KSR is an intriguing component of Ras-dependent signaling pathways. It is a molecule with all of the characteristics of a protein kinase, yet its physiological substrate and its role in signal transduction remain unclear. Therefore, to further elucidate KSR function, we initiated experiments investigating the effect of phosphorylation on KSR activity. Genetic and biochemical studies have indicated that KSR acts downstream of Ras (8
). Therefore, to identify both constitutive and regulatory sites of phosphorylation, we examined the phosphorylation state of KSR in the presence and absence of activated RasV12
and in the presence and absence of growth factor treatment. By protein sequencing data and by loss of the corresponding phosphopeptide after site-directed mutagenesis, we have identified five in vivo phosphorylation sites of KSR. Two constitutive sites of phosphorylation were located in the amino-terminal regulatory domain of KSR and were determined to be Ser297 and Ser392. Analysis of the sequences surrounding these residues indicates that they closely resemble the phosphorylation-dependent binding motif that has been described for the 14-3-3 family of proteins (32
). Indeed, by mutational analysis, our results demonstrate that both of these sites are involved in 14-3-3 binding. Mutation of either residue alone reduced the level of 14-3-3 binding, whereas mutation of both sites in concert completely eliminated the interaction of 14-3-3 with KSR. Although another putative 14-3-3 binding motif is located in the catalytic domain of KSR, we have not found this site (Ser838) to be phosphorylated in vivo, nor have we detected an interaction of 14-3-3 with the KSR catalytic domain, either by 35
S-labeling experiments or by mutational studies.
Determining the role of 14-3-3 binding to cellular proteins has been a complicated matter, and ascertaining the significance of the 14-3-3–KSR interaction appears to be no exception. 14-3-3 represents a highly conserved family of proteins comprised of seven distinct mammalian isoforms that can form homo- or heterodimers (1
). Because 14-3-3 is a specific phosphoserine-binding protein that interacts with a diverse group of proteins, including Raf-1, cdc25, BAD, Bcr, IRS-1, and phosphatidylinositol 3-kinase, it has been implicated in a wide variety of biological processes (1
). For example, 14-3-3 binding to cdc25 inhibits the phosphatase activity of cdc25, thereby preventing entry of cells into mitosis (34
), whereas 14-3-3 binding to BAD prevents the heterodimerization of BAD and BCL-XL
, thus protecting cells from undergoing apoptosis (49
). The role of 14-3-3 binding to Raf-1 is more complex and appears to be twofold: first, in stabilizing the inactive Raf-1 conformation in quiescent cells, and subsequently, in facilitating Raf-1 activation in response to signaling events (30
). Because Ser297 and Ser392 are the major sites of KSR phosphorylated in unstimulated cells, an attractive hypothesis is that in a manner analogous to Raf-1 binding, the binding to KSR may help to maintain KSR in an inactive conformation. The two phosphorylation sites mediating 14-3-3 binding are located on either side of the KSR cysteine-rich CA3 domain (39
). Previous studies from our laboratory have indicated that this domain is critical for KSR function (28
). We have found that the CA3 domain is necessary and sufficient for the cooperative effect that KSR exerts on Ras signaling in Xenopus
oocytes. Further, this domain is responsible for the translocation of KSR to the membrane in the presence of activated Ras. Thus, it is interesting to speculate that binding of a 14-3-3 dimer to the Ser297 and Ser392 sites may serve to sequester the CA3 domain, precluding its interaction with a protein or lipid second messenger that may contribute to KSR activation. If this hypothesis is correct, then exposure of this domain by disrupting 14-3-3 binding would be required for KSR to function. Consistent with this model, phosphorylation of these sites is reduced in the presence of activated Ras, suggesting a Ras-induced dephosphorylation and disruption of 14-3-3 binding at these sites. Alternatively, other models could be invoked in which 14-3-3 binding facilitates the interaction of KSR with other signaling molecules. Clearly, determining the exact role of 14-3-3 binding to KSR requires further investigation.
In the presence of activated Ras, we found that KSR was phosphorylated on Ser297 and Ser392 (albeit at reduced levels), as well as on three additional sites. These sites were identified as Thr260, Thr274, and Ser443, all of which fit the consensus motif for phosphorylation by MAPK (Px[T/S]P [3
]). Thr260 is located immediately upstream of the proline-rich CA2 domain of KSR, Thr274 is contained within the CA2 domain, and Ser443 is found within the serine/threonine-rich CA4 domain (39
). Thr260, Thr274, and Ser443 are phosphorylated in a Ras-inducible manner, and our data strongly indicate that the phosphorylation of these sites is mediated by MAPK. First, we found that blocking MAPK activation by treating cells with the MEK inhibitor PD98059 prevents phosphorylation of Thr260, Thr274, and Ser443 in PDGF-treated cells. Second, KSR was shown to be a substrate of purified MAPK in vitro, and the sites phosphorylated by MAPK in vitro include Thr260, Thr274, and Ser443. Third, we have found that MAPK associates with KSR in a Ras-dependent and growth factor-inducible manner and that the KSR-MAPK interaction correlates with the activation state of MAPK following growth factor treatment. Finally, the phosphorylation and activation of MAPK were shown to be required for the interaction between KSR and MAPK. From these observations, we conclude that activated MAPK kinase associates with KSR and phosphorylates KSR on the Thr260, Thr274, and Ser443 sites. The functional consequence of these phosphorylation events, however, remains unknown, since mutation of the identified phosphorylation sites had no effect on the ability of KSR to augment Ras signaling in the Xenopus
oocyte meiotic maturation assay. Nevertheless, phosphorylation at these sites may be important for other KSR functions. In particular, phosphorylation may play a critical role in modulating the enzymatic activity of KSR; however, such analysis awaits the identification of the KSR substrate.
Interestingly, a critical observation that was revealed during the course of this study was the pronounced effect that the level of protein expression had on the biological function of full-length KSR. Using the Xenopus
oocyte meiotic maturation assay, we found that although full-length KSR augmented Ras-mediated signaling when expressed at low levels, it blocked Ras signaling and MAPK activation when expressed at high levels. Likewise, even though genetic analysis has identified DmKSR as a positive effector of Ras-dependent signaling, overexpression of a full-length Dm-KSR protein blocked R7 photoreceptor formation in the Drosophila
eye. Thus, the interpretation of the biological function of full-length KSR can vary greatly depending on the level of protein expressed. This finding is likely to explain the recent reports that overexpression of full-length KSR inhibits Ras signaling by blocking MEK1 and MAPK activation in mammalian cells (8
). Furthermore, this finding indicates that maintaining KSR protein expression at low or near-physiological levels is critical for investigating the biological function of KSR a positive effector of Ras-dependent signaling. As previously observed, we found that the cooperative activity of KSR is mediated by the amino-terminal domain and that the dominant-negative activity is located in the carboxy-terminal catalytic domain (40
). Furthermore, the effects of the isolated domains on Ras signaling did not vary with the levels of protein expressed, indicating that KSR contains two separable functional domains that, when taken out of the context of the full-length molecule, exert both positive and negative effects on Ras signaling.
In regard to the function of KSR, the results presented here are consistent with our previous model that KSR may act, in part, as a scaffolding protein to propagate signal transmission within the MAPK module (40
). The idea that a signaling protein may provide a scaffolding function within the MAPK module is not unprecedented. For example, the components that control the Saccharomyces cerevisiae
pheromone response and osmoregulatory pathway are coordinated by the yeast scaffolding proteins Ste5 and Pbs2 (5
). Although KSR bears no structural resemblance to Ste5, both KSR and Ste5 appear to bind MEKK (Raf-1 or Ste11), MEK (MEK1 or Ste7), and MAPK (MAPK or Fus3/Kss1) and function to facilitate signaling within this kinase module (5
). Another similarity between KSR and Ste5 is that the yeast MAPK, Fus3, phosphorylates Ste5 (19
). This phosphorylation event has been proposed to inhibit Ste5 function, thus promoting the disruption of the signaling complex. Pbs2, in addition to functioning as a MAPKK, serves as a scaffolding protein by interacting via its proline-rich region with the SH3 domain of the transmembrane osmosensor Sho1. Pbs2 is then activated by the binding of MAPKKK, Ste11, thereby propagating the signal to the MAPK, Hog1 (35
). Although the precise mechanisms by which KSR functions may be different from those of Ste5 or Pbs2, it is clear that KSR is an integral component of an active MAPK signaling complex. Further identification of the components within this complex may reveal the KSR substrate and provide additional clues as to the exact role of KSR in Ras-mediated signal transduction.