Novel component of von Hippel-Lindau tumor suppressor complex and SCF ubiquitin ligase

Abstract

Rbx1, an evolutionarily conserved Cullin-interacting RING-H2 finger protein, has been discovered. Mammalian Rbx1 has been identified as a component of the CUL2-containing VHL complex. An Rbx1 homolog from S. cerevisiae has also been identified as a subunit and activator of the Cdc53-containing SCF Cdc4 ubiquitin ligase required for ubiquitination of the cdk inhibitor Sic1 and for the G1/S cell cycle transition in yeast, providing a link between the multiprotein VHL tumor suppressor complex and cellular ubiquitination. The Rbx1 protein acts as a cellular marker useful (1) in detecting a possible predisposition to certain carcinomas, (2) as a molecular target for treating those carcinomas therapeutically, (3) as a target for inhibition by drugs that manipulate the growth of cells, and (4) as a research tool to better understand the various complex mechanisms of cell ubiquitination, binding of certain activator proteins, fibronectin deposition and other aspects of the cellular division process.

1 - 23 . (Cancelled)  
     
     
         24 . A method for screening for agents that modify Rbx1-dependent stimulation of ubiquitination, the method comprising: 
 forming a complex in vitro that comprises a cofactor protein and one or more proteins selected from the group consisting of a cullin protein, a substrate recognition protein, and a linker protein;    adding a test compound to the complex; and determining if ubiquitination activity by the complex is modified by the test compound, wherein a difference in ubiquitination as compared with a reaction performed in the absence of the test compound indicates that the test compound is an agent that modulates Rbx1-dependent stimulation of ubiquitination.    
     
     
         25 . The method of  claim 24 , wherein the cofactor protein is Rbx1 (SEQ ID NO: 1).  
     
     
         26 . the method of  claim 24 , wherein the complex is disrupted in the presence of the test compound.  
     
     
         27 . the method of  claim 24 , wherein the complex is augmented in the presence of the test compound.  
     
     
         28 . The protein complex of  claim 24  wherein the complex is a ubiquitin ligase protein complex.  
     
     
         29 . The protein complex of  claim 28  wherein the ubiquitin ligase protein complex is selected from the group consisting of SCF and VHL.  
     
     
         30 . The ubiquitin ligase protein complex of  claim 28  wherein the cullin protein is selected from the group consisting of Cdc53, Cullin 1, Cullin 2, Cullin 3, Cullin 4A, Cullin 4B, and Cullin 5.  
     
     
         31 . The ubiquitin ligase protein complex of  claim 28  wherein the substrate recognition protein is selected from the group consisting of β-TRCP, Cdc4, Grr1, VHL and Elongin C binding proteins.  
     
     
         32 . The ubiquitin ligase protein complex of  claim 28  wherein the linker protein is selected from the group consisting of Skp 1 and Elongin C.