Raf-like Ras/Rap-binding domains in RGS12- and still-life-like signalling proteins

Ras proteins play critical roles in regulating cell growth and differentiation, and mutated Ras genes are expressed in a variety of human cancers. Consequently, much interest has centered on the binding partners of Ras, including the Ras-binding domain (RBD) of Raf kinase. Here evidence is presented that domains homologous to the Raf RBD are present in tandem in RGS12, RGS14 and LOCO, and singly in molecules similar to mouse Tiam-1. In addition, RGS12, RGS14 and LOCO are shown to contain single "LGN motifs" that are guanine nucleotide exchange factors specific for the α-subunit of G proteins. These findings indicate "cross-talk" interactions between signalling pathways involving Ras and Rap and pathways involving Rho, Rac and Gα GTPases.

Ras and heterotrimeric G proteins' α subunits are GTPases that play critical roles in the initiation of eukaryotic intracellular signalling pathways. These enzymes cycle between inactive GDPbound forms and active GTP-bound forms. The latter target numerous effector molecules, thereby stimulating the generation of second messenger molecules. The activities of these GTPases are regulated in part by GTPaseactivating proteins (GAPs) that stimulate hydrolysis of GTP, and guanine nucleotide exchange factors (GEFs) that stimulate GDP release [1]. Biomedical interest in these pathways stems mainly from the finding of activated mutant Ras genes in human tumours [2].
Identification of numerous GAPs, GEFs and effector molecules that bind Ras or Gα GTPases has illuminated many of the pathways that radiate from these prolific signalling molecules. Of  [3] we have shown that several GEFs specific for the GTPase Ral p24 contain a Ras GTP -binding "RA domain". These domains were predicted to occur in several distinct signalling molecules contexts. In addition, RA domains were predicted to adopt a ubiquitin-like fold, similar to that known for the Rasbinding domain (RBD) of the Ser/Thrspecific protein kinase Raf1 [4]. Although RA and RBD domains share no significant similarities in sequence, this prediction was borne out by subsequent crystallographic structure determinations [5,6,7].

Raf-like
Here I present revised domain assignments for RGS12 and RGS14 ( Fig.  1) that are GAPs specific for G i α subunits [8,9]. Evidence suggests that these molecules contain tandem domains that are likely to bind, in a GTPdependent manner, one or more of Raslike molecules such as K-Ras, Rap1A, Rap2A, R-Ras and TC21. RGS12 and RGS14 contain "regulators of G protein signalling" (RGS) domains that have been shown to specifically stimulate the GTPase activities of Gα subunits, thereby down-regulating G protein coupled receptor-mediated signalling pathways [10,11,12]. RGS12 isoforms have been shown to contain an N-terminal PDZ domain [13] that interacts with transmembrane receptors and with the C-terminus of an alternatively spliced RGS12 variant [9]. malian RGS12 and RGS14 (10 -13 >E>10 -17 ) in the first round of searching. By round 2, this search also identified a second sub-optimal alignment of the C-terminal half of mouse RGS14 region B (amino acid residues 390-444) with the N-terminal half of fly LOCO region B (amino acid residues 374-428). This implies the presence of tandem repeats within LOCO region B. Although the E-value estimate for this second alignment (E=7.6) would be insufficient as evidence for a single repeat, it is strongly suggestive of tandem repeats within the same sequence.
Round 2 of this PSI-BLAST search also suggested that the putative tandem repeats in LOCO, and by extension in RGS12 and RGS14, are homologues of the Ras-and Rap-binding domains (RBDs) in Raf1 kinases (Fig. 1). Part of LOCO-c1 region B was aligned with Caenorhabditis elegans lin-45 Raf ki-Granderath et al. [14] recently described Drosophila melanogaster LOCO-c1 and LOCO-c2. These are RGS domain containing proteins that are required for glial cell differentiation. LOCO proteins have been shown to be similar to RGS12 and RGS14 throughout their sequences, including their RGS domains and C-terminal conserved regions B, C and D [14]. Database searches were undertaken to investigate whether regions B, C and D are significantly similar to other proteins in the non-redundant protein sequence database held at the NCBI (ftp://ncbi.nlm.nih.gov/blast/db). These searches employed the position-specific and iterative version of BLAST (PSI-BLAST) [15] and an E-value inclusion threshold of 10 -2 .
These newly identified RBD-containing proteins are complex multidomain molecules (Fig. 1). RGS12 and LOCO interact with Gα i subunits [9,14] and RGS12 acts as a Gα i -specific GAP [9]. The finding of LGN motifs in RGS12, RGS14 and LOCO is a sur-prise since this presumed Gα GEF motif [22] might be thought to antagonise the function of their RGS Gα GAP domains. However, it is suggested that these LGN motifs target Gα subunits other than Gα i , such as Gα s , Gα q and Gα 12 .
The finding of Raf1-like RBDs in proteins thought to be Rho/Rac GEFs (Still life, Tiam1 and STEF) and other proteins harbouring Gα GAP and GEF sequences (RGS12, RGS14 and LOCO) suggests hitherto unforeseen "cross-talk" interactions between Ras and Rap signalling pathways and pathways involving Rho, Rac and Gα GTPases. Further investigations are required to determine whether these RBD homologues bind Ras-like GTPases in a GTP-dependent manner and, if so, their relative specificities for Ras and similar molecules.
Addendum from author LGN motifs have recently been documented as "Goloco" motifs (Siderovski DP, Diversé-Pierluissi MA, de Vries L (1999) The Goloco motif: a Gα i/o binding motif and potential guanine-nucleodide exchange factor. Trends Biochem Sci 24:340-341 Acknowledgement CPP is a National Research Council (USA) Senior Associate. nase (amino acid residues 59-191) with an E-value of 7×10 -3 . Support for the prediction of RBD homologous domains in RGS12, RGS14 and LOCO is provided by the finding that mouse RGS14 has already been described as a Rap1/Rap2-interacting protein (Janoueix-Lerosey et al. 1997, unpublished; GenBank identifier 1814396).
A similar search using of mouse RGS14 region B (amino acid residues 264-440) revealed significant similarity, by round 4, to Drosophila Still life (type 1; amino acid residues 1100-1158; E=2×10 -3 ) [16] in a region between its PH and PDZ domains. Molecules with similar domain architectures to Still life, namely mouse Tiam-1 [17] and STEF [18], are also likely to contain single RBD homologues (Fig. 2). A PSI-BLAST search with the region intervening between the N-terminal PH and PDZ domains of STEF showed significant similarity (E=6×10 -4 ) to rat RGS12 by round 2.
A multiple alignment (Fig. 2) shows that human Raf1 Arg89 is conserved, or else substituted with positively charged Lys or His residues, in Still life, Tiam1 and N-terminal RGS12-like RBDs. Arg-89 lies at the centre of the Rap1A-Raf1 RBD binding interface [4] and is substituted for leucine in Drosophila Raf, resulting in a rough eye phenotype [19]. Many of the other Raf1 RBD residues that interact with Ras-like GTPases [4] are not conserved in the newly identified RBD homo-