(Received for publication, August 28, 1995; and in revised form, October 31, 1995)
From the
Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling.
Binding of growth factors to their respective receptors results in the activation of the intrinsic tyrosine kinase. Activation of the receptor tyrosine kinase triggers an increase of autophosphorylation as well as phosphorylation of target proteins(1) . One of the prominent downstream effectors of receptor tyrosine kinases is the Ras oncoprotein, which can be converted from an inactive GDP bound form to the active GTP bound form(2) . The activated Ras protein can elicit a wide range of biological responses including proliferation, differentiation, and neoplastic transformation. One of the best characterized downstream effectors of Ras is the serine/threonine kinase Raf, which itself is a protooncogene(3) . The importance of Raf in Ras function has been demonstrated by the fact that dominant negative mutants of Raf can block Ras-mediated cell growth or transformation(4, 5, 6) . Furthermore, activation of Raf can produce cellular responses similar to Ras(7, 8) .
Three distinct cellular Raf kinases have been isolated(3) . The best characterized is the c-Raf kinase, which is expressed in a wide range of tissues(9) . c-Raf physically interacts with the activated Ras (10, 11, 12, 13, 14) . This Ras-Raf interaction leads to the activation of c-Raf, presumably by recruiting the kinase to the cytoplasmic membrane. At the membrane, Raf becomes activated by a process whose mechanism is poorly understood but that may involve phosphorylation at both tyrosine and serine/threonine residues(15, 16, 17) . Localization to the cytoplasmic membrane apparently plays a critical role in Raf activation as evidenced by the fact that c-Raf artificially targeted to the plasma membrane becomes activated and elicits the biological responses similar to those of the oncogenic mutant of Raf(18, 19) . The other two members of the Raf family, A-Raf and B-Raf, are less well characterized.
One of the critical events in mitogenic growth
factor-induced signal transduction pathway is the activation of the
mitogen-activated protein (MAP) ()kinase, also known as the
extracellular signal-regulated kinase(20) . MAP kinase can be
activated by various extracellular stimuli, including mitogenic growth
factors, cytokines, T-cell antigens, tumor promoters, and hormones
inducing differentiation(20) . MAP kinase is activated by
phosphorylation of a threonine and a tyrosine residue(21) .
These phosphorylations are catalyzed by a single protein kinase known
as MEK, which displays an extremely high substrate selectivity toward
MAP
kinase(22, 23, 24, 25, 26) .
Similarly, MEK is also rapidly activated by agents that stimulate MAP
kinase. c-Raf was the first identified and the best characterized MEK
activator(27, 28, 29) . Biochemical studies
demonstrated that c-Raf phosphorylates two conserved serine residues,
Ser
and Ser
, of
MEK1(30, 31, 32) . These serine
phosphorylations are necessary and sufficient for MEK activation. In
addition to c-Raf, the c-mos protooncogene product and MEK
kinase 1, a yeast STE11 homologue, have been shown to
phosphorylate and activate MEK(33, 34) . Recent data
from several laboratories demonstrated that c-Raf is not the only MEK
activator in growth factor-stimulated
cells(35, 36, 37) . Subsequently, growth
factors were shown to stimulate B-Raf, which then phosphorylates and
activates MEK (38, 39, 40) . Little has been
done with the third member of the Raf family, A-Raf. Although c-Raf is
expressed in a wide range of tissues, both A-Raf and B-Raf expression
are restricted to certain tissues(9) . A-Raf mRNA was detected
at high levels in epididymis, ovary, kidney, and urinary bladder but
was undetectable in brain, lung, and skin(9) . The N-terminal
deletion mutant of A-Raf showed full potential in transformation of
NIH3T3 cells (41) in a manner similar to c-Raf. The possible
involvement of A-Raf in signal transduction is further supported by the
fact that the N-terminal domain of A-Raf specifically interacts with
the activated Ras protein in the yeast two-hybrid system (12) .
In this report, we demonstrated that the kinase domains of A-Raf, B-Raf, and c-Raf interacted with the nonactivatable MEK1S218/222A mutant, which contained alanine substitutions of the activation phosphorylation serine residues 218 and 222, in the yeast two-hybrid system. No such interaction was observed between Rafs and the wild type MEK1 by the same test. We showed that A-Raf indeed can phosphorylate and activate MEK1, and that the kinase activity of A-Raf was stimulated by serum, EGF, and PMA in Hela cells. The EGF-stimulated A-Raf activation in Hela cells was rapid (occurred in 2 min) and transient. Activation of MEK1 by A-Raf required the presence of serine residues 218 and 222. Furthermore, A-Raf did not activate MEK2 in contrast to c-Raf, which activated both MEK1 and MEK2.
To assay for the
phosphorylation of MEK by A-Raf, the kinase-deficient GST-MEK1 was used. The MEK1
mutant has the catalytic essential
lysine residue 97 substituted by an arginine residue and thus cannot
autophosphorylate. 1 µg of GST-MEK1
was phosphorylated
by the immunoprecipitated Raf (30) and analyzed by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Figure 1: Positive clones isolated by the yeast two-hybrid screening using MEK1S218/222A as bait. The filled bars denote the conserved kinase domain CR3 of Raf, whereas the hatched bars denote the N-terminal regulatory domain CR1 and CR2. The Ras-interacting domain is partially overlapped with the N-terminal region of CR1(3, 12) . The numbers on the right indicate the numbers of clones isolated in the screen. The number preceding each line denotes the position of the most N-terminal amino acid residue of each clone, which is represented by a line.
The majority of the positive clones are A-Raf (16 clones) or c-Raf (11 clones), and only two B-Raf clones were isolated. The number of different Raf clones isolated is consistent with the relative levels of Raf mRNA in Hela cells. The shortest clone of A-Raf isolated started at amino acid residue 310. Therefore, it consisted of a sequence that was little more than the kinase domain (Fig. 1). Similarly, the shortest c-Raf clone started at amino acid residue 323 and contained only the C-terminal kinase domain (Fig. 1). Although many clones with various N-terminal truncation were isolated, no positive clone contained sequence less than the kinase domain, suggesting the entire kinase domain structure is necessary and sufficient for the interaction with MEK1S218/222A. This observation is consistent with the notion that the kinase domain of Raf has to bind its substrates, including MEK1. Interestingly, Ras and MEK interact with Raf at different sites.
We
were interested in the observation that no positive clone was isolated
when the wild type MEK1 was used in the identical screening by the
yeast two-hybrid system (Table 1). To test if the wild type MEK1
can interact with Raf in the yeast two-hybrid system, plasmid pAS-MEK1
was cotransformed with A-Raf, B-Raf, and c-Raf clones in the yeast
strain Y190. All transformants were selected on synthetic complete
medium lacking leucine and tryptophan (Fig. 2B), which
selected for the presence of both MEK1- and Raf-containing plasmids.
The transformants were also tested on a selective medium lacking
histidine and supplemented with 50 mM 3-AT, which selects for
positive interaction between the bait (MEK) and the target (Raf) (Fig. 2C). As shown in Fig. 2, positive
interactions between MEK1S218/222A and all three Raf were evident by
the growth of transformants on SC-His, Leu, Trp, 50 mM 3-AT
medium. By contrast, no positive interaction between MEK1 and A-Raf or
B-Raf was observed. The interaction between wild type MEK1 and c-Raf
was much weaker than that of MEK1S218/222A and c-Raf. This result
further supports the notion that the interaction between MEK1 and its
activator, Raf, can be stabilized by the elimination of the
phosphorylation sites in MEK1. A similar screening was performed with
the MEK5 kinase(49) . Extracellular signal-regulated kinase 5
clones were isolated with the inactive MEK5 mutant but not
with the wild type MEK5 (Table 1).
Figure 2: Interaction of MEK1 or MEK1S218/222A with A-Raf, B-Raf, and c-Raf. Yeast Y190 was cotransformed with two plasmids containing either MEK1 or Raf as indicated in A. MEK1m denotes the MEK1S218/222A mutant plasmid. The growth of yeast transformants on SC-Leu, Trp medium, which selects for the presence of both plasmids is shown in B. C represents the growth of the same yeast cells on SC-His, Leu, Trp, +3-AT medium. Positive interaction between the baits (MEK1 or MEK1S218/222A) and targets (A-Raf, B-Raf, or c-Raf) resulted in the transcription activation of the HIS3 gene, thus supporting the growth on SC-His, Leu, Trp, +3-AT medium (C). The MEK3 was included as a negative control.
Figure 3:
Activation and phosphorylation of MEK1 by
A-Raf. A, activation of MEK1 by A-Raf is stimulated by serum.
The A-Raf activity was determined by a coupled kinase assay, in which
A-Raf activated MEK1, which in turn activated extracellular
signal-regulated kinase 1 whose activity was measured by the
phosphorylation of MBP. The phosphorylation of MBP is represented on
the y axis. All reactions contained extracellular
signal-regulated kinase 1. GST-MEK1 was activated by immunoprecipitated
A-Raf from unstimulated cells (column 2) or cells stimulated
with serum for 5 min (column 3) or 10 min (column 4).
GST-MEK1 was omitted as a control (column 5). All the data
presented are a representative of at least three independent
experiments. B, phosphorylation of GST-MEK1. In
order to eliminate MEK1 autophosphorylation, the kinase-deficient
GST-MEK1
was used as a substrate for A-Raf. Purified
GST-MEK1
(1 µg) was incubated with buffer control (lane 1) or immunoprecipitated A-Raf from unstimulated cells (lane 2) or cells stimulated with serum for 5 min (lane
3) or 10 min (lane 4). The arrow indicates
GST-MEK1
.
To
confirm the specificity of the antibody used in the immunoprecipitation
experiments, the antigen peptide (corresponding to the C-terminal 20
amino acid residues of A-Raf) was used for competition in
immunoprecipitation. Preincubation of the anti-A-Raf antibody with
competing peptide completely eliminated A-Raf in the immunoprecipitates (Fig. 4B), whereas competition with a corresponding
c-Raf peptide had no effect on the level of A-Raf precipitated (data
not shown). The peptide also competed with the MEK activating activity
in A-Raf immunoprecipitate. Similarly, phosphorylation of
GST-MEK1 by A-Raf was competed by the antigen peptide,
suggesting that A-Raf or an associated kinase was responsible for the
phosphorylation of MEK1
(Fig. 4A). The low
level phosphorylation of GST-MEK1
in lane 3 of Fig. 4A was likely due to nonspecific activity in the
immunoprecipitate. Our data also indicate that measuring MEK activation
is a more specific assay than measuring phosphorylation of MEK in
determining the Raf activity because a nonspecific kinase in the
immunoprecipitate may phosphorylate but not activate MEK.
Figure 4:
Competition of A-Raf immunoprecipitation. A, the MEK1 kinase activity of A-Raf immunoprecipitate could
be competed by preincubation of antibody with competing peptide.
GST-MEK1 (1 µg) was used for each phosphorylation
reaction. Lane 1, phosphorylation of GST-MEK1
by
A-Raf from unstimulated Hela cells; lane 2, phosphorylation of
GST-MEK1
by A-Raf from cells stimulated with serum; lane 3, same as lane 2 but in the presence of 2
µg of competing peptide; lane 4, GST-MEK1
alone as a control. The arrow indicates
GST-MEK1
. B, competition of A-Raf by antigen
peptide. The immunoprecipitated A-Raf was subjected to Western blotting
with anti-A-Raf antibody and detected by alkaline
phosphatase-conjugated second antibody. Immunoprecipitated A-Raf from
unstimulated Hela cells (lane 1), serum-stimulated cells (lane 2), competition with 2 µg of antigen peptide (lane 3) in immunoprecipitation. The arrow indicates
A-Raf.
Figure 5: Activation of A-Raf by EGF and PMA. A, time course of A-Raf activation by EGF. Hela cells were stimulated with 100 ng/ml EGF for various time periods (indicated by x axis, min). A-Raf was immunoprecipitated and assayed for MEK1 activation. The A-Raf activity in immunoprecipitate of unstimulated cells was set at the value of one. B, time course of A-Raf activation by PMA. C, determination of the relative contributions of A-Raf and c-Raf to MEK1 activation in EGF-stimulated Hela cells. Level of MBP phosphorylation plotted on the y axis reflects the relative MEK-activating activity of immunoprecipitated Raf. Hela cells were stimulated with EGF for 2 min and immunoprecipitated by either anti-A-Raf or anti-c-Raf antibodies. The immunoprecipitates were assayed for GST-MEK1 activation. D, activation of MEK1 by A-Raf requires the presence of serine residue 218 and 222. The arrow indicates the phosphorylated MBP by the coupled kinase assay. All reactions contained extracellular signal-regulated kinase 1. Lanes 1, 2, and 3 represent the basal activity of GST-MEK1, GST-MEK1S218A, and GST-MEK1S222A, respectively. Nonspecific kinase activity in A-Raf immunoprecipitate is shown in lane 4. Lanes 5, 6, and 7 denote the A-Raf-activated activity of GST-MEK1, GST-MEK1S218A, and GST-MEK1S222A, respectively. A-Raf could not activate either GST-MEK1S218A or GST-MEK1S222A (comparing lanes 2 and 6 and lanes 3 and 7), in contrast to the wild type (lanes 1 and 5).
Activation of c-Raf by EGF has been observed in numerous cell types. We compared the relative contributions of A-Raf and c-Raf to MEK activation in Hela cells. B-Raf was not tested because it is expressed at a very low level in Hela cells. The MEK-activating activity of A-Raf and c-Raf was measured in EGF-stimulated Hela cells. Our data demonstrated that c-Raf had a significantly higher MEK activating activity than A-Raf in EGF-stimulated Hela cells (Fig. 5C). Nevertheless, A-Raf constitutes a significant fraction of the MEK-activating activity, approximately 40% of that of c-Raf.
c-Raf activates MEK1 by phosphorylating at serine residues 218 and 222 (30, 31, 32) . We wanted to test if these two serine residues are also required for A-Raf-dependent MEK1 activation. Purified GST-MEK1S218A and GST-MEK1S222A mutants were treated with immunoprecipitated A-Raf, and their ability to be activated by A-Raf was compared with that of the wild type MEK1. Elimination of either serine 218 or 222 completely abolished A-Raf-dependent activation (Fig. 5D). It is worth noting that mutation of either serine residue decreases the basal activity of MEK1, consistent with previous observations(30) . Our data suggest that serine residues 218 and 222 are the phosphorylation residues targeted by A-Raf. The biochemical mechanisms of MEK1 activation by A-Raf is thus similar to that by c-Raf.
Figure 6: Activation of MEK1 but not MEK2 by A-Raf. Activation of GST-MEK1 (0.08 µg) and GST-MEK2 (0.02 µg) by immunoprecipitated A-Raf and c-Raf was determined and compared. A-Raf did not activate MEK2 significantly (comparing columns 3 and 4), whereas the similarly immunoprecipitated A-Raf activated MEK1 (columns 1 and 2). In contrast, c-Raf activated both MEK1 and MEK2 (columns 5-8).
Protein phosphorylation plays critical roles in the regulation of many cellular activities, including growth, differentiation, and metabolism. Protein kinases are key enzymes in signal transduction, typified by the MAP kinase pathway, which involves a cascade of kinases. Identification of upstream regulators and downstream effectors of protein kinases is a challenge for signal transduction research. The yeast two-hybrid system have been widely and successfully used to identify protein-protein interaction(11, 12, 44, 50) . Because many of the components in the signal transduction pathway physically interact with each other, the two-hybrid system may be the ideal approach to the identification of kinase-interacting proteins. However, it has often been difficult to identify interaction between a kinase and its substrates because of the transient nature of the interaction. We reasoned that a kinase may be able to form a stable complex with its substrate if the target phosphorylation residues of the substrates are eliminated. Based on this rationale, we have successfully isolated all members of the Raf family kinases by using the MEK1S218/222A mutant.
The mutant MEK1S218/222A functions as a dominant negative in cultured cells(47) . Similarly, the corresponding mutations in the C. elegans mek-2 also resulted in a dominant negative mutant that blocked the Ras/Raf-dependent vulva induction(48) . MEK1S218/222A could display the dominant negative effect because the mutant MEK1S218/222A may stably bind to its upstream activator, such as Raf, and sequester the Raf from activating the endogenous MEK1. This idea is consistent with our observation that MEK1S218/222A interacted strongly with Raf in the yeast two-hybrid system, whereas the wild type MEK1 failed to isolate Raf by the same screening ( Fig. 2and Table 1).
Using a mutant as the
bait in the yeast two-hybrid system may have a general application in
identifying upstream kinases or downstream substrates. One such example
is that the extracellular signal-regulated kinase 5 was isolated by the
kinase-deficient MEK5 mutant but not by the wild type MEK5 (Table 1). A likely explanation is that the kinase-deficient
MEK5
can form a stable complex with the extracellular
signal-regulated kinase 5 but is unable to phosphorylate extracellular
signal-regulated kinase 5, thus forming a stable complex. In contrast,
the active MEK5 may interact with the extracellular signal-regulated
kinase 5 transiently because the extracellular signal-regulated kinase
5 will dissociate from MEK5 once phosphorylation is completed. Another
example consistent with this idea is the CDK4 kinase. The Rb tumor
suppressor protein is a substrate of CDK4. Kato et al.(51) have observed that the kinase-deficient CDK4 formed a
stable complex with Rb in cultured cells, whereas the wild type CDK4
showed a much weaker interaction with Rb.
Protein phosphorylation is one of the main mechanisms in cellular regulation. Identification of upstream kinase of a phosphoprotein or downstream target of a kinase can provide critical information in signal transduction research. Data from this report suggest the possibility that an upstream kinase may be isolated by the yeast two-hybrid system using mutants that lack the target phosphorylation residues as a bait. Similarly, it may be feasible to identify downstream substrates of a kinase using the kinase-deficient mutant as a bait in the yeast two-hybrid screening. The general application of these ideas remains to be tested with many of the kinases or phosphoproteins available.
MEK1 and MEK2 are the only two identified MAP kinase activators. MAP kinase activity can be stimulated by a wide variety of stimuli, which may be mediated by different MEK activators. Identification of c-Raf as a MEK activator provided an essential link between the growth factor receptor tyrosine kinase and the MAP kinase cascade(27, 28, 29) . Raf is a family of protein kinases consisting of c-Raf, B-Raf, and A-Raf. Recently, B-Raf has been indicated to play an important role in MEK activation. B-Raf activity is rapidly stimulated by growth factors in several cell types(38, 39, 40) . Early transformation experiments of NIH3T3 cells by A-Raf supported a role of this kinase in cell growth regulation. Furthermore, interaction of A-Raf with the activated/oncogenic Ras strongly suggests that A-Raf may function in Ras signaling. Data from this study unambiguously demonstrated the function of A-Raf as a MEK1 activator, indicating a role of A-Raf in mitogenic growth factor and protein kinase C-induced MAP kinase activation.
Activation of MEK1 by A-Raf apparently required the phosphorylation of serine residues 218 and 222, which are also the common phosphorylation sites of different MEK activators including B-Raf, c-Raf, c-mos, and MEK kinase 1. Interestingly, A-Raf preferentially activated MEK1 but not MEK2 (Fig. 6). In contrast, the c-Raf kinase can effectively activate both MEK1 and MEK2 kinases. Although extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 are the only two identified substrates of MEK1 and MEK2, it is possible that MEK1 may have other physiological substrates not shared by MEK2. Currently, it is unclear if MEK1 and MEK2 have the identical physiological functions. Genetic studies in C. elegans and Drosophila demonstrated that a single MEK gene fulfills the critical role in receptor tyrosine kinase signal transduction(48, 52, 53) , suggesting that the mammalian MEK1 and MEK2 may have overlapping as well as distinct functions. Activation of A-Raf may lead to specific activation of MEK1 but not MEK2, possibly eliciting cellular responses different from those elicited by the activation of c-Raf.