(Received for publication, November 14, 1994; and in revised form, December 27, 1994)
From the
Arachidonic acid (20:4(n-6)), which is released by
cells responding to a wide range of stimuli, may play an important role
in intracellular signaling. We now report that incubation of WB cells
with 20:4(n-6) resulted in the appearance of several
tyrosine-phosphorylated cytosolic proteins. Two of the
phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide
gels of approximately 43 and 45 kDa, corresponded in mobility to
phosphorylated species of the 42- and 44-kDa mitogen-activated protein
kinase (MAPK) isoforms. Immunoblots of soluble fractions from
unstimulated WB cells with anti-MAPK antibodies revealed the presence
of the 42- and 44-kDa isoforms of MAPK. Upon incubation with
20:4(n-6), the mobility of both isoforms was retarded,
consistent with their activation by phosphorylation. Chromatography of
soluble fractions from these cells on Mono Q columns revealed early and
late eluting peaks of myelin basic protein kinase activity, which
contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of
MAPK was transient, peaking at 5 min, and was detectable at 5
µM 20:4(n-6). Further studies into the mechanisms
by which MAPK was activated by 20:4(n-6) strongly suggested
the involvement of protein kinase C (PKC). Not only did incubation of
WB cells with 20:4(n-6) result in the translocation of PKC
,
, and
to a particulate fraction, it was found that
the fatty acid failed to activate MAPK in cells pretreated for 26 h
with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC
,
, and
. In addition, fatty acids of the n-3
series were effective activators of MAPK. The present study, to our
knowledge, is the first to report that polyunsaturated fatty acids can
cause the activation of MAPK.
Exogenous polyunsaturated fatty acids (PUFA) ()such
as arachidonic acid (20:4(n-6)) exert a wide range of effects
on cells of diverse origin. These include the regulation of gap
junctional permeability between adherent cells(1) , neutrophil
secretory and NADPH oxidase activities(2, 3) ,
neutrophil migration(4) , insulin secretion (5) ,
expression of cell-surface receptors and adhesion
molecules(3) , gene transcription(6) , and cytotoxic T
cell function(7) . Although a number of studies have reported
that exogenous 20:4(n-6) can modulate the activities of
components of intracellular signaling (8, 9, 10, 11, 12, 13, 14) ,
the mechanisms by which the fatty acid acts still need further
clarification. In addition, 20:4(n-6) is liberated in response
to a diverse range of stimuli through the activation of phospholipase
A
, and 20:4(n-6) thus released and its eicosanoid
metabolites have been proposed to participate in cell
signaling(14, 15) .
The mitogen-activated protein
kinase (MAPK) is a family of kinases which has been reported to play an
important role in intracellular signaling(16) . Members of this
family of kinases, originally found to be activated by mitogens, have
now been found to be activated by a wide variety of mitogenic and
non-mitogenic agents via a cascade of kinases/effector molecules which,
in mammalian cells, include protein kinase C (PKC) and/or
p21, Raf-1, and MEK (MAPK/extracellular
signal-regulated protein kinase (ERK) kinase)(16) . In order to
determine whether some of the actions of 20:4(n-6) could be
mediated by the MAPK cascade, we investigated whether
20:4(n-6) and PUFA of the n-3 series could alter the
activity of MAPK. We report here the novel finding that the n-3 and n-6 series of PUFA stimulated the activity of
MAPK in WB cells.
Incubation of WB cells with 20:4(n-6) (20 µM) for 5 min resulted in tyrosine phosphorylation of a number of cytosolic proteins, including those of 43 and 45 kDa (Fig. 1). Several proteins, notably those of approximately 76, 108, and 138 kDa, reacted strongly with the anti-phosphotyrosine antibody, while others, including those of 43 and 45 kDa, reacted less strongly. The pattern of tyrosine-phosphorylated proteins detected in cells incubated with LPA, EGF, or PMA (Fig. 1) showed no major observable quantitative differences between the four compounds. We have previously reported the presence and activation of the 42- and 44-kDa isoforms of MAPK by LPA, EGF, and PMA in WB cells (18) , raising the possibility that the 43- and 45-kDa proteins shown in Fig. 1could be tyrosine-phosphorylated MAPK isoforms. Members of the MAPK family are phosphorylated on tyrosine and serine/threonine by MEK when activated(16) . To determine whether incubation of WB cells with 20:4(n-6) led to the phosphorylation of the 42- and 44-kDa MAPK isoforms cytosolic fractions obtained from cells incubated with 20:4(n-6) (20 µM) for 5 min were immunoblotted with anti-MAPK antibody R1, which we had used previously to detect phosphorylated MAPK isoforms in WB cells(18) . Incubation of WB cells in the presence of 20:4(n-6) resulted in the appearance of immunoreactive material, which migrated between the 42- and 44-kDa bands, and an additional band of approximately 45 kDa (Fig. 2, lanes 2 and 4). When the same sample was probed with anti-MAPK antibody R2, which detected the 44-kDa MAPK more strongly than the 42-kDa MAPK, the 45-kDa material was revealed more clearly (Fig. 2, lane4). The data thus suggest that the 43- and 45-kDa tyrosine-phosphorylated proteins are likely to be phosphorylated forms of MAPK. To determine whether these fractions contained activated MAPK samples were chromatographed on Mono Q columns. Fig. 3(panelA) shows that cytosolic fractions from WB cells incubated for 5 min with 20:4(n-6) could be resolved into two peaks of MBP kinase activity which corresponded to the peaks induced when cells were incubated with PMA. Immunoblots of the peak fractions revealed that the early and late eluting peaks contained predominately the presence of the 42- and 44-kDa MAPK isoforms (Fig. 3, panelB).
Figure 1: Induction of tyrosine phosphorylation of cytosolic proteins by 20:4(n-6). Cells were incubated with vehicles (lane2), LPA (20 µM, lane3), EGF (10 ng/ml, lane4), PMA (100 nM, lane5), or 20:4(n-6) (20 µM, lane6) for 5 min. Cytosolic fractions were prepared and immunoblotted with 4G10 as described under ``Experimental Procedures.'' Lane1, molecular mass markers. Similar data were obtained in a repeat experiment.
Figure 2: Detection of MAPK isoforms. Cytosolic fractions were prepared as described in legends to Fig. 1, and samples were immunoblotted with anti-MAPK antibodies R1 (lanes1 and 2) or R2 (lanes3 and 4). Lanes1 and 3, vehicle; lanes2 and 4, 20:4(n-6). The 42-kDa MAPK isoform was also detectable in lanes3 and 4 upon longer exposure (not shown). Results are representative of three experiments.
Figure 3:
Chromatography of MAPK isoforms on Mono Q
columns. A, WB cells were incubated with vehicle (),
20:4(n-6) (20 µM) (
) or PMA (100
nM) (
) for 5 min, and cytosolic fractions were
prepared and chromatographed on Mono Q columns as described under
``Experimental Procedures.'' Fractions were assayed for MBP
kinase activity. B, peak fractions from
20:4(n-6)-stimulated samples were immunoblotted with anti-MAPK
antibody R1. Lane1, early eluting peak; lane2, late eluting peak.
To characterize further the activation of MAPK by 20:4(n-6) cytosolic fractions from fatty acid-treated cells were partially purified by adsorption onto phenyl-Sepharose CL4B. These studies showed that the enhancement of kinase activity was transient, peaking at 5 min following the addition of 20:4(n-6) (Fig. 4, panelA). Activation of MAPK was also dependent on the concentration of 20:4(n-6) with a detectable increase in kinase activity observed at a fatty acid concentration of 5 µM (Fig. 4, panelB).
Figure 4:
Activation of MAPK by 20:4(n-6).
Cells were incubated for 0-60 min (A) with
20:4(n-6) (20 µM) or for 5 min (B) with
0-20 µM 20:4(n-6) and kinase activity in
partially purified samples was assayed as described under
``Experimental Procedures.'' (), control; (
),
20:4(n-6). Similar data were obtained in two other repeat
experiments.
Since
PUFA have been shown to activate PKC (12, 19) and that
PKC is one of the upstream regulators of the MAPK cascade(16) ,
we investigated whether PKC was involved in the activation of MAPK by
20:4(n-6) in WB cells. These cells contain PKC ,
,
, and
(Fig. 5, panelsA-C) but not
or
(data not
shown). Visualization of the isozymes was prevented by preincubation of
the antibodies with isozyme-specific peptide antigens (data not shown).
Both 20:4(n-6) and PMA stimulated the translocation of PKC
,
, and
to a particulate fraction (Fig. 5, panels B and C). Densitometric scanning revealed that
20:4(n-6) increased membrane-associated PKC
,
, and
by (mean ± S.E., n = 3) 2.9 ± 0.7 (p < 0.05), 3.1 ± 1.0 (p = 0.054),
and 1.8 ± 0.3 (p < 0.05) fold, respectively, over
control without affecting PKC
(1.14 ± 0.1) (Fig. 5, panelC). Pretreatment of WB cells with PMA (300
nM) for 26 h led to a loss of the majority of
,
,
and
immunoreactivity but not
(Fig. 5, panelA), consistent with PKC
being a PMA-unresponsive
isozyme(12) . This treatment was associated with a loss of
activation of MAPK by both 20:4(n-6) and PMA (Table 1).
Recent studies have implicated PKC
and
as upstream
regulators of the MAPK cascade (20) . (
)Activation
of MAPK by EGF, acting via p21
(21) , was only
slightly reduced in these PKC-depleted cells (Table 1). These
data suggest that activation of MAPK by 20:4(n-6) involved the
activation of PMA-responsive PKC isozymes, and that 20:4(n-6)
and EGF acted through different pathways.
Figure 5:
Depletion and translocation of PKC
isozymes. WB cells were preincubated with PMA (300 nM) or
MeSO (0.01%, v/v) for 26 h to deplete PKC (A),
incubated with PMA (100 nM) for 5 min (B) or with
20:4(n-6) (20 µM) for 2 min (C). Total
PKC (A) or PKC in particulate fractions (B and C) were immunoblotted with isozyme-specific antibodies to PKC
as described under ``Experimental Procedures.'' Results are
representative of three experiments for panelsB and C.
Activation of MAPK by PUFA
was not restricted to the n-6 series of PUFA. Both
20:5(n-3) and 22:6(n-3), found in abundance in fish
oils and in the brain and retina(22) , were also capable of
enhancing the activity of MAPK as determined by the ability of
phenyl-Sepharose CL4B-purified kinase to phosphorylate MBP. Kinase
activities (fmol of P/µg of protein/20 min) after a
5-min incubation with the PUFA (20 µM) were: ethanol, 478;
20:4(n-6), 1134; 20:5(n-3), 1010; 22:6(n-3),
1010. Certain effects of 20:4(n-6) have been proposed to be
dependent on the oxidation of 20:4(n-6) to eicosanoid products
by lipoxygenase(15) . Although both the n-3 PUFA and
20:4(n-6) were capable of enhancing the activity of MAPK,
these PUFA give rise to different eicosanoid products with different
biological actions (see (23) , and references therein). This,
therefore, makes it unlikely that the activation of MAPK by PUFA was
dependent on the formation of eicosanoids.
Both PKC and p21 have been demonstrated to regulate the MAPK cascade in mammalian
cells(16) . While p21
has recently been reported
to activate the MAPK kinase kinase activity of Raf-1 through the
mediation of 14-3-3(24, 25) , it is not clear
whether PKC activates the MAPK cascade via Raf-1, MEK kinase, the
mammalian homologue of yeast STE11(26) , or both. A recent
study has demonstrated phosphorylation of Raf-1 by PKC
on Ser-499
and possibly Ser-259, resulting in the activation of Raf-1 autokinase
and kinase activity toward a peptide substrate(20) . However,
the MAPK kinase kinase activity of Raf-1 was not determined. Another
study failed to detect activation of Raf-1 kinase activity despite
phosphorylation of the kinase by PKC(27) . A Raf-1-independent
pathway has also been identified in a BALB/c-derived cell line
stimulated by phorbol esters (28) . PKC may also activate Raf-1
in a p21
-dependent manner in certain cell
types(29) . Despite these uncertainties regarding the site of
PKC action, the observations that intact PKC was required for the
activation of MAPK by 20:4(n-6), and that the fatty acid
stimulated the translocation of PKC
,
, and
to the
particulate fraction, are consistent with the suggestion that
activation of MAPK by 20:4(n-6) proceeded via PKC. However, we
stress that not all cellular responses to 20:4(n-6) are
blocked by PMA pretreatment since PUFA-induced inhibition of gap
junctional permeability in WB cells was unaffected by PMA pretreatment, (
)suggesting involvement of either PKC
or
PKC-independent mechanisms.
Activation of MAPK by PUFA appears to be a general response in cells exposed to the fatty acids, since we have also observed activation of MAPK by 20:4(n-6) in other cells including human neutrophils, human macrophages, human mesangial cells, human umbilical vein endothelial cells, Monomac 6 cells, and Jurkat cells (data not shown). This raises the possibility that some of the actions of PUFA could be mediated via the MAPK cascade.
Many studies
have reported that stimulation with a diverse range of agonists
activates phospholipase A, resulting in release of
20:4(n-6)(14, 15, 32) . In
stimulated pancreatic B cells, the concentration of unesterified
20:4(n-6) has been reported to be in excess of 50
µM(30) . Furthermore, unesterified fatty acids are
known to be released in pathological conditions such as cardiac
ischemia(31) . This study therefore raises the possibility that
liberated PUFA may be capable of stimulating the activity of MAPK.
Since many of the agonists that activate the MAPK cascade also activate
phospholipase A
(32) , possibly by direct
phosphorylation of phospholipase A
by MAPK(33) , we
speculate that unesterified fatty acids released during agonist
stimulation have the potential to modify or prolong the activation of
MAPK in response to primary stimulants.
This study, to our knowledge, is the first to report the finding that 20:4(n-6) and PUFA of the n-3 series were capable of activating MAPK. We also demonstrated that 20:4(n-6) induced the appearance of several tyrosine-phosphorylated cytosolic proteins. The mechanisms by which 20:4(n-6) induced the tyrosine phosphorylation of these proteins are unknown. Activation of MAPK by 20:4(n-6) was dependent on PKC.