Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules.
Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.
Fujisawa
K
K
Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan.
Madaule
P
P
Ishizaki
T
T
Watanabe
G
G
Bito
H
H
Saito
Y
Y
Hall
A
A
Narumiya
S
S
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006041:Goats; D047908:Intracellular Signaling Peptides and Proteins; D016350:Leucine Zippers; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C498395:Rtkn protein, mouse; C097916:rhophilin; C115635:citron-kinase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein