Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation.
Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.
Max-Planck-Institute for Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Lars
L
Blumenstein
Mohammad Reza
MR
Ahmadian
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013997:Time Factors; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000502:physiology*
D047908:Intracellular Signaling Peptides and Proteins; C061570:protein kinase N; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein