Mutational analysis of Raf-1 cysteine rich domain: Requirement for a cluster of basic aminoacids for interaction with phosphatidylserine

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the p lasma membrane in response to external stimuli. The membrane localization o f Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal re gion of Raf-1 comprising amino acid residues 51-131 and residues 139-184, t he latter of which codes for a zinc containing cysteine-rich domain. The cy steine-rich domain of Raf-1 is also reported to associate with other protei ns, such as 14-3-3, and for selectively binding acidic phospholipids, parti cularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the c ysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interacti on with PS was observed in vitro for all deletion mutants of Raf-1 expresse d either as full-length proteins or as fragments containing the isolated cy steine-rich domain. In particular, the cluster of basic amino acids R-143, K-144, and K-148 appeared to be critical for interaction with PS, since sub stitution of all three residues to alanine resulted in a protein that faile d to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a trunca ted polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These res ults indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipi d bilayer and possibly in protein stability, therefore they might contribut e to Raf-1 localization and subsequent activation.