Here, we study the network of MAPK pathways that transduce different signals and regulate stress response and growth. We find that MAPK proteins are the pivotal nodes that allow their pathways to acquire new components and connections. Through a combination of sequence analysis and experiments, we show that we can continuously change the topology of the MAPK network and its signal processing capabilities by redesigning the MAPK proteins.

For our experiments, we chose two of the five MAPK pathways in S. cerevisiae⁴ that are involved in mating and responding to high osmolarity (Fig. 1a). These two pathways involve the MAPK proteins Fus3 and Hog1. Since another MAPK Kss1 can partially substitute for Fus3 (ref. ⁵), fus3Δ kss1Δ cells are sterile and hog1Δ cells die after hyper-osmolar shock⁶.

To study the evolution of the MAPK pathways, we identified orthologues of their component proteins in fifteen yeast, six animal and one plant species (Figs 1b, ​,2;2; Supplementary Information Fig. S1a). From the fission yeast Schizosaccharomyces pombe⁷ to the higher eukaryotes, the number of MAPKs has increased from three to at least fifteen. As the MAPK family grows, many MAPK interaction partners appear de novo in different lineages. Except for Pbs2, orthologues of all the interaction partners of Fus3 and Hog1 (Fig. 1a) are found only in the yeast lineage, indicating that the specificities of these MAPKs for their partners developed uniquely in yeast (Figs 1b, ​,2).2). This includes scaffolding proteins that nucleate these multi-protein complexes aiding MAPK specificities with their appropriate upstream and downstream partners^8,9. Closely related MAPK paralogues and orthologues show distinct specificities, indicating that new interactions can be acquired with little sequence divergence. For example, in S. cerevisiae the paralogues Kss1 and Fus3 can be activated by Ste7, but only Fus3 can activate Far1 causing cell-cycle arrest¹⁰. Strikingly, we also found that orthologues had switched their specificity from one MAPK in the yeast to another MAPK in the animals (Fig. 2d), providing a clear example of network topology that has changed during evolution.

Despite this plasticity, the MAPKs have been more invariant through evolution than other members of their pathways (Fig. 1b). Which molecular properties have allowed the MAPKs to remain so conserved, while still having a pivotal role in allowing their pathways to rewire and acquire new components during evolution?

The MAPKs are small, compact globular proteins. As with their linear protein sequences, their three-dimensional structures are very similar to one another (Fig. 3). To understand how these proteins achieve their specificities, we first reviewed previous biochemical^11,12, structural^8,13,14 and peptide^10,15 analyses of the roles of certain residues in Fus3 and Hog1 (Fig. 3c, d; Supplementary Information, Section 2). We found that, other than Asp 112 and His 113 in Fus3, these residues cannot function alone as specificity determinants because they are conserved in other MAPK paralogues and their orthologues in other species.

We extracted functional information on residues using a multiple sequence alignment consisting of orthologues of four S. cerevisiae MAPKs (16 Kss1, 15 Fus3, 15 Hog1 and 15 Slt2). The variable residues were almost exclusively on the surface of the proteins (Fig. 3a). Of these residues, we computationally identified a subset putatively responsible for the differences in specificities of Fus3 and Hog1 (Supplementary Information, Figs S2, S3a and Methods). Our sequence analysis suggests two characteristics of MAPKs: first, they are structurally robust to changes in most of their surface residues; second, distinct groups of surface residues stand out on each MAPK as being responsible for specific interactions. The latter finding was unexpected as it may be thought that structurally similar proteins of the same family will use the same surface regions to interact with their corresponding upstream or downstream partners. For instance, we found a group of residues (Pro 80, Phe 83, Glu 84 and Trp 348) that is conserved in Fus3, but absent in Hog1 (Fig. 3b). Other residue positions are well conserved in Hog1 but show great variability in Fus3, signifying neutral drift. Thus distinct patches may be important for the specific interactions of each MAPK with other members of its pathway (Supplementary Information, Figs S2, S3a). If MAPK plasticity stemmed from the underlying flexibility of these patches, then synthetic proteins containing different combinations of these patches should be able to deform the signalling pathways. A chimaera has been generated¹⁶ that, when expressed in mammalian cell lines, directs a stress signal into mitotic output (Supplementary Information, Section 2), suggesting that such deformations may indeed be possible.

To investigate this possibility, we constructed proteins that contained residues from Fus3 and Hog1 in various combinations. We divided both Fus3 and Hog1 into six segments (A, B, C, D, E and F for Fus3 and a, b, c, d, e and f for Hog1) linked by regions conserved in all the MAPKs (blue, Fig. 3c, d), and joined the genes coding for the 64 possible proteins composed of these segments (Supplementary Information, Methods Fig. S2). This achieved a combinatorial redistribution of the surface patches, while also ensuring that internal contacts along the sequence were preserved. The genes were driven by a FUS3 promoter. To test for pheromone output in response to either pheromone or osmolyte (sorbitol) exposure, the 64 hybrids on both low-copy (showing an expression within threefold of the native chromosomal gene; data not shown) and high-copy (showing an expression within eightfold of the native chromosomal gene; data not shown) plasmids were transformed into a haploid fus3Δ kss1Δ strain (Supplementary Information, Table S1). A fluorescent (GFP) reporter was placed under control of the native FUS1 promoter, a faithful reporter of the pheromone pathway (Supplementary Information, Fig. S3b, c). We measured the fold change of fluorescent protein after 2 h of pheromone or sorbitol exposure (Fig. 4; Supplementary Information, Fig. S5a). The strains expressing the hybrids were further assayed for their ability to mate (Fig. 4; Supplementary Information, Fig. S4a and Methods) and arrest their growth on pheromone exposure (Supplementary Table 2). To test for osmolar output in response to either input, hybrids on both low- and high-copy plasmids were transformed into a haploid hog1Δ strain with a fluorescent reporter placed under the native STL1 promoter, a faithful reporter of the osmolar pathway (Supplementary Information, Fig. S3b, d). We measured the fold change of fluorescent protein under the STL1 promoter after 2 h of pheromone or sorbitol exposure (Fig. 4; Supplementary Information, Fig. S5b). The strains with the hybrids were assayed for their ability to grow under high osmolarity conditions (Fig. 4; Supplementary Information, Fig. S4b, c). Hybrids were also assayed in a fus3Δ kss1Δ hog1Δ strain with distinct fluorescent reporters (YFP and CFP) under the FUS1 and STL1 promoters. Without the hybrids, this strain does not show any response to pheromone or sorbitol (Fig. S3e). We measured the time-series of the response for hybrids on low-copy plasmids (Supplementary Information, Figs S6, S7a), and when integrated under the FUS3 promoter in the chromosome (Supplementary Information, Fig. S7b).

Several hybrids show FUS1 promoter (pFUS1) activity in response to either stimulus (Fig. 4a). Another nine (ABcdeF, ABCDeF, aBCdEF, abCDeF, AbCDEF, abCDEf, abCdEF, aBcdEF, AbcDEF) do so when expressed from high-copy plasmids (Supplementary Information, Figs S5a, S8). All low-copy hybrids that showed pFUS1 activity upon pheromone exposure also rescued the cells ability to mate (Fig. 4a). Unstimulated cells carrying ABcdEF and ABcdeF had constitutively active pFUS1 showing about 50% and 20%, respectively, of the fluorescence shown by pheromone stimulated wild-type cells. (Supplementary Information, Fig. S8 and Section 3). This is noteworthy, as producing phospho-mimicking mutations in the activation loop does not constitutively activate MAPKs as it does for the activating MAPK Kinases (MAPKK)^{17, 18, 19}.

One hybrid expressed from a low-copy plasmid, and several hybrids expressed from a high-copy plasmid (Fig. 4a; Supplementary Information, Fig. S5a), also showed pFUS1 activity in response to sorbitol, in fus3Δ kss1Δ cells. Such cross-wiring could occur by either a direct or an indirect mechanism. A direct mechanism would involve the hybrid MAPK being directly activated by Pbs2 and in turn activating Ste12. There are two possible indirect mechanisms: the first is where the hybrid inhibits native Hog1 or Pbs2 while also performing the function of Fus3. This is because strains in which the osmolar pathway is interrupted at the level of Hog1 or Pbs2 (either by deletion or inhibition of these proteins) promiscuously channel the hyper-osmolar signal into a pheromone output (Supplementary Information, Fig. S3d). This promiscuous channelling happens upstream of the MAPK and is abolished by deleting Ste7 (ref. ²⁰). The second indirect mechanism is one in which Hog1 fails to inhibit the hybrid, allowing the osmolar signal to leak through the pheromone pathway, again through Ste7. To discriminate between the direct and indirect mechanisms of cross-wiring, we assayed the hybrid MAPKs in a ste7Δ hog1Δ strain. Without the hybrids, a sorbitol stimulus does not get cross-wired into pheromone output in this strain (Information, Fig. S9a). Three hybrids (ABcdEF, ABcdeF and aBcdeF) showed pFUS1 activity on hyper-osmolar shock in the ste7Δ hog1Δ strain, suggesting that they were activated directly by Pbs2 (Fig. 4a; Supplementary Information, Fig. S9b). For the remaining hybrids, cross-activation was abolished in the ste7Δ hog1Δ strain, implying that the cross-wiring occurred indirectly (Supplementary Information, Fig. S9c).

When expressed from a low-copy plasmid in fus3Δ kss1Δ hog1Δ cells, only the hybrid aBcdeF is capable of cross-wiring a sorbitol input into a pheromone output. Cells carrying aBcdeF are insensitive to pheromone (Fig. 5a, b), implying that this hybrid is directly activated by Pbs2 but not by Ste7. The cross-wiring persisted in the ste7Δ hog1Δ strain. The cross-wiring only occurred in the absence of native HOG1 when aBcdeF was expressed from a low-copy plasmid. Over-expression from a high-copy plasmid rescued this cross-talk in the presence of native HOG1, suggesting either competitive binding to Pbs2 between aBcdeF and Hog1 or direct inhibition of aBcdeF by Hog1.

Several hybrids, when expressed from a low-copy plasmid, show STL1 promoter (pSTL1) activity in response to either input (Fig. 4b; Supplementary Information, Fig. S9d). The hybrid AbCdEf, which faithfully transduces the sorbitol signal almost as efficiently as wild-type Hog1, also cross-wires the pathways by showing pSTL1 activity in response to pheromone exposure (Fig. 5a, b). Another hybrid, aBcdEF, achieves similar cross-wiring but is insensitive to sorbitol itself (Supplementary Information, Fig. S9d). The cross-wiring by aBcdEF occurred only in the absence of native Fus3 and Kss1, suggesting a competitive binding to Ste7 between aBcdEF and Fus3 or Kss1. All low-copy hybrids that showed pSTL1 activity on sorbitol exposure also rescued the ability of the cells to grow under high osmolar conditions (Fig. 4b). Interestingly, certain hybrids rescued the growth of the cells on a high osmolar medium (Supplementary Information, Fig. S4b, c), but did not mediate reporter activity in the cell (Fig. 4b; Supplementary Information, Fig. S5b).

Besides demonstrating the flexibility of MAPKs, our results allow us to draw some conclusions about the modularity and specificity of these proteins.

We find that the MAPK proteins are modular. One in three hybrids were functional, some with as many of their distinguishing residues taken from Fus3 as from Hog1. This strongly suggests that the common conserved residues at the core are sufficient for folding, and the variable exterior residues, where most of the evolution has occurred, control specificity. However, despite their having similar structures and symmetrical roles in their respective pathways, the patches of surface residues used by the paralogues Fus3 and Hog1 to interact with their up and downstream factors seem substantially different. This is highlighted by the hybrids (Fig. 5a, b, c) that are able to interact with both upstream partners, such as ABcdEF (which is one of three hybrids in which sorbitol evoked a pheromone response through Pbs2), or both downstream partners (aBcdeF). The hybrid aBcdeF yields both an osmolar and pheromone response to an osmolar input, but is non-responsive to pheromone (Fig. 5a), implying that it can be activated by Pbs2 alone but can activate both Ste12 and Hot1. As every segment is either from Fus3 or Hog1, the regions on Fus3 and Hog1 that interact with Ste12 and Hot1 must be considerably non-overlapping. Not only do we find that these MAPKs show a modular design, we also note that this design is implemented differently in the two MAPKs in spite of their great structural similarity. Thus, conformational change, which commonly underlies enzyme promiscuity in other synthetic proteins²¹, is unlikely to be the mechanism by which our hybrid kinases achieved their various cross-wirings and specificities.

We can uncover how MAPK proteins achieve specificity. Our results indicate that the segment BEF is required in conjunction with the segments A or D for pheromone input to invoke pheromone output (Fig. 4a). However, as aBcdeF activates the pheromone pathway only on osmolar input, and not on pheromone input, it appears that BF alone is sufficient for pheromone output and that E is important for pheromone input. The segment BF contains all the residues associated with the ‘docking domain’ (Fig. 3c; Supplementary Information, Section 2). These were identified and studied mainly in the context of Fus3 binding to Ste7 and to phosphatases¹⁰. However, that BF appears sufficient for pheromone output suggests it is more important invivo for the Fus3–Ste12 interaction, whereas E is more important for the Fus3–Ste7 interaction. The patch of residues (Pro 80, Phe 83, Glu 84 and Trp 348) identified by our sequence analysis to be unique to Fus3 is contained in BF. The hybrid aBCDEF transduces the pheromone signal to induce high pFUS1 activity but fails to mediate cell-cycle arrest (Supplementary Table 2). Consistently, the strains carrying aBCDEF have a low mating efficiency (Supplementary Information, Fig. S4a). This implicates segment A in Fus3–Far1 interaction. Segment d is necessary for transducing a high osmolar signal into any output (Fig. 4ab). In Fus3, D has a disordered structure and most of its residues undergo neutral drift. This again illustrates how members of this family have specialized different residue patches on their surfaces to achieve analogous specificities rather than refining the residues on some common catalytic loop²².

Our data show that these proteins can find new specificities with relatively few changes in their sequence (as few as seven point-mutations). They can retain their original function while acquiring new interaction partners. This might be why they have increased in number, having repeatedly found new functions after duplication (Supplementary Information, Fig. S10). Indeed our results suggest promiscuous intermediates through which duplicated enzymes could evolve, en route to their new specificities²². We have also gained insights into the specificity determinants of extant proteins. Exploration building on our hybrids can complement traditional biochemical techniques, which are challenged by the transient nature of kinase–substrate interactions. The implication for synthetic design is that pivotal proteins such as these may serve as the best templates, or scaffolds, with which to design new specificities to create new connections in existing pathways.