An evolutionary role of Scaffolds - From Accuracy to Crosstalk Again serving as a prototypical model organism for the discovery of biological

concepts, scaffolding structures of signaling proteins have first been described in S.cerevisiae (concurrently, yet independently researched and reported in close succession by Chol et al.(1994) and Marcus et al.(1994)). Ste5 was observed to tether several components of the pheromone signaling pathway, namely the MAPK cascade comprised of Ste11 (MAPKKK), Ste7 (MAPKK) and Fus3 (MAPK). Since then, the motif has been shown to exist in many different pathways and organisms, respectively (Posas and Saito, 1997; Schaefferet al., 1998; Dickens et al., 1997; Witzel et al., 2012; Dhanasekaran et al., 2007).

Not only does this scaffolding motif appear often in cellular signaling, but it also has been observed to fulfill many different roles and functions. The ever growing number of identified scaffolding proteins and their usages has made our perception of cellular signaling far more complex than previously thought, much like the discovery of microRNAs has impacted the view on gene expression regulation (see e.g. Bartel, 2004, 2009, for reviews).⁵³ This has made the scaffolding motif a very interesting source for fundamental knowledge of signaling systems as well as a promising subject of investigation, especially

53Remark: We already noted that the analogy to an information theoretic channel has been applied to gene expression before (Tkačiket al., 2008b, 2009; Tkačik and Walczak, 2011, e.g.) and recently, the role of microRNAs has been included in such an approach as well (Finn and Searles, 2013; Zheng and Kwoh, 2006).

for the field of synthetic biology. Here we will briefly review some of the central roles that scaffolds have been associated with.

The most basic property of scaffold proteins (and their name-giving function as it was the first to be observed) is the assembly and co-localization of signaling molecules within a pathway. This already allows for several important implications: As reviewed in Goodet al. (2011), it leads to a spatial regulation and coordination of the pathway components (see also Mahanty et al., 1999).

It enables them to “steer” the assembled complex to certain locations in the cell (e.g. the cell membrane or other organelles) where signaling is then to be initiated. While this can be used as well as a “pre-assembly” to provide quicker reactions, scaffolds also regulate the concentrations that are experienced locally by the signaling components, as it brings them into close proximity of one another. This effectively increases reaction rates and efficiency of the pathways in question. Scaffolds in this way help to catalyze reaction cascades. Yet, this could also be achieved by increasing the affinity of binding between adjacent signaling layers.

The concentration of scaffolding proteins has another interesting effect on the regulation of signaling. As was investigated both theoretically and experimentally (Levchenko et al., 2000; Witzel et al., 2012; Chapman and Asthagiri, 2009), there exist optimal ranges of scaffold expression due to either a saturation (low concentrations lead to a bottleneck of signal transmission) or a combinatorial effect (high concentrations allow the assembly of incomplete and thus non-responsive cascades). Thus, again in analogy to microRNAs, regulating the expression of scaffolds allows the organism to regulate and fine-tune its responsiveness to certain stresses.

While the aforementioned functions are of a passive nature, scaffolds have also been shown to actively take part in the signaling process in addition to that. Through their tethering function, they increase the specificity and also sequester the activation of signaling molecules (Good et al., 2009). In that particular case, the pathway would only be activating the MAPK Fus3 weakly without the scaffold protein Ste5, making it essential for the transmission process itself. This is similar to the enhancement of reactions as described

in the previous paragraph, yet going even one step further (Sabbagh et al., 2001): Kss1, a MAPK involved in pheromone signaling and invasive growth, is activated by the same MAPKK as Fus3, namely Ste7. It can take the place of Fus3 in the scaffolding complex of Ste5 (for example when Fus3 is knocked out), but does not need the scaffold itself for its activating function. This separates the two scenarios and makes the scaffold not only an enhancer and catalyst, but an integral part of the signaling structure itself. Other active functions, facilitating allosteric activation of Fus3 (Bhattacharyyaet al., 2006b) and the use of conformational changes to enable signaling (Sette et al., 2000; Zalatan et al., 2012), have been shown for the scaffold Ste5.

While this is already an impressive number of roles that scaffolds have been associated with, the primary one is thought to be the prevention of crosstalk between pathways that make use of the same signaling proteins (Garrington and Johnson, 1999; Whitmarsh and Davis, 1998; Elion, 1998). As with many other prototypical motifs, this function has first been identified in S.cerevisiae.

We introduced the mechanism in chapter 4, where the yeast pheromone path-way and the HOG pathpath-way both make use of Ste11, Ste20 and Ste50, yet still are capable of signaling distinct inputs to distinct outputs. Specificity is achieved by the use of the scaffold proteins Ste5 and Pbs2⁵⁴ that control the flow of information. This management of crosstalk is one of the most important and also most exciting functions of scaffolds as it hands interesting possibilities to synthetic biology (Lai et al., 2015). Re-wiring pathways and thus re-routing information is an interesting concept and possibly evolved many further functionalities (Bhattacharyyaet al., 2006a; Good et al., 2011), yet this would only make sense as a secondary development for an already established system. Additionally, as with the enhancement of signaling efficiency, such specificity could also be achieved through developing separate pathway species with high recognition affinity patterns.

With the many functions and the high conservation of the scaffolding motif in many organisms, the question arises as to what made the re-utilization of

54With both fulfilling additional roles (Pbs2 as a MAPKK (Posas and Saito, 1997) and Ste5 as described in this section).

this structure favorable. As far as their usage might be spread, the (sequence) similarity between different scaffolds is very limited and thus, it seems unlikely that their origin is of particularly close relation. We argue that the benefits of scaffolds are especially based on the structure and its properties, making it appear in many different contexts. Functions like inter-pathway insulation, the branching of information transmission routes and higher regulation patterns are likely to be developed as improvements and extensions of an already estab-lished and favorable design. We found that a potential reason for evolutionary selection can be found by investigating the signal processing properties of prototypical scaffolded signal transmission. In the following sections, we show that scaffolding limits inherent noise and benefits the fidelity that signaling is capable of. In addition, it shapes dose-response alignment in a near-linear way.

An important observation is that, in contrast to many other functionalities reviewed, the here proposed enhancements of insulated signaling by scaffold proteins are not contradictory to other roles that scaffolds have been shown to play. On the contrary, they propose a strong incentive for reusing and refining this structure and thus evolving into the mechanisms that we find nowadays in nature.

5.2. A model comparison for “mixed” and “insulated” information