The Recruitment of Raf-1 to Membranes Is Mediated by Direct Interaction with Phosphatidic Acid and Is Independent of Association with Ras

Abstract

The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonistdependent translocation. Expression of a dominantnegative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.The MAPK 1 cascade is a signaling pathway essential for the regulation of mitogenesis by extracellular signals (1). One of the critical regulatory points in this cascade is the activation of the serine/threonine kinase Raf-1. Inactive Raf-1 exists in a large cytoplasmic complex with molecular chaperone proteins (2). Upon stimulation of cell-surface receptors, Raf-1 becomes associated with membranes and undergoes a complex series of activation steps modulated by the small GTPase Ras (3-5), 14-3-3 proteins (6 -11), and phosphorylation (12-15). Furthermore, the association of Raf-1 with membranes appears to be essential for its activation. Forced membrane recruitment through attachment of the Ras prenylation moiety to the C terminus of Raf-1 has been shown to induce kinase activation by a mechanism reportedly independent of association with Ras (16,17). It was also found that overexpression of constitutively activated Ras proteins results in the recruitment of Raf-1 to membranes (16,17). Although it was clear that Raf-1 associates with activated Ras in vivo, in vitro activation of Raf-1 by Ras has been difficult to demonstrate (18,19). Therefore, it was proposed that Ras mediates recruitment of Raf-1 to membranes (20).The recruitment of Raf-1 to membranes has also been reported to be dependent on its association with phosphatidic acid (21, 24). Disruption of agonist-dependent PLD activity either pharmacologically or by expression of catalytically inactive mutants blocks recruitment of Raf-1 to membranes and Raf-1 activation. Furthermore, addition of exogenous PA reverses the effects of PLD inhibition on Raf-1 translocation and MAPK phosphorylation. Although PA does not activate Raf-1 in vitro or in vivo, addition of exogenous PA can induce recruitment of Raf-1 to membranes in HIRcB cells or Ras-transformed cells (21). Theref...

What Makes the Bioactive Lipids Phosphatidic Acid and Lysophosphatidic Acid So Special?

“…31 P NMR spectra were recorded on a Bruker Avance (Karlsruhe, Germany) 500 widebore spectrometer at 202. 48 MHz, using a 4 mm cross-polarization (CP) MAS NMR probe. Samples were spun at the magic angle (54.7°) at 5 kHz to average the chemical shift anisotropy, and the chemical shift position of (L)PA was recorded relative to 85% H 3 PO 4 .…”

Unique N-region Determines Low Basal Activity and Limited Inducibility of A-RAF Kinase

“…Mutation of one of these residues to alanine (R398A) was sufficient to reduce the ability of RAF proteins to associate with phosphatidic acid. However, this observation is limited to insulin-dependent ERK activation localized on endosomal vesicles (33). Interestingly, in our reconstruction model arginine 398 exactly matches the residue, which interacts with the serine 339 of the N-region segment in C-RAF.…”

“…We and others provided evidence that RAF kinases per se possess high affinities for certain membrane lipids, supporting a new model in which a small fraction of RAF molecules is already associated with the plasma membrane, where Ras-RAF interactions subsequently take place by lateral diffusion in the plane of the membrane (12,33). To investigate whether the mutations in the N-region influence the translocation of C-RAF kinase to cell membranes, we examined the subcellular localization of C-RAF WT and C-RAF mutants listed in Fig.…”

Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner

“…Endogenous PLD activity is significantly increased in several types of cancers [11][12][13][14][15], as well as in cell lines transformed with oncogenes [4,5,8,10,[16][17][18][19]. PLD may participate in the activation of p42/44 ERK via Raf-1 translocation to the plasma membrane [20][21][22][23], as well as in the survival signaling mediated by PI3K/AKT activation [24]. Although the precise mechanisms involving the isoform PLD2 in cell proliferation and survival are still unknown, recent evidence suggests that residues Y 169 and Y 179 serve to recruit the Grb2/Sos complex and that PLD2-Y 169 is involved in Ras/ MAPK activation [25,26].…”

“…Bad, caspase 9, Mdm2) [29][30][31][32]. Interestingly, most of these effectors can also be regulated by PLD [16,21,22,25,[33][34][35][36]. Furthermore, AKT and bacterial PLD interact [37] and PLD plays a critical role in the PI3K-dependent activation of AKT [38][39][40][41][42].…”