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Improta-Brears, T; Ghosh, Sujoy; Bell, Robert M.

doi:10.1023/a:1006981411691

Cited by 63 publications

(35 citation statements)

References 41 publications

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“…5). Taken together, our results with respect to lipid binding performed with purified and functional full-length C-Raf kinases using in vitro reconstitution assay are consistent with observations previously reported (15,16,19). We extended these investigations to include B-Raf kinase, an active mutant of C-Raf (C-Raf-Y340D/ Y341D) and the BXB form of C-Raf.…”

Section: Regulation Of B-and C-raf Kinasessupporting

confidence: 91%

“…Specific Associations of B-and C-Raf Kinase with PS, PA, and Ceramides-Evidence that fragments of Raf proteins, particularly C-Raf-CRD, interact with phospholipids have been presented (15,16). The amino acids in C-Raf-CRD interacting with PS have been mapped as Arg-143, Lys-144, and Lys-148, since substitution of all three basic residues to alanine resulted in a C-Raf variant that failed to interact with PS.…”

Section: Discussionmentioning

confidence: 99%

“…gous to the binding of protein kinase C to lipids, such as PS and diacylglycerol, C-Raf was shown to possess binding affinity for PS but revealed almost no binding to diacylglycerol (5,15,16). Additionally C-Raf was found to bind to PA (19).…”

Section: Purification and Characterization Of Raf Kinases-analo-mentioning

confidence: 99%

“…The C-Raf-CRD was further proposed to bind to phospholipids at the plasma membrane. The first evidence that C-Raf-CRD interacts with phosphatidylserine (PS) (15,16) was not unexpected, since C-Raf-CRD exhibits a structure very similar to the C1-domain of protein kinase C family proteins (17,18), although C-Raf-CRD does not bind tumor promoting phorbol esters and binds only weakly diacylglycerol (5). Taken together, C-Raf-CRD appears to have a complex and multifunctional role in the regulation of C-Raf kinase activity.…”

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confidence: 99%

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Associations of B- and C-Raf with Cholesterol, Phosphatidylserine, and Lipid Second Messengers

Hekman¹,

Hamm²,

Villar³

et al. 2002

Journal of Biological Chemistry

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The serine/threonine kinase C-Raf is a key mediator in cellular signaling. Translocation of Raf to membranes has been proposed to be facilitated by Ras proteins in their GTP-bound state. In this study we provide evidence that both purified B-and C-Raf kinases possess lipophilic properties and associate with phospholipid membranes. In the presence of phosphatidylserine and lipid second messengers such as phosphatidic acid and ceramides these associations were very specific with affinity constants (K D ) in the range of 0.5-50 nM. Raf association with liposomes was accompanied by displacement of 14-3-3 proteins and inhibition of Raf kinase activities. Interactions of Raf with cholesterol are of particular interest, since cholesterol has been shown to be involved, together with sphingomyelin and glycerophospholipids in the formation of specialized lipid microdomains called rafts. We demonstrate here that purified Raf proteins have moderate binding affinity for cholesterol. However, under conditions of lipid raft formation, Raf association with cholesterol (or rafts) increased dramatically. Since ceramides also support formation of rafts and interact with Raf we propose that Raf may be present at the plasma membrane in two distinct microdomains: in raft regions via association with cholesterol and ceramides and in non-raft regions due to interaction with phosphatidylserine and phosphatidic acid. At either location Raf kinase activity was inhibited by lipid binding in the absence or presence of Ras. Ras-Raf interactions with full-length C-Raf were studied both in solution and in phospholipid environment. Ras association with Raf was GTP dependent as previously demonstrated for C-Raf-RBD fragments. In the presence of liposomes the recruitment of C-Raf by reconstituted Ras-farnesyl was only marginal, since almost 70% of added C-Raf was bound by the lipids alone. Thus Ras-Raf binding in response to activation of Ras-coupled receptors may utilize Raf protein that is already present at the membrane.

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Section: Regulation Of B-and C-raf Kinasessupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Purification and Characterization Of Raf Kinases-analo-mentioning

confidence: 99%

mentioning

confidence: 99%

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Associations of B- and C-Raf with Cholesterol, Phosphatidylserine, and Lipid Second Messengers

Hekman¹,

Hamm²,

Villar³

et al. 2002

Journal of Biological Chemistry

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“…Several studies have shown that the RafCRD is essential for Raf-1 activation (Clark et al, 1997;Luo et al, 1997;Roy et al, 1997). This domain has overlapping binding sites for phosphatidylserine (PS), Ras and 14-3-3 (Clark et al, 1997;Daub et al, 1998;Ghosh et al, 1996;Improta-Brears et al, 1999;Mott et al, 1996;Okada et al, 1999;Williams et al, 2000). It has been shown that the interaction of the CRD with 14-3-3 stabilizes the Raf-1/14-3-3 complex and that the incubation of Raf-1 with PS in vitro completely displaces 14-3-3 with a concomitant increase in Raf kinase activity (McPherson et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

2001

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Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, proteinprotein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium¯uoride, or û-glycerophosphate and sodium pyrophosphate, or speci®c PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I 1 or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and signi®cantly alters the distribution of 14-3-3 and Raf-1 across dierent plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated. Oncogene (2001) 20, 3949 ± 3958.

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Quantification of the Raf-C1 Interaction With Solid-Supported Bilayers

et al. 2002

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By use of the quartz crystal microbalance technique, the interaction of the Raf-Ras binding domain (RafRBD) and the cysteine-rich domain Raf-C1 with lipids was quantified by using solid-supported bilayers immobilized on gold electrodes deposited on 5 MHz quartz plates. Solid-supported lipid bilayers were composed of an initial octanethiol monolayer chemisorbed on gold and a physisorbed phospholipid monolayer varying in its lipid composition as the outermost layer. The integrity of bilayer preparation was monitored by impedance spectroscopy. For binding experiments, a protein construct comprising the RafRBD and Raf-C1 linked to the maltose binding protein and a His tag, termed MBP-Raf-C1, was used. Dissociation constants and rate constants of the association and dissociation were obtained for various 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS) lipid mixtures. Independently of the phosphatidylserine (PS) content, the dissociation constants were in the order of 5x10(-7) M, while the on-rate constants were in the range of 2x10(3) (M s)(-1) and the off-rate constants in the range of 1x10(-3) s(-1). The maximum frequency shift increased significantly with increasing amounts of DMPS; this indicates that this negatively charged lipid is the primary binding site for MBP-Raf-C1. Exchange of DMPS for 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) did not alter the thermodynamics and kinetics of protein binding, which implies that the protein interaction is mainly electrostatically driven. Scanning force microscopy (SFM) was employed to render protein adsorption visible and to confirm the assumption of a protein monolayer on the lipid layer. SFM images clearly revealed that the protein binds preferentially, but not solely, to negatively charged phosphatidylserine headgroups. We hypothesize that PS-enriched domains are initial binding sites with high affinity for Raf-C1, but that lateral interactions may account for protein domain growth.

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Cited by 63 publications

References 41 publications

Associations of B- and C-Raf with Cholesterol, Phosphatidylserine, and Lipid Second Messengers

Associations of B- and C-Raf with Cholesterol, Phosphatidylserine, and Lipid Second Messengers

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions

Quantification of the Raf-C1 Interaction With Solid-Supported Bilayers

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