Raf-1 Kinase Possesses Distinct Binding Domains for Phosphatidylserine and Phosphatidic Acid

doi:10.1074/jbc.271.14.8472

Raf-1 Kinase Possesses Distinct Binding Domains for Phosphatidylserine and Phosphatidic Acid

PHOSPHATIDIC ACID REGULATES THE TRANSLOCATION OF Raf-1 IN 12-O-TETRADECANOYLPHORBOL-13-ACETATE-STIMULATED MADIN-DARBY CANINE KIDNEY CELLS (*)

From the (1)Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710,
(2)Glaxo-Wellcome Research, Research Triangle Park, North Carolina 27709, and
(3)Department of Biochemistry, Bowman-Gray School of Medicine, Wake Forest University, Winston Salem, North Carolina 27157

^¶ To whom correspondence should be addressed:
Glaxo Wellcome Inc., Five Moore Dr., Research Triangle Park, NC 27709.
Tel.: 919-990-6145.

Abstract

Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M.(1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295-648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent K ranged from 4.9 ± 0.6 to 7.8 ± 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

Footnotes

↵^§ Supported by National Institutes of Health Signal Transduction Mechanisms and Cell Functions Training Program (CA-09422).
↵¹ The possibility of other mechanisms for activation such as other cofactors have not been excluded.
↵* The work was supported in part by National Institutes of Health Grants CA-48995 and CA-43297 (to L. D.) and GM-38737 and DD-20205 (to R. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵² The abbreviations used are:

PS

phosphatidylserine

PA

phosphatidic acid

PEt

phosphatidylethanol

PC

phosphatidylcholine

PI

phosphatidylinositol

PG

phosphatidylglycerol

PKC

protein kinase C

PLD

phospholipase D

GST

glutathione S-transferase

ELISA

enzyme-linked immunosorbent assay

RafFull

full-length Raf-1

RafCys

cysteine-rich domain of Raf-1

RafC

carboxyl-terminal fragment of Raf-1

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

MDCK

Madin-Darby canine kidney

DMEM

Dulbecco's modified Eagle's medium

TPA

12-O-tetradecanoylphorbol-13-acetate.
- Received September 26, 1995.
- Revision received December 21, 1995.