BIOLOGICAL OVERVIEW

Mechanisms that regulate signal propagation through the ERK/MAPK pathway are still poorly understood. Several proteins are suspected to play critical roles in this process. One of these is Kinase Suppressor of Ras (KSR), a component previously identified in RAS-dependent genetic screens in Drosophila and Caenorhabditis elegans. KSR functions upstream of MEK within the ERK/MAPK module. In agreement with this, KSR facilitates the phosphorylation of MEK (officially termed Downstream of raf1 in Drosophila) by RAF (accepted FlyBase name: Pole hole). KSR associates independently with RAF and MEK, and these interactions lead to the formation of a RAF/MEK complex, thereby positioning RAF in close proximity to its substrate MEK. These findings suggest that KSR functions as a scaffold that assembles the RAF/MEK functional pair (Roy, 2002).

The characterization of KSR with respect to the ERK/MAPK pathway has been undertaken by a number of groups using mKSR1, a murine isoform (for review, see Morrison, 2001). Like RAF, mKSR1 associates with HSP90 and p55/CDC37 as well as with 14-3-3 (see Drosophila Leonardo) proteins (Xing, 1997; Stewart, 1999; Cacace, 1999). Interestingly, mKSR1 has also been shown to interact constitutively with MEK (Denouel-Galy, 1997; Yu, 1998; Muller, 2000) and in a RAS-dependent manner with RAF and ERK/MAPK (Therrien, 1996; Xing, 1997; Cacace, 1999). These results led different groups to propose that mKSR1 might coordinate the assembly of the ERK/MAPK module (for review, see Morrison 2001). However, this hypothesis remains to be tested because the molecular relationship between mKSR1 and the three kinase components of the ERK/MAPK module is currently unknown. Furthermore, functional assays conducted by different groups have produced contradictory results: this results in a fragmented and somewhat controversial view of the role of KSR. A notable case focusses on the catalytic function of mKSR1. It has been reported that mKSR1 can phosphorylate and activate RAF in a TNFalpha- or EGF-dependent manner. Intriguingly, however, these results could not be reproduced by other laboratories. Instead, mKSR1 activity was reported to be independent of its putative catalytic function (Michaud, 1997; Stewart, 1999). Another discrepancy is the observation made by a number of groups that forced expression of mKSR1 strongly and specifically blocks signaling through the ERK/MAPK pathway (Denouel-Galy, 1997; Yu, 1998; Joneson, 1998; Sugimoto, 1998), whereas others showed that mKSR1 strongly cooperated with activated RAS to induce meiotic maturation of Xenopus oocytes (Therrien, 1996). As suggested by Cacace (1999), this contradiction might be attributable to differences in mKSR1 expression levels. Given that mKSR1 interacts with several components of the ERK/MAPK module, it is possible that these components are sequestered from each other when mKSR1 levels are in excess. In any event, it remains unclear whether the information obtained using mKSR1 truly reflects the function of KSR as it had been genetically defined. It is possible that the experimental systems used could simply not support normal mKSR1 function. For example, it is intriguing that in the Xenopus oocyte maturation assay, mKSR1 activity mainly depended on its cysteine-rich motif (CRM; Therrien, 1996; Michaud, 1997), whereas several loss-of-function mutations affecting other parts of the KSR protein (Kornfeld, 1995; Sundaram, 1995; Therrien, 1995) have been identified in Drosophila and C. elegans (Roy, 2002 and references therein).

A major problem hindering the elucidation of the role of KSR is the lack of an assay that faithfully recapitulates its function. A simple transfection protocol has now been used: a KSR-dependent functional assay has been reconstituted in a homologous system, that is, in Schneider (S2) cells using only Drosophila-derived components. KSR has shown to strongly promoted MEK phosphorylation by RAF in a RAS-dependent manner. Strikingly, KSR activity appears to depend mainly on its ability to associate independently with RAF and MEK, thereby allowing it to link the two kinases. Together, these findings suggest that KSR promotes signal propagation through the ERK/MAPK module by coordinating the assembly of a RAF/MEK complex (Roy, 2002).

KSR was originally recognized as an essential component of RAS-mediated signaling pathways in Drosophila and C. elegans (Kornfeld, 1995; Sundaram, 1995; Therrien, 1995). Although the characterization of mouse KSR1 linked it to the ERK module (Morrison 2001), its precise molecular function has not heretofore been elucidated. This study shows that Drosophila KSR is genuinely required for signal transmission through the MAPK pathway at a step upstream of MEK. In agreement with this, KSR directly participates in the phosphorylation of MEK by RAF and this event does not absolutely require the putative catalytic function of KSR. KSR associates independently with MEK and with RAF. These independent interactions lead to the formation of a stable RAF/MEK complex, thereby positioning the activator RAF in close proximity to its substrate MEK. Hence, KSR operationally behaves as a molecular scaffold that assembles the RAF/MEK functional pair. Given that MP1, a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade, apparently coordinates the formation of MEK/ERK complexes (Schaeffer, 1998), it will certainly be interesting to investigate whether KSR and MP1 function together to orchestrate the formation of a complete ERK/MAPK module (Roy, 2002).

Mouse KSR1 has been previously reported to associate constitutively with MEK and in a RAS-dependent manner with RAF. However, the physical consequences of these associations have not been explored further. This study shows that Drosophila KSR also interacts with MEK and RAF, but, in contrast to mKSR1, the Drosophila KSR/RAF association does not appear to depend on RAS activity. A reason for this difference might be that the association between mKSR1 and c-RAF does not resist the immunoprecipitation procedure. However, in the presence of activated RAS other contacts might have formed, which could stabilize the complex. RAS-induced oligomerization and/or other proteins might be involved in stabilizing the mKSR1/c-RAF complex. Regardless of the exact mechanism, these results clearly show that the associations between KSR and RAF and between KSR and MEK are mediated by independent parts of KSR and result in the association of RAF and MEK. Given that these data suggest that RAS is dispensable for these interactions, this implies that an inactive KSR/MEK/RAF ternary complex might exist in nonstimulated cells. Interestingly, STE5 has also been shown to assemble a three-kinase MAPK complex prior to signal transduction (Choi, 1994). This might be a general mechanism used by scaffolding proteins to allow highly efficient switch-like signal transmission. Consistent with this possibility, it was found that endogenous KSR, which apparently represents <1% of endogenous RAF and MEK protein levels, is predominantly associated with RAF and MEK in S2 cells (Roy, 2002).

The CA1 domain, a conserved region of KSR of ~40 amino acids at the N terminus, appears to be involved in connecting the KSR/MEK complex to RAF. It is still unclear whether or not the interaction is direct. Although the mutation affecting the CA1 domain reduces the KSR/RAF association, it does not abrogate it. This suggests that either the mutation does not fully disrupt the interaction with RAF or that another region(s) of KSR makes contact with RAF. This is consistent with the fact that this mutation allows to some extent the formation of a RAF/MEK association, albeit not as effectively as wild-type KSR and that it genetically behaves as a weak loss-of-function mutation (Therrien, 1995). Alternatively, it is possible that the mutation does not affect RAF binding per se, but aberrantly localizes the mutant KSR protein. Although MEK might stabilize the RAF/KSR interaction, it does not primarily mediate the interaction. Indeed, dsMEK RNA does not perturb the KSR/RAF association, and the two mutants, KSR^D800A-D817A and KSR^C922Y, which no longer interacted with MEK, still associated with RAF (Roy, 2002).

Systematic mutagenesis and deletion mutants of KSR should allow the identification of additional regions, if any, involved in the formation of the RAF/MEK complex. Given that mKSR1, like the RAF isozymes, interacts with the 14-3-3 proteins, HSP90, and p55/CDC37 (Xing, 1997; Cacace, 1999; Stewart, 1999), it will be interesting to discover the contribution of these proteins in the KSR/RAF association and in the regulation of the KSR-dependent RAF/MEK complex (Roy, 2002).

Previous work in Drosophila has shown that ksr loss-of-function mutations suppresses activated RAS-mediated signaling, but does not alter activated RAF function (Therrien, 1995). These observations led to the proposal that KSR is required at a step between RAS and RAF or in a pathway that acts in parallel. However, the data were also consistent with the possibility that the activated RAF transgene was expressed to high levels, thereby bypassing the requirement for KSR function. The current results are consistent with the second interpretation. It was found that KSR promotes MEK phosphorylation when low amounts of the RAF construct are transfected, but that this effect declines upon increasing the amounts of transfected RAF. The genetic data also raise the possibility that KSR is involved in the RAS-dependent RAF activation mechanism. This latter process has been the subject of numerous studies, but remains largely enigmatic. A number of laboratories have tested the possibility that KSR directly phosphorylates RAF. Most of these attempts have failed to show catalytic function for KSR. The current data do not support the findings of a catalytic role for KSR, but may provide an explanation for the apparent contradictory evidence. Interestingly, there is a correlation between the ability of KSR to bind MEK and the appearance of a mobility shift in transfected RAF, which appears to be caused by phosphorylation. KSR^D800A-D817A and KSR^C922Y does not induce the mobility shift, whereas KSR^K705M does. This suggests that this event does not depend on the putative catalytic function of KSR, but, rather on its ability to interact with MEK. Moreover, the mobility shift observed for FKBP-RAFc appears to depend on its autocatalytic function because FKBP-RAFc^K498S does not display the mobility shift, even though it heterodimerizes with the FRB-KSRc/MEK complex upon rapamycin treatment. Together, these data suggest that RAF autophosphorylation is strongly stimulated when MEK is brought to RAF by KSR, and thus KSR might be involved indirectly in RAF activation, not by virtue of its catalytic function, but rather by the effect of recruiting MEK to RAF. Consistent with this, the only KSR-dependent phosphorylated residues in KSR that have been identified correspond to RAF autophosphorylation sites (Roy, 2002).

Surprisingly, the simple coexpression of KSR with MEK, RAF, and activated RAS, is sufficient to reconstitute a KSR-dependent assay. The reliability of the assay is supported by the observation that wild-type KSR behaves as a positive component of the pathway, as genetics predicted, and KSR mutations corresponding to previously identified ksr loss-of-function alleles are inert. The critical aspect of the assay is the simultaneous expression of RAF and MEK with KSR. When KSR is overexpressed in a cell line, RAF and MEK also need to be coexpressed to high enough levels to prevent their uncoupling on separate KSR molecules. A similar situation has been observed for JIP-1, which was first thought to be an inhibitor of the JNK pathway. The results thus provide an explanation as to why many laboratories found that KSR inhibits signaling through the ERK pathway when overexpressed in various cell lines. When expressed at very low levels, mKSR1 has been shown to accelerate RAS-dependent Xenopus oocyte meiotic maturation (Therrien, 1996; Cacace, 1999), a process that depends on activation of the ERK module. Intriguingly, mKSR1 activity in this system appears to be mediated mainly by the cysteine-rich motif (CRM; Therrien, 1996; Michaud, 1997). Although this region is probably required for normal KSR activity, it does not account for its entire function. Therefore, this assay probably recapitulates only partially the normal function of KSR. Biochemical analysis of the CRM has revealed that it is involved in targeting mKSR1 to the plasma membrane in a RAS-dependent manner (Michaud, 1997). Nonetheless, it is unclear why the CRM alone is capable of stimulating RAS-dependent Xenopus oocyte maturation. KSR^C398S-C401S is reproducibly less active than wild-type KSR in the S2 cell assay, thus confirming the functional relevance of the CRM. However, the effect is weak. This could be caused by the fact that the main participants were overexpressed, thereby making the system less dependent on signals normally concentrating the various components to the plasma membrane (Roy, 2002).

Intriguingly, besides the lysine to arginine change in subdomain II of the mammalian homologs, KSR proteins have a highly conserved kinase domain (Therrien, 1995). It is formally possible that this domain is enzymatically active, but the proper conditions and/or substrates to detect its activity have not been found. The observation that the two KSR^K705M mutants used in this study are slightly less active than their wild-type counterparts, even though they associate with MEK to the same extent as wild-type KSR, suggests that KSR might possess a catalytic function that is required in concert with its scaffolding property for full activity. Alternatively, it is possible that their lesser activity is due to structural changes in KSR that do not perturb the KSR-MEK interaction but affect the way KSR presents MEK to RAF. Stringent sequence conservation might thus be required to maintain a particular kinase domain conformation to allow highly specific and robust interaction with MEK for the sole purpose of presenting a particular portion of MEK to RAF. Given their structural relatedness and the good homology between their kinase domains, RAF and KSR probably evolved from a common ancestral kinase by gene duplication. One of the descendents of this hypothetical duplication event might have given rise to the three RAF kinase family members, which retained catalytic function, whereas the other descendent might have eventually led to the two KSR genes found in mammals that evolved as scaffolds specialized in bridging RAF and MEK proteins together. Although it is currently unclear whether the functional shift observed for the KSR kinase domain will also be observed in other uncharacterized kinases for a similar purpose, this certainly highlights the importance of showing the catalytic activity of a kinase or any other enzyme before assuming it performs an enzymatic step in a given process (Roy, 2002).

It is becoming increasingly clear that components for several signaling pathways are specifically organized by scaffolding proteins. However, very little is known about the way they operate. The genetic and molecular data gathered so far on Drosophila KSR are consistent with its involvement in signaling efficiency, that is, when KSR is nonfunctional, low amounts of signal reach MAPK. The chain of events is, however, not severed because activated RAF can rescue ksr loss-of-function phenotypes in Drosophila (Therrien, 1995). The results show a role for KSR as a molecular scaffold coordinating the RAF/MEK interaction. This is, however, one part of the signal propagation mechanism through the ERK/MAPK module. Once MEK is activated, it must relay the signal to MAPK. It will be interesting to determine whether KSR also participates in this process or whether another molecule, such as MP1, executes that step independently (Roy, 2002).

GENE STRUCTURE

cDNA clone length - 3697

Bases in 5' UTR - 211

Exons -

Bases in 3' UTR - 585

PROTEIN STRUCTURE

Amino Acids - 966

Structural Domains

KSR was originally identified in RAS-dependent genetic screens in Drosophila and C. elegans (Kornfeld, 1995; Sundaram, 1995; Therrien, 1995). Interestingly, KSR proteins are mostly related to RAF serine/threonine kinase family members (Therrien, 1995), but differ in at least three main aspects: (1) they do not contain the so-called RAS-binding domain found in RAF proteins; (2) they contain a conserved region of ~40 amino acids at their N terminus called Conserved Area 1 (CA1) that is unique to KSR family proteins; and (3) the mammalian homologs contain an arginine residue instead of an invariant lysine residue in kinase subdomain II that is thought to be critical for the phosphotransfer reaction. This peculiarity suggests that KSR proteins might be devoid of kinase activity (Roy, 2002).

date revised: 20 July 2002

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