The Ste5 Scaffold Directs Mating Signaling by Catalytically Unlocking the Fus3 MAP Kinase for Activation

Abstract

The scaffold protein Ste5 is required to properly direct signaling through the yeast mating pathway to the mitogen-activated protein kinase (MAPK), Fus3. Scaffolds are thought to function by tethering kinase and substrate in proximity. We find, however, that the previously identified Fus3-binding site on Ste5 is not required for signaling, suggesting an alternative mechanism controls Fus3’s activation by the MAPKK Ste7. Reconstituting MAPK signaling in vitro, we find that Fus3 is an intrinsically poor substrate for Ste7, although the related filamentation MAPK, Kss1, is an excellent substrate. We identify and structurally characterize a novel domain in Ste5 that catalytically unlocks Fus3 for phosphorylation by Ste7. This domain selectively increases the kcat of Ste7➔Fus3 phosphorylation by 5000-fold but has no effect on Ste7➔Kss1 phosphorylation. The dual requirement for both Ste7 and this Ste5 domain in Fus3 activation explains why Fus3 is selectively activated by the mating pathway, and not by other pathways that also utilize Ste7.

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5- bis phosphate distribution is required for MAPK signaling

“…5A). Taken together, our cytological and biochemical results are consistent with prior evidence (25,26) and recent structural analysis indicating that Fus3 requires allosteric modulation by Ste5 for its activation by Ste7, whereas Kss1 does not (27). We also examined pathway activation in response to pheromone using a transcriptional reporter gene (Fig.…”

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

“…Second, some scaffolding proteins also have catalytic roles, activating different components of the signaling pathway in addition to bringing them to each other (42). This mechanism was most convincingly demonstrated for yeast scaffold Ste5 (64,65). Mathematical modeling showed that when the scaffold tethers the components, bringing them to each other, the scaffold concentration affects the signaling biphasically, increasing it at lower, then reducing it at higher concentrations, when the excess of scaffold over the kinases increases the probability of formation of incomplete complexes, containing only one kinase (38,39).…”

Scaffold Protein Ahk1, Which Associates with Hkr1, Sho1, Ste11, and Pbs2, Inhibits Cross Talk Signaling from the Hkr1 Osmosensor to the Kss1 Mitogen-Activated Protein Kinase

“…Ste5 constrains the signal flow from the pheromone receptor by binding simultaneously to G␤␥ (Ste4/ Ste18), Ste11, Ste7, and Fus3 (57)(58)(59)(60). In the absence of the Ste5 scaffold, the default Ste11-Ste7-Kss1 MAPK cascade, which regulates filamentous growth, is activated (33,61,62). Similarly, in the absence of the Ahk1 scaffold, osmostress activation of Hkr1 leads to cross talk activation of the Ste11-Ste7-Kss1 kinase cascade.…”