This study probes how scaffold proteins and subcellular compartmentalization influence the input-output behavior of a common signaling module, the MAPK cascade. Our results, along with those from previous studies [2
], suggest that the MAPK cascade module is inherently ultrasensitive. In turn, specific mechanisms can either enhance or counteract this default tendency in order to generate switch-like or graded responses, respectively (). A general model that can explain the graded behavior of the yeast mating pathway relates to the effect of Ste5 membrane recruitment on signal propagation through the kinase cascade [25
]. Specifically, because the active form of the MAPKKK Ste11 on its own is relatively inefficient at signaling, it must accumulate to a high threshold level before any significant output occurs. But membrane recruitment of Ste5 enhances propagation of signal from active Ste11 through the kinase cascade (, top), thus allowing low levels of Ste11 activity to produce some output signal. This broadens the range of input levels that can yield a measurable output, making signaling less ultrasensitive and more graded (, bottom).
Interpretive framework and specific models
While it is reasonable to assume that the level of active Ste11 increases roughly in linear proportion to the dose of pheromone stimulus, this appears neither necessary nor sufficient to ensure a graded output. It is not sufficient because cells in which the level of constitutively-active Ste11 is gradually increased (e.g., PGAL1-STE11-Asp3
cells treated with β-estradiol) show an ultrasensitive response. It is not necessary because cells expressing a constant, low level of constitutively-active Ste11 still show a graded response to pheromone (e.g., ste20
cells treated with α factor). Thus, an important determinant of graded behavior is the manner in which pheromone and Ste5 enhance the steps subsequent to Ste11 activation. The next phosphorylation reaction (Ste11 → Ste7) is likely to be the remaining rate-limiting step, because strong signaling is achievable by simply overproducing constitutively-active Ste11 (i.e., unlike upstream components such as Ste20 and Cdc42; see [25
]). Furthermore, signaling methods that show graded behavior require domains in Ste5 that promote both membrane recruitment and binding to Ste11 and Ste7 (see Figure S2
). Thus, it is likely that Ste5 membrane recruitment directly (and separately) promotes both the Ste20 → Ste11 and Ste11 → Ste7 steps. It is less clear whether the final Ste7 → Fus3 step is also stimulated by Ste5 membrane recruitment, as Fus3 activation in vivo is highly dependent on Ste5 [36
] and yet the Fus3 binding site in Ste5 is not required ([30
] and Figure S2
); hence, the precise role of Ste5 in Fus3 activation remains mysterious.
The surprising finding that Ste5 molecules in the cytoplasm cannot reduce ultrasensitivity may indicate that cytoplasmic Ste5 is incapable of promoting processive phosphorylation, contrary to most prior expectations [16
]. Why would this be so? A simple explanation would be that the common view of scaffolds—in which they are fully occupied with kinases that efficiently interact with each other while bound to a single scaffold molecule—is incorrect. Instead, it may be the case that most Ste5 molecules in the cytoplasm are incompletely occupied with kinases (, left). This view was postulated previously based on both experiment and theory [25
], and is supported by recent evidence using fluorescence cross-correlation spectroscopy [24
]. Hence, cytoplasmic scaffolds may largely influence kinases only one at a time, such as by directly modulating their activity [30
], which would negate a role in fostering processivity.
Several possible molecular models could explain how the assembly of scaffolded signaling complexes at the membrane might reduce ultrasensitivity. Membrane recruitment could increase occupancy of the scaffold (, middle), which has been suggested by recent quantitative microscopy [24
], or it could promote signaling in trans
between kinases bound to different scaffold molecules (, right), which has been detected indirectly by complementation between co-expressed Ste5 mutants [45
]. Either mechanism could now permit the scaffold to promote processive phosphorylation reactions largely as was previously assumed to occur on single, cytoplasmic molecules [16
]. More complex alternative models are also possible (Figure S3
). Although our results do not distinguish among these scenarios, they highlight the notion that the relevant molecular context in which scaffold-mediated signaling occurs is likely to be fundamentally different from the simplest models involving fully-occupied scaffolds in the cytoplasm.
A variety of theoretical studies considered the possible effects of scaffolding on the input-output behavior of MAPK cascades [16
], but none of them predicted the experimentally-observed behavior in which pathway ultrasensitivity can be either increased or decreased depending on whether the scaffold is cytoplasmic or membrane-associated, respectively. However, recent mathematical simulations suggested that confinement of signaling proteins into membrane-localized “nanoclusters” may promote graded signaling through the mammalian Raf-MEK-ERK cascade [47
]. While it is unknown if analogous clustering structures exist in yeast, the broader impact of each set of findings is that the assembly of signaling complexes at the plasma membrane can have profound effects on the input-output behavior of a pathway. Modulation of these effects can allow cells to tune the systems-level properties of the signaling pathway in a manner that optimally suits the biological phenomenon being controlled.
Temporal dynamics could also influence input-output behavior. A recent study suggests that the duration of pheromone pathway signaling is sensitive to the dose of input stimulus [48
]. Although the responsible mechanism is unknown, negative feedback loops could contribute. Yet removing individual feedback loops did not eliminate either transient signaling [48
] or graded responses (). Moreover, signaling induced by β-estradiol-regulated pathway activators is persistent, not transient (Figure S1B
), and yet the output response can still be graded (see ). Hence, multiple overlapping control mechanisms may make graded signaling robust.