Function of the MAPK scaffold protein, Ste5, requires a cryptic PH domain
Lindsay S Garrenton1, Susan L Young, Jeremy Thorner
1Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, 94720, USA.
Genes & Development
|July 19, 2006
Summary
The pleckstrin-homology (PH) domain of the Ste5 scaffold protein is crucial for its membrane recruitment and function in initiating mitogen-activated protein kinase (MAPK) signaling pathways. Mutations in this PH domain disrupt signaling, highlighting its essential role.
Area of Science:
- Cellular signaling
- Molecular biology
- Protein-protein interactions
Background:
- Ste5 is a scaffold protein that initiates mitogen-activated protein kinase (MAPK) signaling upon pheromone receptor activation.
- Initial understanding suggested plasma membrane association via Gbetagamma and an N-terminal motif were sufficient for Ste5 function.
Purpose of the Study:
- To investigate the role of the pleckstrin-homology (PH) domain of Ste5 in its membrane recruitment and signaling function.
- To determine if the PH domain is essential for Ste5's interaction with the plasma membrane and subsequent signal initiation.
Main Methods:
- Site-directed mutagenesis of Ste5 to alter residues critical for phosphoinositide binding in the PH domain (R407S K411S).
- In vitro binding assays of the isolated Ste5 PH domain with phosphoinositides.
- Analysis of Ste5 mutant expression, binding interactions (Gbetagamma, Ste11), oligomerization, and membrane recruitment in response to pheromone.
- Restoration of signaling by artificial membrane tethering of the mutant Ste5.
Main Results:
- The PH domain of Ste5 is essential for its recruitment to the plasma membrane.
- Mutations (R407S K411S) in the PH domain that abolish phosphoinositide binding also abolish Ste5 function and membrane recruitment.
- The isolated wild-type PH domain binds phosphoinositides, while the mutant version does not.
- Artificial membrane tethering of the non-functional mutant Ste5 restores signaling, indicating the PH domain's role is in membrane association.
Conclusions:
- The Ste5 PH domain is critical for stable membrane association, which is necessary for initiating MAPK signaling.
- Membrane recruitment mediated by the PH domain allows for the activation of Ste11 (MAPKKK) by Ste20 (MAPKKKK).
- The PH domain's role in membrane localization is a key regulatory step in pheromone-induced MAPK signaling.
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