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Genes Dev

Title:		"Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain"
Author(s):		Garrenton LS; Young SL; Thorner J;
Address:		"Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, 94720, USA"
Journal Title:		Genes Dev
Year:		2006
Volume:		20
Issue:		14
Page Number:		1946 - 1958
DOI:		10.1101/gad.1413706
ISSN/ISBN:		0890-9369 (Print) 0890-9369 (Linking)
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)"
Keywords:		"Adaptor Proteins, Signal Transducing/genetics/*metabolism Amino Acid Sequence Blood Proteins/chemistry Cell Membrane/metabolism GTP-Binding Protein beta Subunits/metabolism GTP-Binding Protein gamma Subunits/metabolism Intracellular Signaling Peptides and;"
Notes:		"MedlineGarrenton, Lindsay S Young, Susan L Thorner, Jeremy eng R01 GM021841/GM/NIGMS NIH HHS/ CA09041/CA/NCI NIH HHS/ GM07048/GM/NIGMS NIH HHS/ T32 GM007232/GM/NIGMS NIH HHS/ T32 GM007048/GM/NIGMS NIH HHS/ GM07232/GM/NIGMS NIH HHS/ GM21841/GM/NIGMS NIH HHS/ T32 CA009041/CA/NCI NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2006/07/19 Genes Dev. 2006 Jul 15; 20(14):1946-58. doi: 10.1101/gad.1413706"