(Received for publication, September 27, 1994; and in revised form, December 1, 1994)
From the
Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.
Raf-1, the product of the c-raf-1 proto-oncogene, is a serine/threonine protein kinase activated by numerous growth factors and cytokines(1, 2, 3, 4, 5, 6, 7, 8) . The stimulation of Raf-1 activity depends partly on the activation of the small G-protein Ras(6, 7, 8, 9, 10, 11, 12, 13) . When activated, Raf-1 phosphorylates and activates the microtubule-associated protein kinase kinases, MEK1 and MEK2(14, 15, 16, 17, 18, 19) . These enzymes in turn phosphorylate and activate the microtubule-associated protein kinases, ERK1 and ERK2(20, 21, 22, 23) . ERKs phosphorylate and regulate the activity of several cytosolic and nuclear proteins(24, 25, 26, 27, 28, 29, 30, 31) . Therefore, Raf-1 is pivotal in transmitting signals from Ras in the plasma membrane to cytosolic and nuclear compartments of the cell.
Raf-1 is composed of two functionally distinct domains, an
NH-terminal regulatory domain and a COOH-terminal kinase
domain(32, 33, 34, 35, 36, 37) .
Active GTP-Ras binds the NH
-terminal domain of Raf-1 in a
region spanning amino acids 50 to
135(38, 39, 40, 41, 42, 43, 44) .
This binding alone does not increase the kinase activity of Raf-1 in vitro(12) , but rather, may function to recruit
Raf-1 to the plasma membrane, which is sufficient to cause its
activation(12, 13) . However, membrane-anchored Raf-1
is further activated by a Ras-independent signal(13) , and in
co-transfection experiments, additional signals besides v-ras are necessary to fully activate Raf-1(6, 45) .
Therefore, these findings suggest that additional Ras-independent
signals are required for the full activation of Raf-1.
Besides
binding GTP-Ras, the NH-terminal domain of Raf-1 has also
been proposed to repress the phosphotransferase activity of the
catalytic domain(33, 46) . For example, replacement of
the NH
-terminal domain with a viral protein, as occurred in v-raf(47) , or its deletion, as represented by
Raf-BXB(35) , is sufficient to cause constitutive activation of
the Raf-1 catalytic domain in fibroblast cell lines. Expression of v-raf or Raf-BXB stimulates cell
transformation(34, 36, 48) , the
transactivation of AP1 regulatory elements(35, 49) ,
and the activation of MEKs and
ERKs(16, 17, 48) . Therefore, based on these
observations and the above Ras-binding studies, two separable signaling
events may be necessary to activate Raf-1. Both events involve
interactions with the NH
-terminal regulatory domain, one
involving GTP-Ras binding, and the other involving modulation of the
inhibitory effect exerted on the catalytic domain.
In this report, we demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Because the defined Ras-binding region is deleted from Raf-BXB, these findings establish that the catalytic domain of Raf-1 is regulated independently of Ras binding in these cells.
Figure 1:
MEK kinase activity of Raf-1 and
Raf-BXB measured from Jurkat T cells. The MEK kinase activity of
Raf-1/Raf-BXB (upper panel) from control (pMEP) and
Raf-BXB cells stimulated with 10 ng/ml PMA, 20 µM okadaic
acid (OA), or 0.1% MeSO carrier (CONT)
for 15 min was measured by an in vitro kinase assay as
described under ``Experimental Procedures.'' NIH 3T3 lysate
was a positive control. Autophosphorylation by rMEK1 was measured by
incubating rMEK1 alone in reaction buffer (rMEK1 CONT).
Samples were immunoblotted with anti-Raf-1 antiserum SP63 (lower
panel).
To measure
Raf-BXB activity exclusively, an expression vector encoding an
epitope-tagged version of Raf-BXB (hemagglutinin antigen; Raf-HABXB) was constructed and stably transfected into Jurkat
T cells. Raf-HABXB activity was examined using a monoclonal antibody
specific for the epitope tag (12CA5; HA). MEK1 kinase
activity was only immunoprecipitated from Raf-HABXB cells; no activity
was found in immunoprecipitates from control cells. The activity of
Raf-BXB was stimulated by PMA and okadaic acid (Fig. 2A). The recombinant MEK1 substrate
phosphorylated by Raf-HABXB became activated, in turn phosphorylating
exogenously added recombinant ERK1 (Fig. 2A). Although
MEK1 has been shown to form a stable complex with the kinase domain of
Raf-1 (56) , the ERK kinase activity detected in these assays
was not from co-immunoprecipitated endogenous MEKs, since the addition
of exogenous recombinant MEK1 to the immune complexes was essential to
detect any activity in our assay system (Fig. 2B).
Figure 2: MEK kinase activity of epitope-tagged Raf-BXB. A, Raf-HABXB MEK kinase activity was measured from stable transfectants stimulated with PMA, okadaic acid (OA), or carrier as described in the legend to Fig. 1by an in vitro kinase assay as described under ``Experimental Procedures.'' Supernatant from the initial reaction was transferred to a new reaction to measure recombinant MEK1 (rMEK1)-catalyzed recombinant ERK1 (rERK1) phosphorylation. Autophosphorylation (AUTO) was measured by incubating rMEK1 alone, and then adding rERK1 substrate. B, assay demonstrating that exogenously added rMEK1 is essential for measuring the coupled ERK kinase activity. After cells were stimulated with 10 ng/ml PMA and 10 µM okadaic acid for 15 min, Raf-HABXB activity was measured as described in A, except with and without the addition of rMEK1.
We next examined the consequence of okadaic acid and PMA together on the activity of Raf-1 and Raf-BXB. Interestingly, the combination of okadaic acid and PMA stimulated less Raf-1 activity than did PMA alone (Fig. 3, left). In contrast, okadaic acid and PMA together synergistically stimulated Raf-HABXB activity (Fig. 3, right). Therefore, the activation of intact Raf-1, but not Raf-BXB, was inhibited by okadaic acid.
Figure 3:
Inhibition of Raf-1 activation and
augmentation of Raf-BXB activation by okadaic acid. Raf-1 activity
measured from Jurkat T cells stimulated with 0.1% MeSO
carrier (CONT), 10 ng/ml PMA, 20 µM okadaic acid,
or the combination for 10 min (left). In a separate
experiment, Raf-HABXB activity was measured from stable transfected
Jurkat T cells stimulated for 15 min as indicated (right).
Raf-1 and Raf-HABXB activities were measured by the activation of rMEK1
catalytic activity toward rERK1 substrate as described under
``Experimental Procedures.''
Engagement of the T cell
antigen receptor-CD3 complex with a monoclonal antibody mimics the
normal physiological stimulus, and has been demonstrated to rapidly and
transiently stimulate Raf-1 activity in peripheral blood T
cells(54) . Therefore, we examined the effect of this stimulus
on Raf-HABXB activity. Raf-HABXB was activated within 30 s of engaging
the CD3 complex, and activation was maximal at 2 min (Fig. 4A, upper panel). After 2 min the
activity subsided, approaching unstimulated activity by 15 min (Fig. 4B, upper panel). The addition of PMA
with anti-CD3 for 2 min even further activated Raf-HABXB (Fig. 4C, left upper panel). As with
Raf-1(4) , treating cells with a Ca ionophore
alone did not stimulate Raf-HABXB activity, or significantly influence
Raf-HABXB activity stimulated by PMA (Fig. 4C, right upper panel). These observed changes in Raf-HABXB
activity were not due to differences in the levels of Raf-HABXB
immunoprecipitated as uniformity of the immunoprecipitations was
confirmed by immunoblotting (Fig. 4, lower panels).
Figure 4: Raf-BXB activity stimulated by engagement of the antigen receptor-CD3 complex. A, early kinetics of Raf-HABXB activation. Stable Raf-HABXB expressing Jurkat T cells were incubated with 1 µg/ml anti-CD3 monoclonal antibody for the time periods indicated and Raf-HABXB activity was measured as in Fig. 2(upper panel). Samples were immunoblotted with anti-Raf-1 serum SP63 (lower panel). B, late kinetics of Raf-HABXB activation. Cells were stimulated and activity measured as in A. C, additive Raf-HABXB stimulation by anti-CD3 and PMA, but not ionomycin and PMA. Stable Raf-HABXB expressing Jurkat cells were stimulated with 1 µg/ml anti-CD3 mAb and/or 10 ng/ml PMA for 2 min (left panel), and with 1 µM ionomycin and/or 10 ng/ml PMA for 15 min (right panel). Activity was measured as in A.
Figure 5: Augmentation of MEK1 and MEK2 activation by Raf-BXB expression. Effect of Raf-HABXB expression on MEK1 and MEK2 activation. The kinase activity of MEK1 and MEK2 was measured as described under ``Experimental Procedures'' from control (pMEP) and Raf-HABXB-expressing Jurkat T cells stimulated with 1 µg/ml anti-CD3 monoclonal antibody for 5 min, or 10 ng/ml PMA, 20 µM okadaic acid (OA), or 1 µM calyculin A (CLA) for 15 min.
Figure 6: Effect of Raf-BXB expression on ERK2 activation. A, the kinase activity of ERK2 was measured as described under ``Experimental Procedures'' from control (pMEP) and Raf-BXB expressing Jurkat T cells stimulated with 10 ng/ml PMA or 20 µM okadaic acid for 15 min. B, effect of Raf-BXB expression on anti-CD3 stimulated ERK2 activation. ERK2 activity was compared in control (pMEP) and Raf-BXB expressing Jurkat T cells stimulated with 1 µg/ml of a control or anti-CD3 monoclonal antibody for various time periods.
Figure 7: Inhibition of Raf-1, but not Raf-BXB activation by increased cAMP. A, effect on Raf-1 and MEK1 activation by PMA. Jurkat T cells were preincubated for 15 min in medium alone, 2 mM (8-Br-cAMP), or 100 µM forskolin and an aliquot of cells was removed to measure cAMP-dependent protein kinase activity. The cells were then stimulated with 10 ng/ml PMA for an additional 15 min. Raf-1, MEK1, and cAMP-dependent protein kinase activities were measured from the same cells as described under ``Experimental Procedures.'' cAMP-dependent protein kinase activities (pmol/min) were: CONT, 17.6; 8-Br-cAMP, 55.7; forskolin (FORSK), 45.1. B, effect on Raf-HABXB. Stable Raf-HABXB expressing Jurkat T cells were preincubated with 2 mM 8-Br-cAMP or 100 µM forskolin and then stimulated with PMA as described above. cAMP-dependent protein kinase activities (pmol/min) were: Experiment 1, CONT, 0.1; 8-Br-cAMP, 3.9. Experiment 2, cont, 2.6; forskolin (FORSK), 10.4.
Figure 8:
Effect
of mutating serine 621 and serine 499 on Raf-HABXB activity. Cells
transiently transfected with vectors encoding wild type (WT),
S499A, and S621
D variants of Raf-HABXB were cultured for 20
h and then stimulated and assayed for MEK kinase activity as described
in the legend to Fig. 2A.
Previous studies have demonstrated that Raf-BXB is
constitutively active when expressed in fibroblast cell
lines(17, 35, 48, 61) . In this
report, we have demonstrated that, in contrast to its behavior in
fibroblasts, Raf-BXB is not constitutively active when expressed in
Jurkat T cells. Our data clearly establishes that in T cells, removal
of the NH-terminal regulatory domain of Raf-1 is
insufficient to activate the catalytic domain. One or more additional
signals, provided by PMA, okadaic acid, or anti-CD3, directed to the
Raf-1 catalytic domain are required for its activation. Interestingly,
engagement of the antigen receptor-CD3 complex stimulated an extremely
rapid and transient activation of Raf-BXB that resembled the time
course of activation of full-length Raf-1(54) . This suggests a
close coupling of this signal to the antigen receptor-CD3 complex. It
is unlikely that Ras provides this signal because the defined
Ras-binding region is deleted from Raf-BXB, the kinase domain of Raf-1
itself does not associate with
Ras(38, 39, 40, 42, 48) ,
and the activating signal is resistant to the effect of increased cAMP,
which inhibits the binding of Raf-1 to
Ras-GTP(39, 57, 58, 59, 60) .
Therefore, based on these observations, we conclude that the activity
of the Raf-1 catalytic domain is regulated independently of Ras
binding, by one or more signals acting directly on the catalytic
domain.
In light of the above observations, we propose that Raf-1
may require two or more separable signaling events for activation. At
least one signal directly regulates the MEK kinase activity of the
catalytic domain of Raf-1. First, by binding Ras-GTP (event 1), Raf-1
is likely translocated to the inner surface of the plasma
membrane(12, 13, 38, 62) . In this
environment, it may receive a Ras-independent signal that modulates the
repression exerted by the NH-terminal regulatory domain
(event 2). Lastly, the importance of an additional signal or signals
that directly activate the Raf-1 catalytic domain is implicated by our
findings in Jurkat T cells (event 3). The last two events may be
coupled and initiated by one signal acting on the catalytic domain.
Therefore, in NIH 3T3 fibroblasts, this model predicts that the last
signal is constitutively activated, explaining why Raf-BXB is active
and transforming in these cells(17, 35, 48) .
The nature and source of the signal regulating the MEK kinase activity of Raf-BXB is unknown. A definitive role for serine phosphorylation in regulating the activity Raf-BXB is currently not evident. Our data excludes the importance of serine 499 as a regulatory site, but suggests that serine 621 is essential for Raf-BXB kinase activity. Whether the phosphorylation of serine 621 regulates the MEK kinase activity of Raf-BXB is unclear.
Interestingly, okadaic acid
inhibits Raf-1 activation while activating Raf-BXB. Increased cAMP also
inhibits the activation of Raf-1, but not Raf-BXB. The resistance of
Raf-BXB to okadaic acid and cAMP antagonism suggests that the
inhibition of Raf-1 is mediated entirely through the
NH-terminal regulatory domain. A precedent for such a
mechanism exists, wherein cAMP-dependent protein kinase phosphorylates
Raf-1 on serine 43 and inhibits its association with
Ras-GTP(39, 57, 58, 59, 60) .
Okadaic acid may inhibit Raf-1 activation by a similar mechanism in
that okadaic acid does stimulate abundant serine and threonine
phosphorylation of Raf-1 and Raf-BXB. (
)
Finally, our data reveals a correlation between the activation of Raf-BXB in Jurkat T cells and it effects on interleukin-2 production. Raf-BXB expression enhances interleukin-2 production only in cells stimulated by engaging the antigen receptor-CD3 complex or treated with phorbol esters(52) . However, considering the modest effect Raf-BXB expression has on stimulated MEK1, MEK2, and ERK2 activities in these cells, it seems plausible that Raf-1 activity may trigger alternative biochemical events that promote interleukin-2 production, such as the activation of NFkB(63) , or other parallel kinase pathways that can regulate the interleukin-2 promoter(64) .