-2-responsive BA/F3 F7 B cells gave the same
result (Fig. 3c). The approximate IC50 for the
effect of SB203580 on this parameter is ~5 µM, similar
to the concentration required for the inhibitory effects on
proliferation (Fig. 1). As expected, wortmannin (Fig. 3) and LY294002
(not shown) also inhibited Ser473 phosphorylation, whereas
rapamycin (not shown) had no effect. The phosphorylation of PKB on
Thr308 was similarly investigated. As the antibody was not
so effective, PKB was first immunoprecipitated, and
phospho-Thr308 was detected by Western blotting. SB203580
inhibited Thr308 phosphorylation in CT6 cells with similar
efficacy to the Ser473 phosphorylation (Fig.
4). Wortmannin, as expected, also
inhibited this Thr308 phosphorylation. To confirm that the
effects of SB203580 on PKB phosphorylation correlated with kinase
activity, assays were performed on immunoprecipitated PKB from
IL-2-stimulated CT6 cells (Fig. 5). The
drug inhibited PKB activation with an IC50 of 3-10
µM, in agreement with its effects on phosphorylation of
the kinase and cell proliferation.
SB203580 Inhibits the Activation of p70S6 Kinase but Not PI
3-Kinase--
Although the above results showed that PKB activation is
inhibited, it is still possible that PKB is one of several mitogenic effector molecules downstream of the actual SB203580 target. Therefore the effect of SB203580 on IL-2-induced activation of the PI
3-kinase/PKB pathway was examined. Exposure of CT6 cells to IL-2 leads
to a reproducible 2-fold increase in anti-p85-precipitable PI 3-kinase activity. This was unaffected by preincubating the cells with SB203580.
In contrast, wortmannin totally inhibited this activity (Fig.
6). Furthermore, direct addition of
SB203580 to PI 3-kinase assays did not have any effect (results not
shown), indicating that PI 3-kinase is not the target of the drug. The
effect of SB203580 on PI 3-kinase/PKB pathway was also examined
indirectly. Several studies have shown that p70S6 kinase is a distal
mediator of PI 3-kinase activity in several systems (15, 25). As
expected, wortmannin (Fig. 6b) and LY294002 (not shown)
inhibited the activation of p70S6 kinase by IL-2, as measured in
immunokinase assays. We observed that SB203580 could also inhibit
IL-2-induced p70S6 kinase activation, although the concentration
required was slightly higher with an IC50 above 10 µM. These observations place the target of SB203580
downstream of PI 3-kinase but upstream of p70S6 kinase. Furthermore, it
suggests that the SB203580 target is a common activator of both PKB and
p70S6 kinase. The most likely candidate is PDK1, which has been
reported to phosphorylate and activate p70S6 kinase. The higher
IC50 for p70S6 kinase activation may reflect the fact that
PDK1 contributes to only one of several phosphorylations required for
p70S6 kinase activation.
SB203580 Can Act as an Inhibitor of PDK1--
The data so far
suggest that PDK1 and/or PDK2, the PKB kinases, are possible targets
for SB203580. The Thr308 kinase, PDK1, is a constitutively
active enzyme (26), but the phosphorylation of PKB on
Thr308 is regulated by PIP3. The kinase for
Ser473, a putative PDK2, is so named because it is also
dependent on PIP3, but PDK2 has not been fully
characterized, and as a result, no direct assays are available to
examine its activity. Because we had evidence that PI 3-kinase activity
and, therefore, PIP3 production are not inhibited (Fig.
6a), we examined whether SB203580 could act as a PDK1
inhibitor. To do this, recombinant kinase was used with recombinant PKB
as the substrate. Activity of the enzyme was assessed by measuring the
incorporation of [32P]phosphate into PKB (Fig.
7a). SB203580 was able to
inhibit the activity of PDK1 in a dose-dependent manner
with an IC50 in the 3-10 µM range, but
CNI-1493, an inhibitor of p38MAP kinase activation (6), did not affect
PDK1 activity. The recombinant PKB used in this assay, but not the
PDK1, had endogenous autokinase activity (results not shown), so we
tested the effect of the drug on this (Fig. 7b). SB203580
was unable to inhibit PKB autokinase activity. The inhibition of PDK1
by SB203580 identifies it as the putative target in the PI 3-kinase
pathway, in agreement with its reported role in p70S6 kinase activation
(27, 28) and supported by our finding above (Fig. 6b).
p38 MAP Kinase Is Not Involved in PKB
Phosphorylation--
Although the above data demonstrate that SB203580
is an inhibitor of the PKB kinases (PDK1 and by inference PDK2), it was important to discriminate between the effects of SB203580 on PKB activation and the p38 MAP kinase pathway. The compound, CNI-1493 can
inhibit the phosphorylation and activation of p38 MAP kinase induced by
IL-2 and lipopolysaccharide (5-7). Therefore the effect of CNI-1493 on
the IL-2-induced phosphorylation of p38 MAP kinase was compared with
that of PKB. This study was performed in BA/F3 F7 cells because the
IL-2-induced phosphorylation of p38 MAP kinase was easier to detect in
these cells compared with T
cells.2 CNI-1493 inhibited
IL-2-induced phosphorylation of p38 MAP kinase in the dose range 0.5 to
5 µM while having no effect on PKB phosphorylation at
Ser473 (Fig. 8). In contrast,
wortmannin inhibited PKB phosphorylation and was consistently found to
enhance the IL-2-induced phosphorylation of p38 MAP kinase (data not
shown), an observation supported by kinase assays (not shown). These
data would discount any role for p38 MAP kinase in the regulation of
PKB either distal or proximal of PI 3-kinase. The data would also
indicate that IL-2-induced PKB and p38 MAP kinase are likely to be on
separate IL-2-mediated signaling pathways.
Since the original description of the pyridinyl imidazole SB203580
as a specific inhibitor of p38 MAP kinase, the drug has been widely
used as a research tool. The correlation drawn between the effects of
SB203580 and a role for p38 MAP kinase has been applied to many systems
with several hundred publications to date. Although the
IC50 for inhibition of p38 MAP kinase activity has been
shown to be approximately 0.3 µM (4, 29), many studies have used SB203580 at higher concentrations, often at 10-30
µM or even up to 100 µM (30, 31). Recently
two studies have shown that SB203580 could inhibit certain subtypes of
the related p54 MAP kinase (JNK/SAPK1) at concentrations of 5-10
µM (8, 9). This is an added complication in the
interpretation of results obtained with SB203580, as p54 MAP kinase is
often activated in tandem with p38 MAP kinase. However, our recently
published findings (5) indicate that the inhibitory effect of SB203580
on proliferation driven by IL-2 was not related to either p38 or p54
MAP kinase function and that the drug was likely to have some other target.
We found that SB203580 in excess of 3 µM had effects on
IL-2-induced hyperphosphorylation of Rb that were similar to those of
the PI 3-kinase inhibitors, wortmannin and LY294002. This led us to
investigate the effects of the inhibitor on the PI 3-kinase/PDK1/PKB pathway. Our data show that SB203580 inhibits the activation of PKB by
blocking the phosphorylation of the kinase on the residues Thr308 and Ser473, that are required for kinase
function. Furthermore, we have shown that SB203580 inhibits PDK1, the
Thr308 kinase, in vitro. The inhibition of
Ser473 phosphorylation by the drug is in agreement with the
recent publication by Balendran et al. (11) that PDK1 also
acts as PDK2 once bound to protein kinase C-related protein kinase-2
(PRK2). The IC50 of SB203580 for the inhibition of PKB
phosphorylation is commensurate with that for the antiproliferative
effects of the drug. The putative role of PKB in mediating
proliferation induced by IL-2 (10) could provide a mechanism for this
effect of SB203580. The compound also inhibited the phosphorylation of
PKB induced by IL-43 or the
antibody-induced cross-linking of
CD45,4 showing that the
effect of SB203580 is not confined to IL-2 signaling. However, our data
disagree with the recent study of Sweeney et al. (32), which
shows no inhibition of insulin-induced PKB activation in adipocytes by
SB203580. A recent study by Shaw et al. also reports no
inhibition of PKB activation in response to cellular stress in SW 3T3
and 293 cells (33). Although both studies used 10 µM
SB203580, a concentration at which we see up to 50% inhibition of
IL-2-induced PKB activity, they were performed on different lineages of
cells from those used in our studies, and furthermore, the
stress-activated PKB responses display much slower kinetics compared
with the mitogen-induced activation in our studies. It is possible that
the cell type or stimulus may modulate the effects of SB203580 on PKB
activation. In our studies, the drug inhibited IL-2 responses in both T
and B cells.
Following our observations that, at higher concentrations, SB203580
inhibited PKB activation, it was important to distinguish between the
effects of the drug on the p38 MAP kinase and PKB pathways. Alessi
et al. (34) show that the p38 MAP kinase substrate, MAPKAP
kinase-2, could also act as a Ser473 kinase. However, this
effect could only be shown with the purified proteins, and the study
found no evidence that this occurred in intact cells. Furthermore, a
recent study by the same group reported that insulin-like growth
factor-1 activated PKB in the absence of p38 MAP kinase activation
(33). These studies support our own finding that the compound CNI-1493,
which blocks p38 MAP kinase activation by IL-2 (5), had no effect on
PKB phosphorylation. In agreement with these observations, we have
previously shown that inhibition of IL-2-induced MAPKAP kinase-2
activation by SB203580 had an IC50 of 0.3 µM
(4), which is 10-20-fold lower that that required to inhibit PKB
phosphorylation. Also, the kinetics of p38 MAP kinase activation is not
consistent with a role in IL-2-induced PKB activation. Phosphorylation
of PKB is maximal at 5 min, whereas activation of p38 MAP kinase takes
15 min (4). Finally, the study of Alessi et al. (34) shows
that MAPKAP kinase-2 did not act as a Thr308 kinase.
Although our data discount a role for p38 MAP kinase in PKB activation,
a recent work by Wang et al. (35) reports that Fas-mediated hyperphosphorylation of Rb is p38 MAP kinase-dependent.
Because the concentrations of SB203580 used in our study totally
inhibit the p38 MAP kinase signaling, the observations of Wang et
al. could provide an explanation for the changes we see in Rb
phosphorylation. However the reduction we see in Rb phosphorylation
does not occur at the lower concentrations of SB203580, which totally
inhibit p38 MAP kinase activity in our cells (IC50 = 0.3-0.5 µM (4)). The need to use higher concentrations
of SB203580 dissociates the mitogen-induced hyperphosphorylation
of Rb from the p38 MAP kinase pathway in our system. Furthermore,
it would be interesting to see if an inhibitor of p38 MAP kinase
activation, CNI-1493 has a similar effect on Fas-induced Rb hyperphosphorylation.
The effect of SB203580 on the PI 3-kinase-mediated activation of PKB
supports the reported role for these kinases in cellular proliferation
and survival. In particular, the identification of PDK1 as a putative
target of SB203580 provides a possible mechanism for this effect.
However in the context of IL-2 signaling, the effect of SB203580
appears to be solely on cell cycle progression, as we have found no
evidence of increased apoptosis in treated cells. It would be
interesting to see if the phosphorylation of Bad, which can be mediated
by PKB (36, 37), is affected by SB203580. The inhibitory effect of
SB203580 on the activation of p70S6 kinase in vivo would
agree with recent studies that have identified this kinase as a
substrate for PDK1 (26, 28). Additionally, PKB has been reported to
play a role in p70S6 kinase activation. Although data from
membrane-targeted PKB support this putative role in p70S6 kinase
activation (38), Dufner et al. (39) find this activity only
in the membrane-targeted form of PKB and argue that it may not reflect
the normal mechanism of p70S6 kinase activation. The observations made
in our study, however, remain consistent with a model of PI 3-kinase
signaling in which PDK1 is upstream of both 70S6 kinase and PKB.
Besides the established downstream effectors of PI 3-kinase-signaling
discussed above, the pathway has also been implicated in the regulation
of cell cycle proteins (10). Our findings on Rb regulation agree with
these observations. The lack of effect of SB203580 (or wortmannin and
LY294002) on p27kip1 degradation, however, was surprising,
since previous work on Kit-225 T cells suggested a role for PKB in
IL-2-induced p27kip1 degradation (10).
These observations raise questions concerning studies that have used
the inhibitory effects of SB203580 as a confirmation of the involvement
of p38 MAP kinase in a given cellular activity. Although previous
reports (29, 40) and our own work (4) would suggest that at
concentrations of <1 µM the effects of SB203580 can
still be correlated with inhibition of p38 MAP kinase activity, we now
submit that at higher concentrations, extreme caution must be exercised
in interpreting data. Our observations do not establish a requirement
for PDK1 or PKB activity in Rb phosphorylation but suggest a role for
these kinases and their distal effectors in Rb regulation. Furthermore,
the data do not preclude a role for novel targets of SB203580 in
mediating Rb phosphorylation and T cell proliferation.
In summary, we have putatively identified PDK1/PDK2 as targets
for the pyridinyl imidazole p38 MAP kinase inhibitor SB203580, which
could, at least in part, explain the antiproliferative effect of the
drug. The observation that SB203580 can inhibit PI 3-kinase/PDK1/PKB pathway could have major implications for the interpretation of data
obtained with this drug.
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INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
c subunits. Several intracellular signaling pathways are
known to be activated by IL-2, including the p42/44 mitogen-activated
protein kinase (MAP kinase, also known as ERK2/1), the p38 and p54 MAP
kinases (also called stress kinases, or HOG and JNK, respectively), the
phosphatidyl inositol 3' (PI) 3-kinase pathway and the Jak/STAT (signal
transducer and activator of transcription) pathways. Our earlier
studies using the MEK (mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase) inhibitor PD098059 (1) and those
of others (2, 3) have indicated that the p42/44 MAP kinase pathway is
not required for IL-2-driven proliferation. In contrast, a pyridinyl
imidazole inhibitor of p38 MAP kinase, SB203580, inhibited IL-2-driven
T cell proliferation with an IC50 of 3-5 µM,
suggesting a possible role for p38 MAP kinase in this process (4).
Recently, we have further investigated the role of p38 MAP kinase in
proliferation by mapping the subdomains of the IL-2 receptor
chain
involved in the activation of the kinase. As previously shown for
p42/44 MAP kinase, activation of p38 and p54 MAP kinases required the
acidic rich A region of the IL-2 receptor
chain (5). However, the A
region is not required for proliferation (2, 5), indicating that
neither p38 MAP kinase nor p54 MAP kinase is essential for this
function. Furthermore, CNI-1493 (6, 7), an inhibitor of p38 and p54 MAP
kinase activation by IL-2 was unable to inhibit proliferation (5).
Surprisingly, SB203580 was still able to inhibit proliferation in the
absence of IL-2 stimulated p38 MAP kinase activation. It has already
been reported that SB203580 does inhibit p54 MAP kinase activity (8,
9), but the possibility that the anti-proliferative effects of SB203580
may be mediated by effects on p54 MAP kinase can also be discounted by
the studies of Hunt et al. (5).
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EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
monoclonal antibody was a generous gift from Professor D. Cantrell (Imperial Cancer Research Fund, London). Rabbit antisera to
p38 MAP kinase was from Professor J. Saklatvala (Kennedy Institute of
Rheumatology, London). Second-layer antibodies (horseradish
peroxidase-conjugated) were purchased from DAKO (DAKO A/S Denmark).
Recombinant PDK1 and recombinant PKB were kindly provided by Dr. L. Stephens (Babraham Institute, Cambridge, UK) and Dr. D. Alessi (Dundee,
UK). CN1-1493 was from Dr. K. Tracey (Picower Institute of Medical
Research New York).
-mercaptoethanol supplements. 2-5 × 106 rested
CT6 cells were resuspended in 2 ml of RPMI, 5% fetal calf serum and
preincubated with inhibitors or vehicle control as indicated in figure
legends. Cells were then stimulated with 20 ng/ml recombinant human
IL-2 for 5 min at 37 °C and pelleted in a minifuge for 30 s,
medium was aspirated, and the pellet was lysed in the appropriate buffer. BA/F3 cells stably expressing deletion mutants of IL-2 receptor
chain (a generous gift from Professor T. Taniguchi, Tokyo, Japan)
were maintained in glutamine containing RPMI further supplemented with
5% fetal calf serum and 0.2 µg/ml G418 (Calbiochem-Novabiochem) as
described previously (13). Human peripheral blood mononuclear cells
were prepared from buffy coat leukophoresis residues (North London
Blood Transfusion Service, Colindale, London UK) and activated with 50 ng/ml OKT3 for 48 h. The cells were then washed extensively, rested overnight, and washed again before activating with IL-2; such
cell preparations were >90% T cells (14). Cellular proliferation assays were performed by measurement of [3H]thymidine
incorporation as described previously (12).
was immobilized on 25 µl of protein
G-Sepharose overnight (or 1.5 h) and washed in Buffer A (50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM
EGTA, 0.5 mM Na3VO4, 0.1%
-mercaptoethanol, 1% Triton X-100, 50 mM sodium
fluoride, 5 mM sodium pyrophosphate, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, pepstatin,
leupeptin, and 1 µM microcystin). The immobilized
anti-PKB was then incubated with 0.5 ml of lysate (from 5 × 106 cells) for 1.5 h and washed three times in 0.5 ml
of Buffer A supplemented with 0.5 M NaCl, two times in 0.5 ml of Buffer B (50 mM Tris-HCl, pH 7.5, 0.03% (w/v)
Brij-35, 0.1 mM EGTA, and 0.1%
-mercaptoethanol), and
twice with 100 µl of assay dilution buffer; 5× assay dilution buffer
is 100 mM MOPS, pH 7.2, 125 mM
-glycerophosphate, 25 mM EGTA, 5 mM sodium
orthovanadate, 5 mM DTT. To the PKB enzyme immune complex
was added 10 µl of assay dilution buffer, 40 µM protein
kinase A inhibitor peptide, 100 µM PKB-specific substrate
peptide, and 10 µCi of [
-32P]ATP, all made up in
assay dilution buffer. The reaction was incubated for 20 min at room
temperature with shaking, then samples were pulse spun, and 40 µl of
reaction volume were removed into another tube to which was added 20 µl of 40% trichloroacetic acid to stop the reaction. This was mixed
and incubated for 5 min at room temperature, and 40 µl was
transferred onto P81 phosphocellulose paper and allowed to bind for
30 s. The P81 pieces were washed three times in 0.75% phosphoric
acid then in acetone at room temperature.
-32P
incorporation was then measured by scintillation counting.
(U5) and further with 25 µl of protein G-Sepharose for the final 1 h. The pellets were washed three times in 0.5 ml of PI 3-kinase assay buffer. The pellet was then resuspended in 25 µl of kinase assay buffer. To this, 10 µl of a 1 mg/ml
mixture of phosphatidylinositol and phosphatidylserine (made up in
100 mM HEPES, pH 7.5, and sonicated just before use) was
added. The mixture was then preincubated at room temperature for 10 min, and the reaction was started by the addition of 15 µl of ATP
mixture (340 µl of water, 4.2 µl of 1 M
MgCl2, 16 µl of 100 mM ATP) supplemented with
5 µCi of [
-32P]ATP. The reaction proceeded for 15 min and was stopped by the addition of 100 µl of 1 M HCl
and vortexing, adding a further 200 µl of a 1:1 chloroform:methanol
and vortexing again, and microfuge-spinning the tubes for 5 min. The
lower layer was removed and dried in vacuo (or at 60 °C
on dry block) then redissolved in 10 µl of 4:1 chloroform:methanol
before spotting onto silica plates. The plate was developed in a
preequilibrated vertical tank with chloroform, methanol, 28% ammonium
hydroxide, water (180:140:10.8:27.5) for 3 h (or overnight)
followed by phosphorimaging analysis (Fuji FLA-2000).
-32P]ATP), and the reaction was allowed to continue
for 30 min at room temperature. The reaction was stopped by the
addition of reducing sample buffer and boiling for 5 min. After
separation on a peptide gel as described before (15), radioactivity
incorporated into peptide was quantitated by phosphorimaging.
(both >98% pure) were prediluted in enzyme dilution buffer
(1 mM DTT, 0.1 M NaCl, 1 mM EGTA,
20 mM HEPES). PDK1 assays were performed with 1 µM EE-PKB and 50 nM EE-PDK1 in the presence
of appropriately diluted lipid vesicles, 0.5 µM ATP, and
1 µCi of [
-32P]ATP in assay buffer (8 mM
MgCl2, 0.12 M NaCl, 1.2 mM DTT, 1.2 mM EGTA, 0.01% azide) supplemented with protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
aprotinin, 10 µg/ml leupeptin) in a final volume of 5 µl. The
reaction was allowed to continue for 5 min at 30 °C and stopped by
boiling with 10 µl of 1.5 times SDS sample buffer (with 5 mM EDTA). The PKB autokinase assays were performed as above
for PDK1 but in the absence of PDK1 and PIP3. Samples were
then resolved on a 10% SDS-polyacrylamide electrophoresis gel and
quantitated by phosphorimaging (Fuji FLA-2000).
![]()
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Fig. 1.
Effect of SB203580 on IL-2-induced
proliferation. Effect of SB203580 on the proliferation of CT6
(diamonds), BA/F3 cell line F7 (squares), and
PBMC/T cells (circles). Rested cells were preincubated with
0-30 µM SB203580 or vehicle control for 1 h and
cultured for 24 h in the presence of 20 ng/ml IL-2. 0.5 µCi/well
of [3H]thymidine was added for the last 6 h.
Thymidine incorporation was measured by scintillation counting, and the
data points are expressed as the mean of triplicate measurements ± S.E. The data show [3H]thymidine incorporation as a
percentage of [3H]thymidine incorporated into cells
without inhibitor. The average IL-2-induced thymidine incorporation
(cpm ± S.E.) for the different cell lines were 166,977 ± 7,465 for CT6 cells, 93,219 ± 3,537 for PBMCs, and 130,571 ± 661 for the BA/F3 F7 cells. Data are representative of at least
three separate experiments.

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Fig. 2.
Effect of SB203580 on cell cycle
proteins. Resting CT6 cells (a, b,
c) and PBMC (d and e) were
preincubated with the indicated doses of SB203580, wortmannin,
LY294002, or vehicle control for 1 h before stimulating with 20 ng/ml IL-2 or vehicle control for 3 h (c-Myc) or 20 h (Rb and
p27kip). Cells were then lysed and prepared for total
cellular c-Myc (a) Rb (b and d) or
p27kip (c and e) measurement by
Western blotting as described under "Experimental Procedures." The
data are representative of three separate experiments. pRb,
hypophosphorylated; ppRb, hyperphosphorylated Rb.

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[in a new window]
Fig. 3.
Effect of SB203580 on serine 473 phosphorylation of PKB. CT6 cells (a), activated human
T cells (b), and BA/F3 F7 cells (c) were preincubated with
0-30 µM SB203580 or vehicle control for 1 h or 100 nM wortmannin for 15 min before stimulating with 20 ng/ml
IL-2 or vehicle control for 5 min. PKB phosphoserine 473 (upper
panels) was detected by Western blotting. Membranes were stripped
and reprobed for total PKB to confirm equal loading (lower
panels). The data are representative of three separate
experiments.

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[in a new window]
Fig. 4.
Effect of SB203580 on phosphorylation of
threonine-308 on PKB. Rested CT6 were preincubated with the
indicated doses of SB203580 or vehicle control for 1 h or 100 nM wortmannin for 15 min before stimulating with 20 ng/ml
IL-2 or vehicle control for 5 min. Cells were then lysed, and PKB was
immunoprecipitated (IP) with anti-PKB
antiserum or
irrelevant control antibody. Threonine 308 phosphorylation was assayed
by Western blotting (upper panel). Membranes were stripped
and reprobed for total PKB to confirm equal loading (lower
panel).

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[in a new window]
Fig. 5.
SB203580 inhibits IL-2-induced PKB
activity. Rested CT6 cells per sample were preincubated with
indicated concentrations of SB203580, wortmannin, or vehicle control
before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min.
Immunokinase assays were performed as described under "Experimental
Procedures." Panel a is representative of three separate
experiments. b, the accumulated data from the three
experiments is represented graphically showing inhibition of PKB
activity as a percentage of uninhibited, IL-2-activated controls.
Error bars equal ± S.D. (n = 3).

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Fig. 6.
Effect of SB203580 on the activation of
PI 3-kinase and p70S6 kinase. Rested cells were preincubated with
indicated concentrations of SB203580, wortmannin, or vehicle control
before stimulating with 20 ng/ml IL-2 or vehicle control for 5 min.
Representative immunokinase assays for PI 3-kinase showing fold
activation over unstimulated cells (a) and p70S6 kinase with
% inhibition of IL-2-stimulated activity labeled on top of peptide gel
panel (b) are shown. The PI 3-kinase experiment
is representative of two, and the p70S6 kinase experiment is
representative of three repeat experiments.

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Fig. 7.
SB 203580 inhibits PDK1 activity.
Recombinant PDK1 activity was assayed after preincubation with 0-30
µM SB203580 or 5 µM CNI-1493 using
recombinant PKB as substrate (a). PKB autokinase activity
was measured using recombinant PKB in the absence of PDK-1 or
PIP3 (b). Reactions were performed as described
under "Experimental Procedures." Data is expressed as the
percentage of total activity obtained with a 5-min incubation in the
absence of inhibitor. The figures are each representative of three
separate experiments.

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Fig. 8.
p38 MAP kinase is not required for PKB
phosphorylation. BA/F3 F7 cells were stimulated with 20 ng/ml IL-2
for 15 min following preincubation with 0-5 µM CNI-1493
or vehicle control. Cell extracts were then separated by
SDS-polyacrylamide gel electrophoresis and transferred onto
polyvinylidene difluoride. The membrane was cut in half and
immunoblotted for the phosphorylation of p38 MAP kinase (upper
panel) and PKB (lower panel). The upper
panel was reprobed for p38 MAP kinase (not shown) and showed equal
loading on all tracks.
![]()
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank Professors Feldmann, Saklatvala, and Cantrell and Drs. Davies, Dean, and Finch for reading the manuscript and S. Johns for typing the manuscript. We also thank Professor Taniguchi, Dr. K. Tracey, and Drs. Alessi and Stephens for their gifts of reagents.
| |
FOOTNOTES |
|---|
* 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.
Supported by grants from the BBSRC (UK), Rhone Poulenc-Rorer, The
Arthritis and Rheumatism Campaign (UK), and the Wellcome Trust.
§ To whom all correspondence should be addressed: Kennedy Instititute of Rheumatology, 1 Aspenlea Rd., London W6 8LH, UK. Tel.: 44-208 383 4444; E-mail: b.foxwell@cxwms.ac.uk.
2 F. V. Lali, A. E. Hunt, S. J. Turner, and B. M. J. Foxwell, unpublished observations.
3 A. Hunt and B. M. J. Foxwell, unpublished observations.
4 L. Hayes, unpublished observations.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: IL, interleukin; MAP, mitogen-activated protein; PKB, protein kinase B; PDK, 3-phosphoinositide-dependent protein kinase; Rb, retinoblastoma; PI, phosphatidylinositol; JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; MOPS, 4-morpholinepropanesulfonic acid; DTT, dithiothreitol; PIP3, phosphatidylinositol 3,4,5-trisphosphate.
| |
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