Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells.

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.

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 charac-terized 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 -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 -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 mitogenactivated protein (MAP) 1 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 218 and Ser 222 , of MEK1 (30 -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 -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 EGFstimulated 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.

MATERIALS AND METHODS
Cell Culture-Hela cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). Cells were cultured to 80 -90% confluence and then stimulated with 100 nM PMA (Sigma), 100 ng/ml EGF (Sigma), 10 ng/ml tumor necrosis factor (Calbiochem), or 0.5 uM ionomycin (Calbiochem). For serum stimulation, Hela cells were cultured to 50% confluence and starved in minimal essential medium with 0.1% fetal bovine serum for 24 h. The serum-starved cells were stimulated with 10% fetal bovine serum in minimal essential medium.
Immunoprecipitation-Cells were washed twice with ice-cold phosphate-buffered saline before harvest. The washed cells were scraped into NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, 0.5% Nonidet P-40, 0.5 mM Na 3 VO 4, 50 mM NaF, 0.2 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml chymostatin, 1 g/ml trypsin inhibitor, 5 g/ml aprotinin), sonicated for 10 s, and centrifuged at 15,000 ϫ g for 10 min at 4°C. The cell lysates were incubated with 1 g of anti-Raf antibody for 2 h, and then protein A agarose (Pierce) was added for an additional hour with shaking. The immunoprecipitates were washed three times with NETN buffer and once with 20 mM HEPES, pH 7.5, 0.05% 2-mercaptoethanol, 0.2 mM EDTA. The anti-A-Raf, B-Raf, and c-Raf antibodies (Santa Cruz Biotechnology) were raised against the synthetic peptides corresponding to the C-terminal 20 amino acid residues of each protein. For peptide competition, 2 g of antigenic peptide was incubated with 1 g of affinity purified antibody for 10 min before immunoprecipitation. Western blotting was performed with anti-Raf antibodies (1:1000 dilution).
Kinase Assay-Recombinant human extracellular signal-regulated kinase 1 was expressed as GST fusion (42). MEK1, MEK2, and corresponding mutants were also expressed as GST fusions and purified as described previously (43). Immunoprecipitated Raf was used to activate 0.08 g of GST-MEK1 in 20 l of kinase buffer (18 mM HEPES, pH 7.4, 10 mM magnesium acetate, 50 M ATP). The reactions were incubated at 30°C for 30 min with gentle shaking. The samples were briefly spun in a microfuge, and 10 l of the activated GST-MEK1 (0.04 g) was used to activate 0.3 g of extracellular signal-regulated kinase 1 (43). When GST-MEK2 was used, 0.02 g of recombinant GST-MEK2 was used for in vitro activation by immunoprecipitated Raf.
To assay for the phosphorylation of MEK by A-Raf, the kinasedeficient GST-MEK1 * was used. The MEK1 * mutant has the catalytic essential lysine residue 97 substituted by an arginine residue and thus cannot autophosphorylate. 1 g of GST-MEK1 * was phosphorylated by the immunoprecipitated Raf (30) and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Yeast Two-hybrid Screening-The coding sequences of wild type MEK1 and MEK1S218/222A were subcloned into the BamHI site of pAS-CYH vector (44) to produce pAS-MEK1 and pAS-MEK1S218/222A. These vectors express the fusion of the DNA binding domain of Gal4 and the entire MEK protein. The pAS-MEK1 and pAS-MEK1S218/ 222A were introduced into the yeast strain Y190 (MATa leu2-3, 112 ura3-52 trp1-901 his3-⌬200 ade2-101 gal4 gal80 LYS2::Gal-HIS3 Gal-lacZ cyh r ) by the lithium acetate method (45). The transformants were selected on yeast synthetic complete medium lacking the amino acid tryptophan (SC-Trp). A two-hybrid cDNA library prepared from Hela cells (Clonetech) was then introduced into the yeast Y190 cells containing the bait vector to screen for MEK1 interactive proteins. Transformants were selected on yeast dropout medium lacking histidine, leucine, and tryptophan and containing 50 mM 3-amino-1,2,4 triazole (SC-His, Leu, Trpϩ 3-AT) for 5-7 days. Hisϩ colonies, which denote yeast cells that contain both the bait and interacting targets, were purified and tested for lacZ expression by ␤-galactosidase staining. In order to isolate the library plasmid, the positive yeast colonies were streaked on SC-Leu plates containing cycloheximide (2.5 g/ml), which selects for cells containing only the library plasmids and not the bait plasmid (pAS-MEK1S218/222A). Total chromosomal DNA was prepared from positive clones and electroporated into DH5␣ Escherichia coli cells to recover the library plasmid. Positive clones were further analyzed by DNA sequencing.

Substitution of the Phosphorylation Sites in MEK1 Facilitates Its Interaction with A-Raf in the Yeast Two-hybrid System-Several MEK activators have previously been identified,
including c-Raf, B-Raf, c-mos, and MEK kinase 1 (27-29, 33, 34, 38 -40). Weak interaction between c-Raf and MEK1 has been observed (46). To identify MEK1 interacting proteins, we performed a screening using MEK1 as a bait by the yeast two-hybrid system. When the wild type MEK1 was initially used as a bait in the screening for interacting proteins, no positive clone was isolated among several libraries screened (Table I). Activation of MEK1 requires the phosphorylation of serine residues 218 and 222. Mutation of these two serine residues produced a dominant negative mutant of MEK1 that can block the MAP kinase signal transduction pathway in cultured cells (47) as well as in the Caenorhabiditis elegans vulval development (48). This dominant negative effect of MEK1S218/222A could be due to the possibility that the mutant MEK1 blocks the function of upstream activators by forming a stable complex with the activators. The MEK1S218/222A mutant was subcloned into the yeast pAS-CYH vector and used as a bait to screen the Hela cell cDNA library by the yeast two-hybrid system. Among 1.2 ϫ 10 7 colonies screened, 29 positive clones were isolated and purified. DNA sequence analysis demonstrated that every one of the positive clones encoded a member of the Raf family kinases (Fig. 1).
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 I). 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 I).
Phosphorylation and Activation of MEK1 by A-Raf-Both c-Raf and B-Raf have been demonstrated to phosphorylate and activate MEK1. However, little is known about the role of A-Raf in MEK activation. We immunoprecipitated A-Raf with a specific antibody that recognizes the C-terminal 20 amino acid residues of A-Raf that are distinct from c-Raf and B-Raf. When the immunoprecipitated A-Raf was tested in a coupled in vitro kinase assay, A-Raf activated the recombinant GST-MEK1 (Fig. 3A). In the in vitro kinase assay, the immunoprecipitated A-Raf was used to phosphorylate and activate the purified GST-MEK1. The activated GST-MEK1 was then used to activate the purified recombinant extracellular signal-regulated kinase 1, whose activity was measured by the [ 32 P-␥]ATP incorporation into MBP, an extracellular signal-regulated kinase 1 substrate. A-Raf activity in Hela cells was acutely increased by stimulation of the cells with serum. Maximal activation of A-Raf was observed 5 min after serum stimulation. To test if A-Raf phosphorylates MEK1, the kinase-deficient MEK1 * mutant (to reduce background by eliminating autophosphorylation) was subjected to in vitro phosphorylation by immunoprecipitated A-Raf. Fig. 3B shows that A-Raf could phosphorylate GST-MEK1 * and that the phosphorylation activity was stimulated by serum. It is worth noting that the effect of serum on A-Raf phosphorylation of MEK1 * (Fig. 3B) was much greater than the corresponding increase of MEK1 activity (Fig. 3A). This was likely due to the phosphorylation of MEK1 * by the residual amount of MAP kinase co-immunoprecipitated with Raf. MAP kinase phosphorylates MEK1 on threonine residues 292 and 386 but does not activate 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.
Activation of A-Raf by PMA and EGF-We wished to determine if growth factor can activate A-Raf. Several extracellular stimuli including tumor necrosis factor ␣, PMA, ionomycin, EGF, and UV treatment were tested in stimulation of A-Raf activity. Treatment of Hela cells with tumor necrosis factor ␣, ionomycin, or UV irradiation did not cause significant activation of A-Raf (data not shown). In contrast, A-Raf activity was strongly activated in cells treated with EGF and to a lesser extent with PMA (Fig. 5, A and B). EGF activated A-Raf by as much as 16-fold, which was much greater than the effect of serum (Fig. 3A). EGF and PMA both activated A-Raf activity in a rapid and transient manner (Fig. 5, A and B). However, stimulation of cells by EGF resulted in an increase of A-Raf activity stronger than that by PMA. Furthermore, maximal activation by EGF was observed at 2 min, whereas maximal activation by PMA occurred at 5 min after stimulation. The time course of A-Raf activation was similar to that of c-Raf (data not shown). These results indicate that A-Raf may play an important role in growth factor mediated MEK activation in Hela cells. We have also found that EGF stimulated A-Raf activity in human epidermoid carcinoma A431 cells (data not shown).
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 -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.
A-Raf Preferentially Activates MEK1 over MEK2-MEK1 and MEK2 are two closely related MAP kinase activators. The functional redundancy between MEK1 and MEK2 is not clear. We tested the activation of MEK2 by A-Raf in vitro. Purified recombinant GST-MEK2 was incubated with immunoprecipitated A-Raf. The activity of treated GST-MEK2 was determined by the extracellular signal-regulated kinase activation assay. We were surprised to find that A-Raf did not activate MEK2 (Fig. 6, columns 3 and 4). In a parallel experiment, the   FIG. 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. similarly immunoprecipitated A-Raf-activated MEK1 effectively (Fig. 6, columns 1 and 2). Immunoprecipitated c-Raf, however, activated MEK2 as effectively as MEK1 (Fig. 6, lanes [5][6][7][8]. Similar observation that immunoprecipitated B-Raf from EGF-stimulated Swiss3T3 cells could activate both MEK1 and MEK2 was also obtained (data not shown). These data suggest that unlike c-Raf, A-Raf is specific to MEK1 in vitro. The immunoprecipitated A-Raf has also been tested in the activation of MEK3 and MEK5 to determine if A-Raf may activate other members of the MEK family kinases. Neither MEK3 nor MEK5 was activated by A-Raf (data not shown).

DISCUSSION
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 proteinprotein 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  2 and 6 and  lanes 3 and 7), in contrast to the wild type (lanes 1 and 5). 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. 2 and Table I).
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 I). A likely explanation is that the kinase-deficient MEK5 * can form a stable complex with the extracellular signalregulated 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 twohybrid 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 -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 in-cluding 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.