# DA-Raf synergistically binds to the plasma membrane and Ras to suppress ERK signaling

## TL;DR

DA-Raf binds to the cell membrane and Ras more effectively than other Raf proteins, blocking the ERK signaling pathway.

## Contribution

DA-Raf's unique structure allows it to outcompete Raf proteins in binding to Ras, suppressing ERK signaling.

## Key findings

- DA-Raf lacks CR2 and CR3, preventing its autoinhibitory conformation and favoring plasma membrane localization.
- Basic amino acids in DA-Raf's domains are crucial for phosphatidylserine interaction and Ras binding.
- DA-Raf's stable membrane association enables it to block the Ras–ERK pathway by outcompeting Raf proteins.

## Abstract

DA-Raf efficiently associates with the plasma membrane phosphatidylserine via the RBD and CRD domains and thus predominates over Raf proteins in the binding to Ras, thereby preventing the ERK pathway.

The small GTPase Ras on the plasma membrane (PM) activates the ERK pathway (Raf–MEK–ERK signaling pathway) to regulate a variety of cellular, physiological, and pathological events. DA-Raf1 (DA-Raf) is a splicing isoform of A-Raf and contains the Ras-binding domain and the Cys-rich domain but lacks the conserved region 2 (CR2) and CR3 containing the kinase domain. Accordingly, DA-Raf dominant-negatively regulates Raf proteins to prevent the Ras–ERK pathway. We elucidate here the mechanisms of how DA-Raf conducts its dominant-negative function on Raf proteins. Because DA-Raf lacks the CR2 and CR3, it was incapable of adopting the autoinhibitory closed conformation and thereby favorable for PM localization. Basic amino acids in DA-Raf Ras-binding domain, and those in the Cys-rich domain, were essential for the interaction with phosphatidylserine in the PM. This interaction favored the cooperative binding of DA-Raf to active Ras, which predominated over that of Raf proteins, leading to the stable PM association of DA-Raf. Consequently, DA-Raf exerts its dominant-negative function on Raf proteins to prevent the Ras–ERK pathway.