Thus, the effects of PA are not a consequence of a direct activation of Raf. of the phosphatidic acid binding domain name was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events. Although the idea that phosphatidic acid (PA)3is an important lipid second messenger seems to be widely accepted, the actual functions of this lipid in transmission transduction are still poorly comprehended. Several physiological functions for PA have been proposed in the past. These include regulation of protein and lipid phosphorylation (1-3), regulation of cAMP degradation (4), activation of oxidative processes (3,5), and modulation of membrane traffic (6-9). Many of these functions are mediated by the direct conversation of PA with MK-8719 specific target proteins. Thus, PA appears to function in a manner analogous to other MK-8719 lipid second messengers (i.e.by promoting the binding of target proteins to specific regions of the cell membrane). Direct binding of PA has been demonstrated in a small subset of proteins. The interactions of PA with Raf-1 have been mapped to a 35-amino acid stretch within the kinase domain name (10-13), whereas the binding of PA to mTOR entails Arg2109, a residue located in the vicinity of the rapamycin binding domain name (14,15). Putative binding sites for the cyclic nucleotide phosphodiesterase PDE4D3 (4) and the protein-tyrosine phosphatase SHP-1 (16) have also been described. In general, these PA binding domains bear little sequence similarity to each other, except for the fact that they all contain at least one polybasic motif. Previous work from our laboratory has exhibited that PA binding is essential for the recruitment of Raf-1 to the plasma membrane and its subsequent activation (12,13). This conclusion was based on the findings that treatment with exogenous PA induced a rapid, transient translocation of Raf-1 to the plasma membrane and that mutation of key basic residues located in the putative PA binding region (PABR) of Raf-1 prevented Raf-1 translocation and activation. We also showed that interference with PA production or expression of a PA scavenger peptide based on the Raf-1 PABR resulted in the inhibition of insulin-dependent ERK phosphorylation (13). More recent work has exhibited a second PA target in the Ras/Raf/ERK cascade: the Ras guanine nucleotide exchange factor Sos, which binds PA via its pleckstrin homology domain name (17). Thus, PA production appears to be an absolute requirement for the coupling of the ERK cascade in cell membranes. However, neither MEK1/2 nor ERK1/2 possess well defined membrane binding domains. Thus, the question of how the remaining components of the cascade assemble on lipid membranes remains an open question. The scaffolding proteins KSR1 and KSR2 (kinase suppressor of Ras 1 and 2) bear substantial structural similarities with the Raf family of protein kinases. KSR1 binds MEK1, ERK1/2, and perhaps Raf-1 (18-20). The C-terminal domain name of KSR1 is usually remarkably similar to the kinase domain name of Raf-1 (21,22). KSR1 has been shown to bind lipids via an N-terminal cysteine-rich domain name (23). Moreover, KSR1 contains a sequence homologous to the PABR of Raf-1. Thus, we hypothesized that PA modulates the scaffolding function of KSR proteins by promoting their association to PA-enriched regions that contain the remaining elements of the ERK cascade. CSH1 In this paper, we compare the interactions of PA with the PABR of Raf-1 and KSR1. Our data demonstrate direct, specific binding of PA to the PABR of both proteins. Furthermore, we provide substantial evidence in support of the hypothesis that PA modulates the binding of KSR1 to membranes and that this binding is essential for the scaffolding role of KSR in the coupling of the ERK kinase cascade. == MATERIALS AND METHODS == Cells, cDNAs, and Lipids UsedHIRcB cells were cultured in Dulbecco’s altered Eagle’s medium/F-12, 10% fetal calf serum made up of 100 nmmetothrexate. NIH-3T3 and HEK-293 cells were cultured in Dulbecco’s altered Eagle’s medium, 10% fetal calf MK-8719 serum. Plasmids encoding mKSR1 were kindly supplied.
