ralGDS family members interact with the effector loop of ras p21.

Abstract

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69%6 amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant ofras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop ofras p21. Since (2,17,24,32). ras p21 has GDP/GTP-binding and GTPase activities and cycles between the GDP-bound inactive and GTP-bound active forms. The GDP-bound inactive form can be activated by guanine nucleotide exchange proteins (24, 32) which promote the exchange of GDP for GTP, thereby converting ras p21 to the GTP-bound active form. Presumably the GTP-bound active form of ras p21 interacts with effector proteins that can mediate ras p21-dependent processes such as growth factor-stimulated cell proliferation.Identification of effector proteins of the active form of ras p21 has been difficult. One candidate effector protein of ras p21 is Raf, a cytoplasmic serine/threonine protein kinase that has been previously shown to act downstream of ras p21 (8,15,21). It has been recently demonstrated that Raf interacts with the GTP-bound but not with the GDP-bound form of ras p21 (18,20,29,(33)(34)(35)41), that Raf binds to the effector loop of ras p21 (20,(33)(34)(35)41), and that Raf inhibits the GTPase-activating activity of GTPase-activating protein (GAP), which is known to interact with the effector loop of ras p21 (35, 41). These results have indicated that Raf is an effector protein of ras p21, consistent with previous observations that Raf acts downstream of ras p21 in signaling pathways that mediate both the differentiation and mitogenic responses to receptor tyrosine kinases (8,15,17,21,24,30,36 (33,34,41), and that Bcl-2 binds to R-ras p23 (11). We report here the identification of a novel protein that interacts with ras p21 in the yeast two-hybrid system. Thi' p"rotpin, termed RGL (ralGDS-like), is highly homologous with rat' guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24, a member of small G-protein superfamily (1). Our results indicate that the ras p21-binding domain of RGL binds to ras p21 through the effector loop of ras p21 and that this domain is highly conserved in ralGDS. These results prompted us to examine whether this new family of proteins (RGL and ralGDS) could be effector proteins of ras p21. Since ralGDS has been well characterized (1), we used ralGDS to examine this possibility. We tested three characteristics that could be considered to be criteria for identification of an effector protein of ras p21: (i) the protein must interact with the GTP-bound active form of ras p21 but not with the GDPbound inactive form, (ii) it must interact with ras p21 through the effector loop of ras p21, and (iii) ideally the protein should inhibit the ...

rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21

“…The amino acids 602-768 of RGL contained RID [20]. pMAL/RID (pMAL/RGL(602-768)) was constructed by the following procedures.…”

“…This oligonucleotide was designed to have the EcoRI site at the 5' end and the BamHI site at the 3' end. pBSKS/RID [20] was digested with XhoI and EeoRI. RID and the Myc epitope were ligated with pGEM7z which was digested with XhoI and BamHI to generate pGEM7z/RID, pMAL-c was digested with PstI and the protruding 3' overhang was removed by T4 DNA polymerase, pGEM7z/RID was digested with ShpI and SacI to obtain the RID-Myc fragment.…”

“…We have previously found that the amino acid 602-768 of RGL is a ras p21-interacting domain (RID) by the yeast twohybrid system [20]. We first examined the direct binding of small G proteins such as ras p21, rapl p21, racl p21 or ral p24 to RID using the purified proteins.…”

“…We have identified RGL (ralGDS like) as a new effector protein of ras p21 [20]. RGL has 70% amino acid homology with ralGDS [21].…”

“…RGL has 70% amino acid homology with ralGDS [21]. The C-terminal domain of RGL and ralGDS has been found to associate with the GTP-bound form of ras p21 through the effector loop of ras p21 [20,22,23]. ralGDS stimulates GDP/GTP exchange of ral p24 [21].…”