title: Loop 6 of RhoA confers specificity for effector binding, stress fiber formation, and cellular transformation.
abstract: Rho family GTPases regulate multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These effects are achieved through the interaction of GTP-bound proteins with various downstream targets. A series of RhoA/Rac1 and Rho/Ras chimeras was generated to map the domain(s) of RhoA involved in its association with two classes of effector kinase, represented by PRK2 and ROCK-I. Although the switch 1 domain was required for effector binding, the N terminus of Rho (residues 1-75) was interchangeable with that of Rac. This suggested that the region of Rho that confers effector binding specificity lay further C-terminal. Subsequent studies indicated that the "insert domain"(residues 123-137), a region unique to Rho family GTPases, is not the specificity determinant. However, a determinant for effector binding was identified between Rho residues 75-92. Rac to Rho point mutations (V85D or A88D) within loop 6 of Rac promoted its association with PRK2 and ROCK, whereas the reciprocal Rho(D87V/D90A) double mutant significantly reduced effector binding capacity. In vivo studies showed that microinjection of Rac(Q6IL/V85D/A88D) but not Rac(Q6IL) induced stress fiber formation in LLC-PK epithelial cells, suggesting that loop 6 residues conferred the ability of Rac to activate ROCK. On the other hand, the reciprocal Rho (Q6IL/D87V/D90A) mutant was defective in its ability to transform NIH 3T3 cells. These data suggest that although Rho effectors can utilize a Rho or Rac switch 1 domain to sense the GTP-bound state of Rho, unique residues within loop 6 are essential for determining both effector binding specificity and cellular function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zong
authors.forename: N
authors.initials: N
authors.lastname: Raman
authors.forename: L A
authors.initials: LA
authors.lastname: Mickelson-Young
authors.forename: S J
authors.initials: SJ
authors.lastname: Atkinson
authors.forename: L A
authors.initials: LA
authors.lastname: Quilliam
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002461:Cell Line, Transformed; D019204:GTP-Binding Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D018374:LLC-PK1 Cells; D051379:Mice; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013552:Swine; D054460:rho-Associated Kinases