Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42.
We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).GST-Cdc42 but not by GDP.GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPgammaS.GST-Cdc42 but not GDP.GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.
Fukata
M
M
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-01, Japan.
Kuroda
S
S
Fujii
K
K
Nakamura
T
T
Shoji
I
I
Matsuura
Y
Y
Okawa
K
K
Iwamatsu
A
A
Kikuchi
A
A
Kaibuchi
K
K
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D019281:Dimerization; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D008970:Molecular Weight; D018411:Spodoptera; D020764:cdc42 GTP-Binding Protein; D020703:ras GTPase-Activating Proteins
D000199:Actins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C101203:IQ motif containing GTPase activating protein 1; D020703:ras GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein