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

Hekman, Mirko; Hamm, Heike; Villar, Ana V.; Bader, Benjamin; Kuhlmann, Jürgen; Nickel, J. Curtis; Rapp, Ulf R.

doi:10.1074/jbc.m200576200

Cited by 95 publications

(100 citation statements)

References 73 publications

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“…Some of these, like phosphatidylethanolamine and phosphatidylinositol, inhibit their kinase activity. [49] It is possible that the amount of Raf available for activation and its degree of activation could be regulated by the phospholipid composition of the membranes where Ras is acting. Similarly, the presence of regulatory proteins like RKIP, a phosphatidylethanolamine-binding Raf inhibitor, [50] could also be dependent on the membrane composition.…”

Section: Ras: An Actor On Many Stagesmentioning

confidence: 99%

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

2010

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Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site-specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating ''house-keeping'' functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras-ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space-related regulatory processes. These findings may open a gate for aiming at the Ras-ERK pathway in a spatially restricted fashion, in our quest for new anti-tumor therapies.

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Section: Ras: An Actor On Many Stagesmentioning

confidence: 99%

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

2010

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“…Preparation of Liposomes and Biosensor Measurements-Large unilamellar vesicles were prepared by the extrusion method using a LiposoFast extrusion apparatus (Avestin Inc.) as described (21). To determine quantitatively the interactions between BAD and different 14-3-3 isoforms and for BAD interactions with liposomes, the surface plasmon resonance (SPR) technique was applied.…”

Section: Methodsmentioning

confidence: 99%

Reversible Membrane Interaction of BAD Requires two C-terminal Lipid Binding Domains in Conjunction with 14-3-3 Protein Binding

Hekman

Albert

Galmiche

et al. 2006

Journal of Biological Chemistry

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BAD is a Bcl-2 homology domain 3 (BH3)-only proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Binding of BAD to mitochondria is thought to be exclusively mediated by its BH3 domain. We show here that BAD binds to lipids with high affinities, predominantly to negatively charged phospholipids, such as phosphatidylserine, phosphatidic acid, and cardiolipin, as well as to cholesterol-rich liposomes. Two lipid binding domains (LBD1 and LBD2) with different binding preferences were identified, both located in the C-terminal part of the BAD protein. BAD facilitates membrane translocation of Bcl-X L in a process that requires LBD2. Integrity of LBD1 and LBD2 is also required for proapoptotic activity in vivo. Phosphorylation of BAD does not affect membrane binding but renders BAD susceptible to membrane extraction by 14-3-3 proteins. BAD can be removed efficiently by 14-3-3, -, -and less efficiently by other 14-3-3 isoforms. The assembled BAD⅐14-3-3 complex exhibited high affinity for cholesterol-rich liposomes but low affinity for mitochondrial membranes. We conclude that BAD is a membraneassociated protein that has the hallmarks of a receptor rather than a ligand. Lipid binding is essential for the proapoptotic function of BAD in vivo. The data support a model in which BAD shuttles in a phosphorylation-dependent manner between mitochondria and other membranes and where 14-3-3 is a key regulator of this relocation. The dynamic interaction of BAD with membranes is tied to activation and membrane translocation of Bcl-X L .

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“…Therefore, we propose that Rap1 prevents Raf-1 activation by positioning it away from tyrosine kinases that are required for Tyr-341 phosphorylation. One of the functions of Tyr-341 phosphorylation might be to provide a regulatable interaction with proteins or lipids to participate in proper targeting of Ras/Raf-1 (62,63).…”

Section: Phosphorylation Of Tyr-341 Is Required For Proper Raft Localmentioning

confidence: 99%

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

Carey

Watson

Pessin

et al. 2003

Journal of Biological Chemistry

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Activation of Raf-1 by Ras requires recruitment to the membrane as well as additional phosphorylations, including phosphorylation at serine 338 (Ser-338) and tyrosine 341 (Tyr-341). In this study we show that Tyr-341 participates in the recruitment of Raf-1 to specialized membrane domains called "rafts," which are required for Raf-1 to be phosphorylated on Ser-338. Raf-1 is also thought to be recruited to the small G protein Rap1 upon GTP loading of Rap1. However, this does not result in Raf-1 activation. We propose that this is because Raf-1 is not phosphorylated on Tyr-341 upon recruitment to Rap1. Redirecting Rap1 to Ras-containing membranes or mimicking Tyr-341 phosphorylation of Raf-1 by mutation converts Rap1 into an activator of Raf-1. In contrast to Raf-1, B-Raf is activated by Rap1. We suggest that this is because B-Raf activation is independent of tyrosine phosphorylation. Moreover, mutants that render B-Raf dependent on tyrosine phosphorylation are no longer activated by Rap1.The mitogen-activated protein (MAP) 1 kinase family regulates diverse physiological processes including cell growth, differentiation, and death. Activation of one of these MAP kinases (the extracellular signal-regulated kinase or ERK) is initiated by the recruitment of the MAP kinase kinase kinase Raf-1 to the small G protein Ras, a resident plasma membrane protein.The Ras family consists of three members (Ha-Ras, Ki-Ras, and N-Ras) (1) that display overlapping but distinct patterns of expression and function (2). Ras is tethered to the membrane via a carboxyl CAAX motif containing a cysteine followed by two aliphatic amino acids (A) and a carboxyl-terminal amino acid (X) that directs the attachment of a farnesyl moiety (3, 4). In addition to farnesylation, a second signal assists in correct membrane targeting. For Ha-Ras and N-Ras, this second signal is a palmitoyl moiety that is introduced on a neighboring cysteine. In Ki-Ras, this second site is a polybasic domain.

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

Cited by 95 publications

References 73 publications

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

Reversible Membrane Interaction of BAD Requires two C-terminal Lipid Binding Domains in Conjunction with 14-3-3 Protein Binding

The Requirement of Specific Membrane Domains for Raf-1 Phosphorylation and Activation

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