Methods for Modulating an Immune Response by Modulating Krc Activity

Abstract

This invention demonstrates that KRC molecules have multiple important functions as modulating agents in regulating a wide variety of cellular processes including: inhibiting NFkB transactivation, increasing TNF-alpha induced apoptosis, inhibiting JNK activation, inhibiting endogenous TNF-alpha expression, promoting immune cell proliferation and immune cell activation (e.g., in Th1 cells and/or Th2), activating IL-2 expression e.g., by activating the AP-1 transcription factor, and increasing actin polymerization. The present invention also demonstrates that KRC interacts with TRAF. Furthermore, the present invention demonstrates that KRC physically interacts with the c-Jun component of AP-1 to control its degradation. The present invention also demonstrates that KRC is downstream of several lymphocyte membrane receptors, including TNFR, TCR and TGFβR. Upon TNFR signaling, KRC associates with the adaptor protein TRAF2 to inhibit NF K B and JNK-dependent gene expression. Upon TCR stimulation, KRC expression is rapidly induced and KRC physically associates with the c-Jun transcription factor to augment AP-1 dependent gene transcription. KRC knock-out (KO) T cells have impaired production of AP-1-dependent genes such as CD69 and IL-2. Upon TCR stimulation KRC also associates with the Th2-specific transcription factor GATA3, and T cells lacking KRC have impaired production of GATA3 dependent Th2 cytokines, IL-4, IL-5 and IL-13. Finally, upon TGFβ receptor signaling, KRC physically associates with the transcription factor SMAD3 to activate IgA germline transcription in B cells, since KRC KO B cells have impaired IgA production and germline Igα (GLα) gene transcription. Methods for identifying modulators of KRC activity are provided. Methods for modulating an immune response and KRC-associated disorders using agents that modulate KRC expression and/or activity are also provided.

1 . A method for identifying a compound which modulates an interaction between a first and a second polypeptide comprising: 
 (a) contacting a cell having a first polypeptide comprising a binding portion of a KRC polypeptide and a second polypeptide comprising a binding portion of a polypeptide selected from the group consisting of: GATA3, SMAD, or Runx2 in the presence and the absence of a test compound; and    (b) determining the degree of interaction between the first and the second polypeptide in the presence and the absence of the test compound,    to thereby identify a compound which modulates an interaction between a first and a second polypeptide.    
     
     
         2 . The method of  claim 1 , wherein the first polypeptide comprises at least one KRC zinc finger domain.  
     
     
         3 . (canceled)  
     
     
         4 . The method of  claim 1 , wherein the second polypeptide is a SMAD2 polypeptide.  
     
     
         5 . The method of  claim 1 , wherein the second polypeptide is a SMAD3 polypeptide.  
     
     
         6 . The method of  claim 1 , wherein the first polypeptide is derived from an exogenous source.  
     
     
         7 . The method of  claim 1 , wherein the second polypeptide is derived from an exogenous source.  
     
     
         8 . The method of  claim 1 , wherein the cell is a yeast cell.  
     
     
         9 . The method of  claim 8 , wherein determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the compound to modulate growth of the yeast cell on nutritionally selective media.  
     
     
         10 . The method of  claim 8 , wherein determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the compound to modulate expression of a reporter gene in the yeast cell.  
     
     
         11 . The method of  claim 1 , wherein determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate the coimmunoprecipitation of the first polypeptide and the second polypeptide.  
     
     
         12 . The method of  claim 1 , wherein determining the ability of the test compound to modulate the interaction of the first polypeptide and the second polypeptide comprises determining the ability of the test compound to modulate signaling via a signal transduction pathway involving KRC in the cell.  
     
     
         13 . The method of  claim 12 , wherein at least one of TNFα production, IL-2 production, AP-1 activity, Ras and Rac activity, actin polymerization, ubiquitination of AP-1, ubiquitination of TRAF, ubiquitination of Runx2, degradation of c-Jun, degradation of c-Fos degradation of SMAD, degradation of Runx2, degradation of GATA3, GATA3 expression, Th2 cell differentiation, Th2 cytokine production, IgA production, GLα transcription (Igα chain germline transcription), and/or osteocalcin gene transcription is measured.  
     
     
         14 . The method of  claim 12 , wherein ubiquitination or degradation of c-fos, c-Jun, SMAD3, GATA3 or Runx2 is measured.  
     
     
         15 . The method of  claim 12 , wherein AP-1, TRAF2 or Runx2 ubiquitination is measured.  
     
     
         16 . The method of  claim 1 , wherein the binding of first and second polypeptide is inhibited.  
     
     
         17 . The method of  claim 1 , wherein the binding of first and second polypeptide is stimulated.  
     
     
         18 . A method of identifying a compound that modulates a mammalian KRC biological activity comprising: 
 (a) contacting cells deficient in KRC or a molecule in a signaling pathway involving KRC with a test compound; and    (b) determining the effect of the test compound on the KRC biological activity, the test compound being identified as a modulator of the biological activity based on the ability of the test compound to modulate the biological activity in the cells deficient in KRC or a molecule in a signaling pathway involving KRC to thereby identify a compound that modulates a mammalian KRC biological activity.    
     
     
         19 . The method of  claim 18 , wherein the biological activity of KRC is selected from the group consisting of modulation of: modulation of a TGFβ signaling pathway, modulation of ubiquitination of AP-1, modulation of ubiquitination of TRAF, modulation of ubiquitination of Runx2, modulation of the degradation of c-Jun, modulation of the degradation of c-Fos, modulation of degradation of SMAD, modulation of degradation of Runx, modulation of degradation of GATA3, modulation of GATA3 expression, modulation of Th2 cell differentiation, modulation of Th2 cytokine production, modulation of IgA production, modulation of GLα transcription, or modulation of osteocalcin gene transcription.  
     
     
         20 . The method of  claim 18 , wherein the cells are in a non-human animal deficient in KRC or a molecule in a signal transduction pathway involving KRC and the cells are contacted with the test compound by administering the test compound to the animal.  
     
     
         21 . A method of identifying compounds useful in modulating a biological activity of mammalian KRC comprising: 
 a) providing an indicator composition comprising mammalian KRC or a molecule in a signal transduction pathway involving KRC;    b) contacting the indicator composition with each member of a library of test compounds;    c) selecting from the library of test compounds a compound of interest that modulates a biological activity of KRC or the molecule in a signal transduction pathway involving KRC; to thereby identify a compound that modulates a biological activity of mammalian KRC, wherein the biological activity of KRC is selected from the group consisting of: modulation of ubiquitination of Runx2, modulation of degradation of SMAD, modulation of degradation of Runx, modulation of degradation of GATA3, modulation of GATA3 expression, modulation of Th2 cell differentiation, modulation of Th2 cytokine production, modulation of IgA production, modulation of GLα transcription, and modulation of osteocalcin gene transcription.    
     
     
         22 . The method of  claim 21 , wherein the indicator composition is a cell that expresses KRC, and at least one molecule selected from the group consisting of: GATA3, SMAD, and Runx2 protein.  
     
     
         23 . The method of  claim 21 , wherein the indicator composition is a cell free composition.  
     
     
         24 - 45 . (canceled)  
     
     
         46 . A non-human animal, in which the gene encoding the KRC gene is misexpressed.  
     
     
         47 . The animal of  claim 46 , wherein the animal is a transgenic animal.  
     
     
         48 . The animal of  claim 47 , wherein the transgenic animal is a mouse.  
     
     
         49 . The animal of  claim 46 , wherein the KRC gene is disrupted by removal of DNA encoding all or part of the KRC protein.  
     
     
         50 . The animal of  claim 49 , wherein the animal is homozygous for the disrupted gene.  
     
     
         51 . The animal of  claim 49 , wherein the animal is heterozygous for the disrupted gene.  
     
     
         52 . The animal of  claim 46 , wherein the animal is a transgenic mouse with a transgenic disruption of the KRC gene.  
     
     
         53 . The animal of  claim 52 , wherein the disruption is an insertion or deletion.  
     
     
         54 . A transgenic mouse comprising in its genome an exogenous DNA molecule that functionally disrupts a KRC gene of said mouse, wherein the mouse exhibits a phenotype characterized by impaired Th2 cell development, decreased Th2 cytokine production, impaired TGFβR signaling in B cells, decreased IgA secretion and decreased transcription of the GLα gene, relative to a wildtype mouse.