The microinjection of anti-RKIP antibodies raised against the full-length RKIP protein efficiently activated an AP-1-dependent reporter gene. This induction was due to the activation of MEK, since it could be suppressed by two structurally different MEK inhibitors, U0126 and PD98059 (Fig. a). This showed that the expression of the reporter gene is controlled by the ERK pathway and supports our previous conclusion that RKIP inhibits this pathway by downregulating the activation of MEK by Raf-1 (26
). The induction of the reporter gene could be completely prevented by coinjection of an RKIP expression vector (26
), indicating that the RKIP antibodies specifically neutralized RKIP function. These antibodies are therefore useful tools for investigating the molecular mechanism by which RKIP works. The RKIP antiserum interfered with the binding of Raf-1 and MEK to RKIP (Fig. b). This effect was specific, as (i) the corresponding preimmune serum had no effect and (ii) the RKIP antibodies did not prevent the binding of Raf-1 to 14-3-3. Furthermore, the RKIP antibodies reversed the inhibitory effect of RKIP on MEK phosphorylation by Raf-1 (Fig. c). These results indicated that the inhibitory effect of RKIP on MEK activation by Raf-1 depends on RKIP binding to Raf-1 and/or to MEK.
FIG. 1 RKIP inhibits the ERK pathway by preventing MEK activation. (a) Rat-1 cells were microinjected with a TRE-LacZ reporter plasmid and affinity-purified RKIP antibodies or preimmune immunoglobulin G (IgG) and treated as indicated. The MEK inhibitors PD98059 (more ...)
Therefore, we analyzed the role of RKIP in the formation of ternary protein complexes with Raf, MEK, and ERK in more detail (Fig. ). Immobilized GST-MEK could bind Raf-1, ERK, and RKIP (Fig. a). However, while GST-MEK could bind both ERK and RKIP simultaneously (Fig. a, panels 2 and 3), Raf-1 and RKIP seemed to compete for binding (Fig. a, panels 1 and 2). Consistent results were obtained when GST-RKIP, GST-ERK, or GST–Raf-1 beads were used for binding assays. RKIP decreased the binding of Raf-1 to GST-MEK beads (Fig. a, panel 1) and the binding of MEK to GST–Raf-1 beads (Fig. d, panel 1). In both cases, RKIP competed for binding. In contrast, RKIP did not interfere with the association of ERK with GST-MEK beads (Fig. a, panel 3) or of MEK with GST-ERK beads (Fig. c, panel 1). When GST-RKIP beads were used as bait, Raf and MEK mutually diminished their binding to GST-RKIP (Fig. b, panels 1 and 2), whereas MEK and ERK mixed together bound with an efficiency similar to that of each individual protein alone (Fig. b, panels 1 and 3). In summary, these experiments demonstrated that MEK and ERK can bind to RKIP at the same time but the binding of Raf-1 to RKIP and that of MEK are mutually exclusive. Further, these data suggest that the binding of Raf-1 or MEK to RKIP may compete with their binding to each other and thus interfere with the formation of Raf-1–MEK complexes.
FIG. 2 Analysis of the composition of RKIP protein complexes. (a) GST-MEK beads were incubated with RKIP, Raf, and MEK in the indicated combinations. GST-RKIP beads (b), GST-ERK beads (c), or GST-Raf-1 beads (d) were incubated with recombinant purified proteins (more ...)
This possibility was tested. The analysis of the kinetics of MEK phosphorylation by Raf-1 revealed that RKIP diminished the Km
but not the Vmax
of the reaction, indicating a competitive type of inhibition (Fig. a). Control proteins, such as GST and 14-3-3, had no effect, and since RKIP is not a Raf-1 substrate, it did not compete for phosphorylation (data not shown). We have previously shown that the association of Raf-1 and MEK is required for efficient MEK phosphorylation and activation (12
). Therefore, we tested whether RKIP could disturb the physical interaction between Raf-1 and MEK. For this purpose we coexpressed Raf-1 and GST–MEK-1 in Sf-9 insect cells and purified the GST–MEK-1–Raf-1 complex by adsorption to glutathione Sepharose beads. The Raf-1–GST–MEK complex was incubated with increasing amounts of purified RKIP. After a washing, the composition of the complex was examined by Western blotting (Fig. b). The addition of RKIP resulted in RKIP binding and a concomitant displacement of Raf-1 from the GST-MEK beads, thus confirming a competitive mode of inhibition. These data suggest that at least two populations of Raf-1 can be distinguished, one that is associated with MEK and competent for MEK phosphorylation and another that is bound to RKIP and disabled for MEK phosphorylation. To test this prediction, Raf-1 was produced in Sf-9 insect cells and recovered either by affinity adsorption to GST-RKIP beads or by immunoprecipitation with Raf antibodies from serial dilutions of the same lysate (Fig. c). When assayed for MEK phosphorylation, the kinase activity of RKIP-associated Raf-1 was severely impaired compared to an equivalent amount of immunoprecipitated Raf-1. These results confirmed the hypothesis that Raf-1 bound to RKIP is inactive as MEK kinase.
FIG. 3 RKIP inhibits Raf-1 by a competitive mechanism. (a) Lineweaver-Burk plot of Raf-1 inhibition by RKIP. Activated GST–Raf-1 was used to phosphorylate GST–MEK-1 in the presence of increasing amounts of RKIP, as indicated. Phosphorylation (more ...)
These results also suggested that only the fraction of Raf-1 which is not bound to RKIP is available for activation. Therefore, we examined whether Raf-1 dissociates from RKIP during activation. For this purpose, RKIP and Raf-1 were coexpressed in COS-1 cells (Fig. a). Raf-1 coprecipitated with RKIP in quiescent cells. Stimulation of the cells with tetradecanoyl phorbol acetate (TPA) plus epidermal growth factor caused an increase in Raf-1 kinase activity which correlated with a decrease of RKIP association. At later time points, as Raf-1 catalytic activity declined, the levels of Raf-1 coprecipitating with RKIP increased again. To investigate whether the changes in RKIP association are related to the activation status of Raf-1, the binding of purified RKIP to inactive and activated GST–Raf-1 beads was determined (Fig. b). Activated GST–Raf-1 was produced in Sf-9 insect cells coinfected with RasV12 and Lck, which results in a robust activation of the catalytic activity. GST–Raf-1 proteins were purified by adsorption to glutathione Sepharose beads and incubated with recombinant RKIP produced in E. coli
. Less RKIP bound to activated GST–Raf-1, indicating that Raf-1 activation weakens the affinity towards RKIP. This finding, however, did not seem to depend on the kinase activity of Raf-1 per se. Kinase-negative Raf-1 mutants, such as RafK375W (11
) or RafS621A (17
), as well as activated Raf-1 mutants, such as RafS259D (17
) or the isolated kinase domain BXB, bound to RKIP at levels comparable to that of the wild-type Raf-1 (reference 26
and data not shown). We also tested whether activation affected the binding of MEK and ERK to RKIP. Purified MEK and ERK were phosphorylated in vitro with recombinant Raf-1 or Raf-1 plus MEK, respectively, and incubated with GST or GST-RKIP beads. The binding reaction products were washed, separated on SDS gels, and immunoblotted with the appropriate antisera. We did not observe any differences in binding between activated and nonactivated forms (data not shown). However, since only small fractions of MEK and ERK become phosphorylated (1
), we also carried out the phosphorylation in the presence of [γ-32
P]ATP in order to avoid misinterpretation due to low phosphorylation efficiencies (Fig. c and d). The blots were autoradiographed to detect phosphorylated MEK and ERK and were subsequently stained with the cognate antisera to visualize total protein bound. Under these conditions, binding of phosphorylated MEK and ERK to RKIP was evident.
These data were consistent with Raf-1 being the main regulatory target of RKIP. To further examine the molecular basis for the observed competitive mode of RKIP inhibition, we mapped the domains in the Raf-1 kinase domain, BXB, which are necessary for RKIP and MEK binding (Fig. a). BXB deletion mutants were expressed as GST fusion proteins in E. coli
and were examined for binding to purified RKIP or MEK in vitro. Surprisingly, the required binding domains were different. Raf-1 kinase subdomains VIb to VIII were essential for MEK binding, whereas RKIP bound to subdomains I and II. The latter region contains the ATP binding site, but RKIP did not compete for ATP (data not shown). Likewise, RKIP and Raf-1 bound to different domains in MEK-1 (Fig. b). As previously reported (2
), Raf-1 bound to MEK-1 constructs containing the proline-rich region, whereas RKIP bound to the N-terminus of MEK-1. Thus, RKIP's ability to dissociate Raf-MEK complexes does not seem to involve a direct competition for the same binding sites. Rather, it must be due to an allosteric reduction of the binding affinity induced by RKIP or to mutual steric hindrance that excludes simultaneous binding of RKIP and Raf to MEK or of RKIP and MEK to Raf-1, respectively. When we mapped the binding sites of Raf-1 and MEK-1 to RKIP (Fig. c), the RKIP domain required for MEK binding could be clearly located, while Raf-1 interacted with multiple domains in RKIP. Notably, removal of the RKIP carboxy terminus up to the Bsp
EI site enhanced Raf-1 association, whereas further deletion up to the Ppu
MI site decreased Raf-1 binding again. These data suggest that the interaction between Raf-1 and RKIP is complex, involving a main site of binding to amino acids 77 to 108 in the Bsp
MI fragment, as well as minor contacts with several other domains. The partial overlap between the MEK and Raf-1 binding sites, however, is consistent with the observation that RKIP cannot bind Raf-1 and MEK simultaneously (Fig. ).
In summary, all these data suggested that RKIP mutants that are defective for Raf-1 binding should also be compromised as inhibitors of the ERK pathway. To examine this possibility, we generated RKIP deletion mutants suitable for expression in mammalian cells. The analysis of Raf-1 binding to the RKIP deletion mutants in mammalian cells was consistent with the in vitro mapping of the main Raf-1 binding site to amino acids 77 to 108 (Fig. a). The N93 and the C93 RKIP mutants, which both disrupt this domain, failed to coimmunoprecipitate with Raf-1. However, C93 RKIP still contains the MEK binding domain. When tested for suppression of Raf-mediated AP-1 induction, only N93 RKIP showed a clear decrease in inhibitory activity (Fig. b). Since N93 RKIP is the only mutant that lacks both the Raf-1 and MEK interaction domains, we conclude that either Raf-1 or MEK binding is sufficient for suppression of the ERK pathway.
FIG. 6 RKIP binding to Raf-1 or MEK is sufficient for inhibition. (a) Coimmunoprecipitation of RKIP deletion mutants with Raf-1. FLAG–Raf-1 and hemagglutinin (HA)-RKIP or HA-RKIP deletion mutants were coexpressed in COS cells. Lysates were immunoprecipitated (more ...)