Living cells receive vast amounts of environmental information, and a central question is how the cell’s system of signal transduction proteins is able to specifically process this information. This problem is particularly acute given that many closely related molecules (e.g. kinases, phosphatases, etc.) are involved in diverse, functionally distinct signaling pathways. An emerging paradigm is that, in many cases, signaling pathways are organized by scaffold proteins. Scaffolds are proteins that interact with multiple members of a pathway and are thought to function as “wiring” elements that by tethering pathway components into complexes and localizing them to specific sites in the cell, direct the flow of signaling information. Scaffolds are proposed to both enhance interactions between the correct signaling proteins and to insulate them from interactions with competing proteins (Bhattacharyya et al., 2006a
; Bhattacharyya et al., 2006b
; Burack et al., 2002
; Burack and Shaw, 2000
One of the first identified examples of a signaling scaffold is the Ste5 protein from Saccharomyces cerevisiae
, which plays an essential role in signal transmission through the yeast mating pathway. When yeast are stimulated by mating pheromone from the opposite mating type, signal is transmitted from the mating receptor (Ste2) via a heterotrimeric G-protein (Gpa1, Ste4 and Ste18) to a mitogen-activated protein (MAP) kinase cascade. MAP kinase cascades are composed of three kinases that successively phosphorylate and activate one another: signal passes from a MAP kinase kinase kinase (MAPKKK) to a MAP kinase kinase (MAPKK) and finally to a MAP kinase (MAPK). In the mating pathway, signal is transmitted from the MAPKKK Ste11 to the MAPKK Ste7 to the MAPK Fus3. The Ste5 scaffold, although it has no catalytic domains (eg. kinase domains), is required for the mating response. Ste5 was initially identified as a scaffold protein because, by yeast two-hybrid assays, it was shown to have binding sites for all three MAPK cascade members (Ste11, Ste7, and Fus3) (Choi et al., 1994
) and the Gβ protein, Ste4 (Whiteway et al., 1995
). Interaction with Ste4 localizes the Ste5 complex to the membrane upon stimulation, allowing Ste11 to be activated by a membrane-localized (PAK) kinase, Ste20. Additionally, interaction of Ste5 with the kinases in the cascade is thought to promote their successive phosphorylation.
The need for robust mechanisms for controlling signaling specificity is particularly important for the mating pathway because of the potential for cross-signaling with other related MAPK pathways that use overlapping signaling components. For example, the filamentous growth pathway, which is activated by nitrogen starvation, requires kinases shared with the mating pathway: the MAPKKK Ste11 and the MAPKK Ste7 (although it does not require the scaffold Ste5). During the mating respose, signaling to Ste7 is primarily transmitted to the MAPK Fus3, while in the filamentation pathway signaling is transmitted to the MAPK Kss1. Here we focus on the critical question of how activated Ste7 chooses between the two MAP kinases, Fus3 and Kss1, which are 55% identical (). Why does Ste7 that is activated by pheromone stimulation phosphorylate Fus3, whereas Ste7 that is activated by nitrogen starvation phosphorylate only Kss1? What is the role of the Ste5 scaffold in this specificity choice?
Ste5 scaffold protein is required for mating pathway signaling
Despite the importance of Ste5 as a canonical example of a scaffold protein, little is understood about the biochemical mechanisms that scaffolds use to regulate MAPK signaling specificity. The simplest model for how a scaffold might promote phosphorylation of one substrate versus another is through tethering – by increasing the proximity and effective concentration of components in the scaffold complex. Tethering, appears to be important for certain key aspects of Ste5 function: mutation of the binding sites for the Ste11 and Ste7 kinases disrupts signal transmission, while re-recruitment of these proteins to the Ste5 complex via heterologous engineered protein-protein interactions or covalent fusion can partially rescue signaling (Harris et al., 2001
; Park et al., 2003
The mechanism by which Ste5 directs signaling from the MAPKK Ste7 to the MAPK Fus3, however, is far less clear. Is the scaffold needed to colocalize these kinases or does it play some other role? Previous work identified and characterized a binding site for Fus3 within Ste5. This ~30 amino acid peptide (288–316) binds Fus3 with an affinity of 1µM, and it stimulates partial Fus3 autophosphorylation (it promotes one of two phosphorylation events required for Fus3 activation) (Bhattacharyya et al., 2006a
). Surprisingly, however, mutation of this Fus3 binding site does not block mating but actually increases mating output (as measured by transcription), suggesting that this site plays more of a tuning role, modulating signaling dynamics (Bhattacharyya et al., 2006a
). Nonetheless, the scaffold as a whole is still absolutely required for signaling to Fus3. Thus, it appears that there may be another site in Ste5 that controls Fus3 activation and that the scaffold may be playing a more active or catalytic role in controlling signal transmission to this MAP kinase.
Here we have purified components of the mating and filamentous growth MAP kinase pathways (Ste7, Fus3, Kss1, and Ste5) in order to understand the role of scaffolds in specifying in MAPKK➔MAPK signal transmission. We find that Fus3 is intrinsically a poor substrate for activated Ste7, while Kss1 is intrinsically a very good substrate. A ~200 residue segment of Ste5, however, is sufficient to permit Ste7 phosphorylation of Fus3 but has no effect on Kss1 phosphorylation. This Ste5 fragment is distinct from the previously identified Fus3 binding site, and crystallographic studies show that it is an independently folding domain which we refer to as Ste5-ms (minimal scaffold). The Ste5-ms domain binds tightly to Ste7, but only very weakly to Fus3. However, mutational and kinetic studies show that the Ste5-ms fragment can catalytically unlock the Fus3 MAPK so that it is now a good substrate for Ste7. This domain specifically increases the kcat for the Ste7➔ Fus3 reaction by ~5000-fold, while it has no effect on the kcat or KM of the Ste7➔ Kss1 reaction. Fus3 appears to have evolved a structure that is “locked” to prevent stray activation by isolated forms of Ste7 (generated by non-mating inputs). Phosphorylation of Fus3 occurs only in the combined presence of Ste7 and Ste5, and this mechanism explains why Fus3 is only activated by mating input.