Sec14p-like domains in NF1 and Dbl-like proteins indicate lipid regulation of Ras and Rho signaling

The small GTPases Ras and Rho, which perform central roles in eukaryotic signal transduction, cycle between active GTP-bound and inactive GDP-bound forms via interactions with GTPase-activating proteins (GAPs) and with guanine nucleotide exchange factors (GEFs) [1]. As a result, mutations in GAPs or GEFs are often associated with malignant transformation and have been the subject of intensive investigation. GAPs and GEFs typically possess multiple domains, some of which bind to membranes or other proteins allowing the GAPs and GEFs to act as adaptor proteins. Here, we report the discovery of domains in GAPs and GEFs that are homologous to the lipid-binding domain of the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p, suggesting a possible link between the binding of lipids by these proteins and the regulation of Ras and Rho GTPases. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder affecting 1 out of every 3,500 individuals [2]. The NF1 protein neurofibromin — as suggested by the variable clinical manifestations of NF1 mutations — appears to possess multiple functional domains; however, only a GAP domain specific for GTPbound Ras has thus far been identified and characterized [3]. This has hindered our understanding of the molecular and cellular functions of this large, 2839 amino-acid protein. During an investigation of the uncharacterized predicted globular domains of homologues of the Rho GAP RhoGAP5, we found that these proteins share subtle yet statistically significant sequence similarity with neurofibromin, mouse Dbs (a Dbllike proto-oncogene product [4]), the apoptosis regulator Nip2 [5] and S. cerevisiae Sec14p and related proteins [6] (Figure 1; see figure legend for details on statistical significances of database searches). A subset of these Sec14p homologues, excluding neurofibromin and Dbs, are reported in Pfam [7] (CRAL_TRIO, PF00650) and Prosite [8] (profile PS50191) entries. The Pfam alignment, however, contains plant isoflavone reductases that are homologues not of Sec14p but of short-chain dehydrogenases, as demonstrated by PSI-BLAST searches (unpublished observations). Functional and structural interpretation of these sequence relationships was enhanced by the recently-determined crystal structure of Sec14p in a complex with n-octylβ-D-glucopyranoside [9]. In vitro, Sec14p mediates the exchange of phosphatidylinositol and phosphatidylcholine between membrane bilayers [10]. The Sec14p structure consists of two domains: an amino-terminal α-helical domain and a carboxy-terminal lipid-interacting α/β domain. Conserved regions in the alignment corresponding to the carboxy-terminal domain generally map to the β sheet, suggesting that these constitute a lipid-binding surface for many, if not all, of these proteins (Figures 1,2). If so, then the hydrophobic residues corresponding to Met177 and Phe212 in Sec14p, which are particularly well conserved, are likely to be key participants in lipid interactions. Site directed mutagenesis has demonstrated that Lys239 [9], which forms a salt-bridge with Glu207 (Figure 2), is necessary for Sec14p Magazine R195