(Received for publication, May 22, 1995; and in revised form, August 9, 1995)
From the
We detected expression of two Raf isoforms, c-Raf and A-Raf, in neonatal rat heart. Both isoforms phosphorylated, activated, and formed complexes with mitogen-activated protein kinase kinase 1 in vitro. However, these isoforms were differentially activated by hypertrophic stimuli such as peptide growth factors, endothelin-1 (ET1), or 12-O-tetradecanoylphorbol-13-acetate (TPA) that activate the mitogen-activated protein kinase cascade. Exposure of cultured ventricular myocytes to acidic fibroblast growth factor activated c-Raf but not A-Raf. In contrast, TPA produced a sustained activation of A-Raf and only transiently activated c-Raf. ET1 transiently activated both isoforms. TPA and ET1 were the most potent activators of c-Raf and A-Raf. Both utilized protein kinase C-dependent pathways, but stimulation by ET1 was also partially sensitive to pertussis toxin pretreatment. c-Raf was inhibited by activation of cAMP-dependent protein kinase although A-Raf was less affected. Fetal calf serum, phenylephrine, and carbachol were less potent activators of c-Raf and A-Raf. These results demonstrate that A-Raf and c-Raf are differentially regulated and that A-Raf may be an important mediator of mitogen-activated protein kinase cascade activation when cAMP is elevated.
The exposure of cells to mitogenic agents that act through
protein tyrosine kinase receptors activates a cascade of protein
kinases including c-Raf, mitogen-activated protein kinase kinase (MEK), ()and mitogen-activated protein kinases
(MAPKs)(1, 2) . This protein kinase cascade provides a
link between growth factor stimulation and the transcriptional changes
that occur in the cell nucleus(3, 4) . More recently,
the activation of receptors coupled to heterotrimeric G-proteins has
also been shown to activate MEK, p42
, and
p44
(5, 6) . The upstream events in this
signaling cascade may also involve c-Raf(7) . Thus, activation
of c-Raf may integrate signals from tyrosine kinase receptors and
G-protein-coupled receptors.
c-Raf was originally identified as the cellular homolog of an oncogene of a murine sarcoma virus, but the regulation of c-Raf activity and its role in normal cellular functions have, until recently, remained unclear(8) . c-Raf has been shown to interact directly with Ras.GTP (9, 10, 11, 12) . This interaction translocates c-Raf to the membrane(13, 14) and, coupled with other events that may include phosphorylation, activates c-Raf(15, 16, 17) . Activated c-Raf directly phosphorylates MEK1 on two serine residues within a conserved regulatory region between the ``DFG'' and ``A(/S)PE'' motifs(18) . This identification of MEK1 as a physiological substrate allows assay of Raf activation(14, 19) .
c-Raf may not be the only MEK activator in the eukaryotic cell. Recent studies have shown that c-Raf is not the major upstream activator of MAPKs in rat fibroblasts(20, 21) , adipocytes(22) , and PC12 cells(23, 24) . c-Raf is a member of a family of related protein kinases that includes A-Raf and B-Raf(25, 26, 27) . In contrast to the ubiquitous expression of c-Raf, the expression of A-Raf and B-Raf is restricted (28) . Although the expression of truncated, constitutively active forms of A-Raf and B-Raf leads to activation of MAPK(29) , the regulation of these isoforms has not been well-characterized. It is possible that different Raf isoforms may regulate different cellular events. Thus, B-Raf has been proposed as a major activator of MEK in nerve growth factor-stimulated PC12 cells (24) and brain extracts(30, 31) .
The
exposure of ventricular myocytes to growth promoting stimuli such as
tumor-promoting phorbol esters, endothelin-1 (ET1),
-adrenergic agonists, peptide growth factors, or
mechanical stretch activates a series of genetic changes that leads to
cell hypertrophy rather than cell division(32, 33) .
Many of these agonists have also been shown to activate MEK and MAPK in
these cells (34, 35, 36, 37) .
Recent studies suggest that the MAPK cascade may play a role in the
transcriptional changes associated with hypertrophy(38) . In
this study, we have examined the expression and activation of Raf
isoforms in cultured neonatal rat ventricular myocytes. We show that
ventricular myocytes express c-Raf and A-Raf and that hypertrophic
stimuli differentially activate these two Raf isoforms.
The detergent-soluble supernatants (approximately 1.5-2.0 mg protein/ml) were retained. When activation of A-Raf was directly compared to activation of c-Raf, each supernatant was divided equally. Antibodies that recognize c-Raf or A-Raf (3 µl) were added to each aliquot (90 µl) and these incubated with mixing at 4 °C for 2 h. Protein A-Sepharose was added, and the incubation was continued for 1 h. The immunoprecipitates were washed three times with Buffer A and twice with Buffer B.
Figure 1: Neonatal rat heart ventricles express c-Raf and A-Raf, but not B-Raf. Proteins were separated by SDS-PAGE and transferred to nitrocellulose as described under ``Experimental Procedures.'' The nitrocellulose membranes were probed with antibodies to c-Raf (panels A and D) or A-Raf (panels B and E) or B-Raf (panel C), and the blots were developed using the ECL method. A and B, detergent-soluble extracts of neonatal rat ventricles (approximately 10 mg protein/ml) were incubated with antibodies to c-Raf (lanes 1 and 2) or A-Raf (lanes 3 and 4) at a final concentration of 5 µg/ml as described under ``Experimental Procedures.'' In lanes 2 and 4, the appropriate competing peptide immunogen (c-Raf in lanes 2 and A-Raf in lanes 4) was added at a final concentration of 2 µg/ml. C, detergent-soluble extracts (approximately 10 mg protein/ml) of adult rat brain (lanes 1 and 2) or neonatal rat ventricles (lanes 3 and 4) were incubated with antibodies to B-Raf at a final concentration of 5 µg/ml. In lanes 2 and 4, the B-Raf competing peptide immunogen was added at a final concentration of 2 µg/ml. D and E, detergent-soluble extracts of neonatal rat ventricles (approximately 10 mg protein/ml) were incubated with antibodies to c-Raf (panel D, lanes 1 and 2) or A-Raf (panel E, lanes 1 and 2). The proteins associated with the antibodies are shown in lanes 1 and 2 or those remaining in the supernatant are shown in lanes 1` and 2`. In lanes 2 and 2`, the appropriate competing peptide immunogen was added at a final concentration of 2 µg/ml. The molecular masses (kilodaltons) of standard proteins are shown to the left of each panel. The specific bands for c-Raf, A-Raf, and B-Raf are indicated by the arrows to the right of each panel. Experiments were carried out on three separate occasions with similar results.
The specificity of the immunoreactions was assessed by a number of procedures. First, inclusion of the appropriate immunogenic peptide during the immunoprecipitation procedure competed with Raf binding to the antibody (Fig. 1A, lane 2; Fig. 1B, lane 4). Second, incubation of blots with antibody and the appropriate immunogenic peptide prevented the binding of the antibody to the cognate Raf protein (results not shown). Third, there was no cross-reaction of the c-Raf immunoblotting antibody with the protein immunoprecipitated by the A-Raf antibody (Fig. 1A, lane 3) nor was there cross-reaction of the A-Raf immunoblotting antibody with the protein immunoprecipitated by the c-Raf antibody (Fig. 1B, lane 1). Finally, immunoprecipitation cleared >90% of c-Raf (Fig. 1D) or A-Raf (Fig. 1E) from the extracts.
Figure 2: c-Raf and A-Raf from neonatal rat heart ventricles interact with MEK1. Detergent-soluble extracts of neonatal rat ventricles (approximately 10 mg protein/ml) were incubated with 0.3 mg (5 nmol) of GST-MEK1(R97/A291/A385) (lanes 1), 0.1 mg (5 nmol) of GST (lanes 3), or buffer in the place of these proteins (lanes 4) as described under ``Experimental Procedures.'' In addition, 0.3 mg of GST-MEK1(R97/A291/A385) was incubated with buffer alone (lanes 2). The proteins associated with subsequently added glutathione-Sepharose (lanes 1-4) or remaining in the supernatant (lanes 1`-4`) were separated by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose membranes were probed with antibodies to c-Raf (panel A) or A-Raf (panel B), and the blots were developed using the ECL method. The molecular masses (kilodaltons) of standard proteins are shown to the left of each panel. The specific bands for c-Raf and A-Raf are indicated by arrows to the right of each panel. Experiments were carried out on three separate occasions with similar results.
Figure 3:
ET1 activates c-Raf and A-Raf. Cultured
ventricular myocytes were treated with 100 nM ET1 (ET) at 37 °C for the times indicated, then A-Raf and
c-Raf were immunoprecipitated from the detergent-soluble cell lysates
as described under ``Experimental Procedures.'' A,
activity of the immunoprecipitated c-Raf () or A-Raf (
) was
measured in a coupled assay as MEK activating activity. B, the
immunoprecipitated c-Raf was immunoblotted (upper panel), and
the kinase activity of c-Raf was measured directly by the
phosphorylation of GST-MEK1(R97/A291/A385) (lower panel).
Blank reactions in which c-Raf antibody was incubated with Buffer A and
Protein A-Sepharose, then used in the subsequent assays, were always
performed (lane B). The positions of c-Raf and the mutant
GST-MEK1 are indicated by the arrows to the right of
each panel. C, the immunoprecipitated A-Raf was immunoblotted (upper panel) and the activity of A-Raf was measured directly
by the phosphorylation of GST-MEK1 (R97/A291/A385) (lower
panel). Blank reactions in which A-Raf antibody was incubated with
Buffer A and Protein A-Sepharose, then used in the subsequent assays,
were always performed (lane B). The positions of A-Raf and the
mutant GST-MEK1 are indicated by the arrows to the right of each panel. Experiments on c-Raf activation were carried out on
five separate occasions, and these results were compared with A-Raf
activation carried out on three of those five
occasions.
We compared the time courses for activation of c-Raf and A-Raf in ET1-stimulated cells. ET1 activated A-Raf measured using the coupled assay (Fig. 3A, open symbols). A-Raf activities were maximal at 3-5 min but were lower than those for c-Raf (49 ± 4% of maximal c-Raf activities, n = 3). A-Raf activity decreased to control levels within 30 min of exposure (21 ± 8% of peak A-Raf activities, n = 3). We confirmed that the amounts of A-Raf immunoprecipitated from these cells did not vary (Fig. 3C, upper panel). The assay of A-Raf by its phosphorylation of GST-MEK1(R97/A291/A385) confirmed that MEK1 is phosphorylated by immunoprecipitated A-Raf (Fig. 3C, lower panel).
Figure 4:
TPA differentially activates c-Raf and
A-Raf. Cultured ventricular myocytes were treated with 1 µM TPA at 37 °C for the times indicated then A-Raf and c-Raf
immunoprecipitated from the detergent-soluble cell lysates as described
under ``Experimental Procedures.'' A, activity of
the immunoprecipitated c-Raf () or A-Raf (
) was measured in
a coupled assay as MEK activating activity. The kinase activity of
c-Raf (panel B) or A-Raf (panel C) was directly
measured by the phosphorylation of GST-MEK1(R97/A291/A385). Blank
reactions in which c-Raf antibody (panel B) or A-Raf antibody (panel C) was incubated with Buffer A and Protein A-Sepharose,
then used in the subsequent assays, were always performed (lane
B). The position of the mutant GST-MEK1 is indicated by the arrow to the right of each panel. Experiments on
c-Raf activation were carried out on five separate occasions, and these
results were compared with A-Raf activation carried out on three of
those five occasions.
A-Raf was also activated as shown by the coupled assay (Fig. 4A, open symbols) or by the direct phosphorylation assay (Fig. 4C). A-Raf was activated to a lesser extent than c-Raf (75 ± 8% of c-Raf activity at 3 min, n = 4) and was slower than c-Raf activation (Fig. 4A, open symbols). Activation of A-Raf was more sustained (69 ± 3% of maximal response at 30 min exposure, n = 4) than the activation of c-Raf so that the activity of A-Raf exceeded that of c-Raf after a 30-min exposure (178 ± 48% of c-Raf activity at 30 min, n = 4).
Figure 5:
aFGF activates c-Raf but not A-Raf.
Cultured ventricular myocytes were treated with 25 ng/ml aFGF at 37
°C for the times indicated. A-Raf and c-Raf were then
immunoprecipitated from the detergent-soluble cell lysates as described
under ``Experimental Procedures.'' A, activity of
the immunoprecipitated c-Raf () or A-Raf (
) was measured in
a coupled assay as MEK activating activity. B, the kinase
activity of c-Raf was measured directly by the phosphorylation of
GST-MEK1(R97/A291/A385). Blank reactions in which c-Raf antibody was
incubated with Buffer A and Protein A-Sepharose, then used in the
subsequent assays, were always performed (lane B). The
position of the mutant GST-MEK1 is indicated by the arrow at
the right of the panel. Experiments on c-Raf activation were
carried out on four separate occasions, and these results were compared
with A-Raf activation carried out on two of those four
occasions.
Figure 6: TPA and ET1 were the most potent activators of c-Raf and A-Raf identified in ventricular myocytes. Cultured ventricular myocytes were treated with 1 µM TPA, 100 nM ET1, 50 µM phenylephrine (PE), 20% (v/v) FCS, 25 ng/ml aFGF (FGF), or 100 µM carbachol (CB) at 37 °C for 3 min. Activity of c-Raf (solid bars) or A-Raf (open bars) immunoprecipitated from the detergent-soluble cell lysates was measured in a coupled assay as MEK activating activity as described under ``Experimental Procedures.'' Results (means ± S.E.) are expressed as a percentage of the TPA response measured on each separate preparation of myocytes and are an average of 3-15 independent observations.
Figure 7: Classical and novel PKC isoforms are involved in the activation of c-Raf and A-Raf. Cultured ventricular myocytes were either incubated in serum-free medium (solid or open bars) or pretreated with 1 µM TPA for 24 h (crosshatched or diagonally striped bars), then exposed to serum-free medium (CON), 100 nM ET1, 1 µM TPA, or 20% (v/v) FCS at 37 °C for 3 min. Activity of c-Raf (panel A) or A-Raf (panel B) immunoprecipitated from the detergent-soluble cell lysates was measured in a coupled assay as MEK activating activity as described under ``Experimental Procedures.'' The experiment was performed on three separate occasions with similar results.
In contrast to c-Raf, basal A-Raf activity was not enhanced by TPA pretreatment (Fig. 7B). ET1, TPA, and FCS activation of A-Raf were all decreased by TPA pretreatment, although FCS activation was least sensitive (Fig. 7B). Thus, classical/novel PKCs are essential for TPA and ET1 signaling to A-Raf. FCS may also utilize PKC-independent pathways.
Figure 8: Pertussis toxin inhibits activation of c-Raf and A-Raf by ET1 and FCS. Cultured ventricular myocytes were either incubated in serum-free medium (solid or open bars) or pretreated with 150 ng/ml pertussis toxin for 24 h (crosshatched or diagonally striped bars), then exposed to serum-free medium (CON), 100 nM ET1, 1 µM TPA, or 20% (v/v) FCS at 37 °C for 3 min. Activity of c-Raf (panel A) or A-Raf (panel B) immunoprecipitated from the detergent-soluble cell lysates was measured in a coupled assay as MEK activating activity as described under ``Experimental Procedures.'' The experiment was performed on three separate occasions with similar results.
Figure 9: Elevated PKA activity preferentially inhibits activation of c-Raf by ET1. Cultured ventricular myocytes were treated with serum-free medium (CON), 100 µM CPT-cAMP, 100 nM ET1 (ET) or with 100 µM CPT-cAMP in the presence of 100 nM ET1 (CPT-cAMP + ET) at 37 °C for 3 min. Activity of c-Raf (solid bars) or A-Raf (open bars) immunoprecipitated from the detergent-soluble cell lysates was measured in a coupled assay as MEK activating activity as described under ``Experimental Procedures.'' The experiment was performed on four separate occasions with similar results.
Two protein kinase activators of MEK have been identified in somatic cells, namely Raf and MEKK(1, 58) . Although both Raf and MEKK may act downstream of Ras(59, 60) , recent evidence indicates that MEKK participates in the activation of stress-activated protein kinase pathways(61, 62, 63) . Either Raf or MEKK may be important in the regulation of the Ras-dependent hypertrophic responses previously observed(55) , but recent studies have demonstrated changes in gene expression typical of the hypertrophic response after transfection with activated forms of c-Raf(64) .
We have
observed that exposure of the ventricular myocyte to aFGF activated
c-Raf, but not A-Raf. The reason for this differential activation of
Raf isoforms by aFGF is unclear and requires further investigation. The
functional effectors of Raf in addition to Ras and Src remain to be
identified(15, 16, 17) . Although lipids may
interact with the Cys-rich Zn finger of the Raf
molecule, diacylglycerol and phosphatidylserine do not appear to
activate c-Raf in vitro(67) . Recently, c-Raf activity
has shown to be further enhanced by a lipid factor(68) . It may
be that the differential activation of A-Raf and c-Raf reflects
different requirements for effectors (either species or
concentrations). The interaction of c-Raf with other proteins has been
suggested(14) . Thus, 14-3-3 proteins interact with
c-Raf(69) , and this prevents the deactivation of c-Raf by
phosphatases(70) .
The activation of c-Raf by ET1 in
ventricular myocytes was sensitive to inhibition by PKA. This is
consistent with previous studies which demonstrated the inhibition of
the MAPK pathway by
PKA(71, 72, 73, 74, 75) .
Ser of c-Raf is directly phosphorylated by PKA, and this
decreases the affinity of interaction between Ras and c-Raf (71) . This provides negative regulation of c-Raf activity and
cross-talk between signal transduction cascades(54) . The
N-terminal regions of both B-Raf and A-Raf do not have a site analogous
to this consensus PKA phosphorylation site(25, 26) .
However, B-Raf is also inhibited by PKA activation(23) , and
other sites of phosphorylation (such as in the kinase domain) may also
contribute to the PKA-mediated inhibition of c-Raf (76) and
possibly B-Raf. We found that A-Raf was less sensitive than c-Raf to
inhibition by PKA. The activation of MAPK by ET1 is also relatively
insensitive to inhibition by PKA in ventricular myocytes,
and A-Raf potentially provides a signaling pathway for MAPK
activation that is not inhibited when cAMP concentrations are elevated.
TPA was the
most potent activator of both c-Raf and A-Raf in cultured ventricular
myocytes. The activation of c-Raf was rapid and transient whereas
activation of A-Raf was slower and sustained. Because the classical and
novel isoforms of PKC are major targets of phorbol ester action in the
eukaryotic cell(83) , a role for PKC in the activation of both
Raf isoforms is implied. In ventricular myocytes, activation of
PKC-, PKC-
, and PKC-
by TPA is rapid (<30 s for
maximal response(52, 84) ) and precedes activation of
A-Raf or c-Raf. A number of studies have demonstrated activation of
c-Raf by PKC(85, 86, 87) , and PKC-
directly activates c-Raf by phosphorylation of Ser499(87) which lies within a regulatory region between the
``DFG'' and ``A(/S)PE'' motifs(88) . A-Raf
does not have a residue analogous to Ser
, and its direct
regulation by PKC has not been examined.
Both c-Raf and A-Raf have been identified to interact specifically with HRas after screening a randomly primed mouse embryo cDNA library in the yeast-two hybrid system(10) . An alternative route of Raf activation may involve a PKC-dependent activation of Ras (e.g. activation of Ras guanine nucleotide exchange factors to promote formation of Ras.GTP)(89) . Other sites for PKC-independent actions of phorbol esters cannot be ruled out(90, 91) . Differences in the mechanism of activation of c-Raf and A-Raf by phorbol esters may help to explain the differences in their time courses of activation.
Exposure of ventricular myocytes to ET1, a
potent hypertrophic agonist (80, 81) , leads to rapid
(within 30 s) stimulation of hydrolysis of membrane
phosphatidylinositols(37) , equally rapid
activation of PKC-
and PKC-
(92) , and activation of
MEK and MAPK (34, 37, 92) . ET1 activated
c-Raf and A-Raf to 70-90% of the levels achieved by TPA. Both Raf
isoforms followed similar rapid and transient time courses of
activation in response to ET1. Depletion of novel and classical PKC
isoforms which inhibited the ET1-stimulated MAPK activation (37) also inhibited c-Raf and A-Raf activation. These findings
are consistent with a hypothesis that activation of PKC leads to
activation of both Raf isoforms. Therefore, there must be a major
PKC-dependent pathway from the ET1 receptor to Raf activation in these
cells. In addition, ET1 elicits tyrosine phosphorylation of Shc in
astrocytes (93) which leads to its association with GRB2 (93) and provides a Ras-dependent mechanism for activation of
Raf by endothelin.
Cross-talk between G- and
G
-coupled pathways is implied from the findings that
pretreatment with either TPA or pertussis toxin down-regulates
activation of A-Raf. Thus, activation of PKC downstream from both the
stimulation of phosphatidylinositol hydrolysis and G
is necessary for activation of A-Raf by ET1. Alternatively,
activation of phosphatidylinositol hydrolysis by ET1 may be stimulated
through G
as well as G
. The anomalous
activation of c-Raf by TPA pretreatment prevents similar attempts at
interpretation.
A potent Raf-independent pathway of MAPK activation is suggested by our studies with phenylephrine which activates MAPK and MEK(34, 57, 92) . Here, we demonstrate that phenylephrine is a poor activator of both A-Raf and c-Raf. Future studies must identify which pathways of activation of MEK are utilized by agonists such as phenylephrine. It is equally important to clarify the different mechanisms of activation of A-Raf and c-Raf and to assess the quantitative contribution of each Raf isoform to activation of the MAPK cascade.