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Molecular and Cellular Biochemistry

, Volume 198, Issue 1–2, pp 171–178 | Cite as

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

  • Teresa Improta-Brears
  • Sujoy Ghosh
  • Robert M. Bell
Article

Abstract

Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of 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 region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results 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 lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

phospholipids phospatidylserine interaction Raf-1 protein kinase mutational analysis cysteine-rich domain 

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Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Teresa Improta-Brears
    • 1
  • Sujoy Ghosh
    • 2
  • Robert M. Bell
    • 2
  1. 1.Department of Pharmacology and Cancer BiologyDuke University Medical CenterDurhamUSA
  2. 2.Glaxo WellcomeResearch Triangle ParkUSA

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