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 Kd 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.
Highlights
Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine
Expression of GST-Raf-1 Fusion Proteins—Fusion proteins consisting of different fragments of human Raf-1 attached to GST were expressed in E. coli BL-21 strain using the pGEX 4T-2 vector, with the exception of full-length Raf-1 (RafFull), cysteine-rich domain of Raf-1 (RafCys), and the carboxyl-terminal fragment of Raf-1 (RafC), which were expressed from the pGEX-2T vector
Binding of a COOH-terminal Fragment of Raf-1 to phosphatidic acid (PA)—In a previous article [23], we reported that residues 128 –196 of human Raf-1 kinase, containing the cysteine-rich domain, was capable of translocating to PS-enriched liposomes in vitro
Summary
Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine Based on in vitro direct binding assays and immunoprecipitation assays from cultured cells, the amino terminus of Raf-1 is known to associate directly with another protooncogene product, p21Ras, and the regions on Raf-1 critical for high affinity interaction with p21Ras have been identified [5,6,7,8,9,10]. For protein kinase C, a zinc-containing cysteine-rich domain is believed to play a key role in the activation of the enzyme by binding to the acidic phospholipid, phosphatidylserine, and to diacylglycerol [22]. In contrast to protein kinase C, the binding of the Raf-1 cysteine-rich domain to PS is not modulated by either diacylglycerol or phorbol esters
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