TY - JOUR
T1 - Tiam1 mediates Ras activation of Rac by a PI(3)K-independent mechanism
AU - Lambert, John M.
AU - Lambert, Que T.
AU - Reuther, Gary W.
AU - Malliri, Angeliki
AU - Siderovski, David P.
AU - Sondek, John
AU - Collard, John G.
AU - Der, Chaning J.
N1 - Funding Information:
The pCGN-RacQ61L, pCDNA3-H-RasQ61L, pCDNA3-H-Raswt (ref. 28) and pCGN-Raf-N1 (Raf-RBD)18 mammalian expression vectors have been described previously, as have the Gal4-Elk1, Gal4-Jun-1(amino acids 1–254) and 5×Gal4-Luc reporter plasmids28. To generate pCGN-Tiam1-RBD, the Ras-binding domain of mouse tiam1 (amino acids 760–837 of the SwissProt sequence Q60610 (TIAM_MOUSE)) was amplified by PCR and cloned into the BamH1 site of pCGN-hygro, which was provided by M. Ostrowski (Ohio State University, OH). pCDNA3-Myc-Tiam1wt was provided by G. Bollag (Onyx Pharmaceuticals, Richmond, CA). To generate pCDNA3-Myc-Tiam1-RBD*, a SpeI-BamH1 fragment of pCDNA3-Myc-Tiam1wt was ligated into pBluescriptII S/K (Stratagene, La Jolla, CA) and then nucleotides encoding residues corresponding to amino acids 796–799 (KTHQ) of h-tiam1 were mutated to encode AADE using the Quick Change Mutagenesis kit (Stratagene) in accordance with the manufacturer’s instructions. The fidelity of mutagenesis reactions was verified by automated sequencing and the resulting mutated SpeI–BamH1 fragment was ligated back into the parental vector. pCDNA3-p110-CAAX was provided by J. Downward (Imperial Cancer Research Fund, London, UK). The c-Fos-Luc reporter plasmid was provided by C. Hauser (Burnham Foundation, La Jolla, CA). The pGEX-2T bacterial expression vector encoding GST–PAK1 (amino acids 70–132) was provided by B. Mayer (University of Connecticut, CT). Antibodies used were: anti-Tiam1 (C-16; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Myc (9E10; Santa Cruz Biotechnology), anti-H-Ras (146-03E4; NCI/BCB Repository, Bethesda, MD), anti-Rac1 (23A8; Upstate Biotechnology), anti-HA (12CA5; Covance Inc., Princeton, NJ), and anti-Akt and anti-phospho-Akt(Ser 473) (catalogue numbers 9272 and 9271S, respectively; Cell Signalling Technology (Beverly, MA).
PY - 2002/8
Y1 - 2002/8
N2 - Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch1. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2). Once activated, Rac stimulates signalling pathways that regulate actin organization, gene expression and cellular proliferation. Rac also functions downstream of the Ras oncoprotein in pathways that stimulate membrane ruffling3, growth transformation4, 5, activation of the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase6, activation of the NF-κB transcription factor and promotion of cell survival7, 8. Although recent studies support phosphatidylinositol 3-OH kinase (PI(3)K)-dependent mechanisms through which Ras might activate Rac (refs 9, 10), the precise mechanism remains to be determined. Here we demonstrate that Tiam1, a Rac-specific GEF, preferentially associates with activated GTP-bound Ras through a Ras-binding domain. Furthermore, activated Ras and Tiam1 cooperate to cause synergistic formation of Rac-GTP in a PI(3)K-independent manner. Thus, Tiam1 can function as an effector that directly mediates Ras activation of Rac.
AB - Rac is a member of the Ras superfamily of GTPases and functions as a GDP/GTP-regulated switch1. Formation of active Rac-GTP is stimulated by Dbl family guanine nucleotide exchange factors (GEFs), such as Tiam1 (ref. 2). Once activated, Rac stimulates signalling pathways that regulate actin organization, gene expression and cellular proliferation. Rac also functions downstream of the Ras oncoprotein in pathways that stimulate membrane ruffling3, growth transformation4, 5, activation of the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase6, activation of the NF-κB transcription factor and promotion of cell survival7, 8. Although recent studies support phosphatidylinositol 3-OH kinase (PI(3)K)-dependent mechanisms through which Ras might activate Rac (refs 9, 10), the precise mechanism remains to be determined. Here we demonstrate that Tiam1, a Rac-specific GEF, preferentially associates with activated GTP-bound Ras through a Ras-binding domain. Furthermore, activated Ras and Tiam1 cooperate to cause synergistic formation of Rac-GTP in a PI(3)K-independent manner. Thus, Tiam1 can function as an effector that directly mediates Ras activation of Rac.
UR - http://www.scopus.com/inward/record.url?scp=0036051325&partnerID=8YFLogxK
U2 - 10.1038/ncb833
DO - 10.1038/ncb833
M3 - Article
C2 - 12134164
AN - SCOPUS:0036051325
SN - 1465-7392
VL - 4
SP - 621
EP - 625
JO - Nature Cell Biology
JF - Nature Cell Biology
IS - 8
ER -