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

Abstract

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.

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

“…However, as the aforementioned crystal structure is based on a dephosphorylated protein, the importance of the serine residues within the Nregion is less understood. In striking contrast to Raf-1 S338 phosphorylation, phosphorylation of the evolutionary conserved S446 in B-Raf is neither stimulated by nor dependent on Ras-GTP/B-Raf interaction (Mason et al, 1999;Brummer et al, 2002) and is not affected by lipid raft disruption (Carey et al, 2003). This suggests not only that S446 phosphorylation occurs prior to interaction with Ras-GTP but also that Raf-1 and B-Raf are phosphorylated in different subcellular locations and/or by distinct N-region kinases.…”

“…Induction of Ras-GTP by extracellular signals results in the recruitment of pre-activated B-Raf to the plasma membrane where it is fully activated by activation segment kinases. This assumption is supported by the observation that a membrane-targeted B-Raf-CAAX protein displays constitutive MAPKKK and high transforming activities (Papin et al, 1998;Carey et al, 2003). Phosphorylation of the activation segment destabilizes the inactive conformation of the kinase domain (Wan et al, 2004) and renders B-Raf fully active.…”

“…Negative charges within this region are required for activation and enhance the affinity of Raf-1 towards its substrate MEK (Xiang et al, 2002). Raf-1 activation involves an inducible two-step mechanism leading to Ras-dependent phosphorylation of the N-region residues S338/339 and Y340/341 at the plasma membrane (Mason et al, 1999;Carey et al, 2003). In contrast, the N-region of B-Raf is permanently negatively charged owing to the presence of the D448/449, which occupy positions equivalent to Y340/341 in Raf-1, and the constitutive phosphorylation of the S338 equivalent (S446) (Mason et al, 1999;Brummer et al, 2002).…”

Inverse Rap1 and Phospho-ERK Expression Discriminate the Maintenance Phase of Tolerance and Priming of Antigen-Specific CD4+ T Cells In Vitro and In Vivo

“…Collectively, these data suggest that Rap1 may be up-regulated and recruited to the immune synapse upon stimulation of tolerized T cells with Ag and that this may result in down-regulation of ERK recruitment and activation, possibly as a result of Raf-1 sequestration. This would lead to the uncoupling of the Ras-ERK-MAPK pathway from the TCR as has been observed in tolerant cells (43)(44)(45).…”

Plasma membrane regulates Ras signaling networks

“…62 Significantly, when Raf-1 is directed to H-Ras occupied lipid rafts, the activation is minimal. 63 This can be attributed to the exposure of Raf-1 to a distinct group of proteins and lipids present in the plasma membrane. Temporal regulation of Raf activation by Ras is an understudied topic.…”