Faculty Opinions recommendation of Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain.

Abstract

Ste5, the prototypic mitogen-activated protein kinase (MAPK) scaffold protein, associates with plasma membrane-tethered Gbetagamma freed upon pheromone receptor occupancy, thereby initiating downstream signaling. We demonstrate that this interaction and membrane binding of an N-terminal amphipathic alpha-helix (PM motif) are not sufficient for Ste5 action. Rather, Ste5 contains a pleckstrin-homology (PH) domain (residues 388-518) that is essential for its membrane recruitment and function. Altering residues (R407S K411S) equivalent to those that mediate phosphoinositide binding in other PH domains abolishes Ste5 function. The isolated PH domain, but not a R407S K411S derivative, binds phosphoinositides in vitro. Ste5(R407S K411S) is expressed normally, retains Gbetagamma and Ste11 binding, and oligomerizes, yet is not recruited to the membrane in response to pheromone. Artificial membrane tethering of Ste5(R407S K411S) restores signaling. R407S K411S loss-of-function mutations abrogate the constitutive activity of gain-of-function Ste5 alleles, including one (P44L) that increases membrane affinity of the PM motif. Thus, the PH domain is essential for stable membrane recruitment of Ste5, and this association is critical for initiation of downstream signaling because it allows Ste5-bound Ste11 (MAPKKK) to be activated by membrane-bound Ste20 (MAPKKKK). PMID: 16847350 Funding information This work was supported by: NCI NIH HHS, United States Grant ID: T32 CA009041 NIGMS NIH HHS, United States Grant ID: GM21841 NIGMS NIH HHS, United States Grant ID: GM07232 NIGMS NIH HHS, United States Grant ID: T32 GM007048 NIGMS NIH HHS, United States Grant ID: T32 GM007232 NIGMS NIH HHS, United States Grant ID: GM07048 NCI NIH HHS, United States Grant ID: CA09041 NIGMS NIH HHS, United States Grant ID: R01 GM021841 More Less keyboard_arrow_down