Methods and compositions for treating abnormal cell growth related to unwanted guanine nucleotide exchange factor activity

Abstract

Methods and compositions are described that affect the GTPase activity of members of the Ras superfamily, preferably Rac, such compositions include guanine nucleotide exchange factors that modulate the GTPase activity, preferably in the presence of GEF enhancers, exemplary guanine nucleotide exchange factors being Rac-GEF and Tiam-1, which are encoded by certain nucleic acid sequences that are herein described, along with uses for the guanine nucleotide exchange factors and the nucleic acid sequences including screening for ligands which recognize Rac-GEF, regulators of Rac-GEF activity, and methods of treating pathological conditions associated or related to a Ras superfamily GTPase, including Rac.

What is claimed:  
     
         1 . An isolated Rac-GEF polypeptide or a biologically-active fragment thereof.  
     
     
         2 . An isolated Rac-GEF, or a biologically-active fragment thereof, of  claim 1 , wherein said polypeptide has a guanine nucleotide exchange activity, a specific binding affinity for a guanine nucleotide depleted Rac, or a cellular oncogenic transforming activity.  
     
     
         3 . An isolated Rac-GEF or a biologically-active fragment thereof of  claim 1  which is of human.  
     
     
         4 . An isolated Rac-GEF of  claim 1  comprising amino acid 1 to amino acid 711, as set forth in FIG. 1 (SEQ. ID NO:2).  
     
     
         5 . An isolated biologically-active fragment of Rac-GEF of  claim 4  which comprises amino acids 273-605.  
     
     
         6 . An isolated Rac-GEF, or a biologically-active fragment thereof, of  claim 1 , which is substantially purified.  
     
     
         7 . An isolated nucleic acid comprising a nucleotide sequence coding for a Rac-GEF polypeptide.  
     
     
         8 . An isolated nucleic acid of  claim 7 , wherein said coded for polypeptide has a guanine nucleotide exchange activity, a specific binding affinity for a guanine nucleotide depleted Rac, or a cellular oncogenic transforming activity.  
     
     
         9 . An isolated nucleic acid of  claim 7  which is human.  
     
     
         10 . An isolated nucleic acid of  claim 7 , wherein the nucleic acid sequence codes for amino acid 1 to amino acid 711, as set forth in FIG. 1 (SEQ. ID NO:2).  
     
     
         11 . An isolated nucleic acid of  claim 7 , wherein the nucleotide sequence is operably linked to an expression control sequence.  
     
     
         12 . An isolated nucleic acid of  claim 7 , wherein the nucleic acid comprises a naturally-occurring nucleotide sequence.  
     
     
         13 . An isolated nucleic acid of  claim 7 , wherein the nucleic acid codes for said polypeptide without interruption.  
     
     
         14 . An isolated nucleic acid of  claim 7 , wherein the nucleic acid is DNA or RNA.  
     
     
         15 . An isolated nucleic acid of  claim 7 , wherein the nucleic acid further comprises a detectable label.  
     
     
         16 . An isolated nucleic acid of  claim 7 , except where one or more amino acid positions are substituted or deleted, or both, and the polypeptide coded for by the nucleic acid is biologically-active.  
     
     
         17 . An isolated nucleic acid of  claim 16 , wherein the biological activity is a guanine nucleotide exchange activity, a specific binding affinity for a guanine nucleotide depleted G-protein, or a cellular oncogenic transforming activity.  
     
     
         18 . An isolated nucleic acid of  claim 16 , wherein the one or more substituted amino acid positions are substituted by conservative amino acids.  
     
     
         19 . An isolated nucleic acid of  claim 16 , wherein the one or more substituted amino acid positions is in the Dbl homology domain or the pleckstrin homology domain.  
     
     
         20 . An isolated nucleic acid comprising a nucleotide sequence which hybridizes, or whose nucleic acid complement hybridizes, under stringent conditions to base pairs of nucleotide sequence 900-1482 as set forth in FIG. 1 (SEQ. ID NO:1).  
     
     
         21 . An isolated nucleic acid  claim 20  comprising at least 95% nucleotide sequence identity to the nucleotide sequence set forth in  claim 20 .  
     
     
         22 . An isolated nucleic acid of  claim 20 , wherein said nucleic acid codes for a polypeptide having a guanine nucleotide exchange activity, a specific binding affinity for a guanine nucleotide depleted Rac, or a cellular oncogenic transforming activity.  
     
     
         23 . An isolated nucleic acid comprising a nucleotide sequence which is unique to Rac-GEF.  
     
     
         24 . An isolated nucleic acid comprising a nucleotide sequence which hybridizes, or whose nucleic acid complement hybridizes, under stringent conditions to the unique nucleotide sequence of  claim 23 .  
     
     
         25 . An isolated nucleic acid of  claim 24  which codes for a polypeptide having a guanine nucleotide exchange activity, a specific binding affinity for a guanine nucleotide depleted Rac, or a cellular oncogenic transforming activity.  
     
     
         26 . A method of expressing in transformed host cells, a Rac-GEF polypeptide coded for by a nucleic acid, comprising culturing transformed host cells containing a nucleic acid according to  claim 7  under conditions effective to express the polypeptide.  
     
     
         27 . A method of expressing, in transformed host cells, a polypeptide coded for by a nucleic acid, comprising culturing transformed host cells containing a nucleic acid according to  claim 20  under conditions effective to express the polypeptide.  
     
     
         28 . A method of  claim 26 , further comprising isolating the polypeptide.  
     
     
         29 . A method of  claim 26 , further comprising modulating expression of the polypeptide.  
     
     
         30 . An isolated polypeptide produced by a method of  claim 26 .  
     
     
         31 . An isolated polypeptide produced by a method of  claim 27 .  
     
     
         32 . A transformed host cell containing a nucleic acid of  claim 7 .  
     
     
         33 . A transformed host cell containing a nucleic acid of  claim 20 .  
     
     
         34 . A vector comprising a nucleic acid of  claim 7 .  
     
     
         35 . A vector comprising a nucleic acid of  claim 20 .  
     
     
         36 . A method of modulating an activity of a Rac polypeptide comprising, administering an effective amount of a Rac-GEF polypeptide or a biologically-active fragment thereof, or an effective amount of a compound which modulates the activity of the Rac-GEF.  
     
     
         37 . A method of  claim 36 , wherein the Rac-GEF, or biologically-active fragment thereof, comprises an amino acid sequence which has a specific binding activity for a guanine nucleotide depleted state of said Rac.  
     
     
         38 . A method of modulating an activity of a Rac polypeptide comprising; 
 introducing a nucleic acid of  claim 21  into said cell under conditions whereby said nucleic acid is expressed in an effective amount to modulate said activity of Rac in said cell.    
     
     
         39 . A method of  claim 38  wherein said nucleic acid oncogenically transforms said cell.  
     
     
         40 . A method of isolating a molecule that binds to a guanine nucleotide-depleted state of a Rac polypeptide comprising; 
 contacting a Rac polypeptide with a medium comprising said molecule under conditions effective for said molecule to bind to said Rac polypeptide; and    separating said Rac polypeptide to which said molecule has bound from said medium.    
     
     
         41 . A method of  claim 40 , wherein said molecule is Rac-GEF.  
     
     
         42 . A method of  claim 40 , wherein said molecule has a molecular weight of about 82.5 kilodaltons.  
     
     
         43 . A method of  claim 40 , further comprising separating said molecule from said Rac polypeptide.  
     
     
         44 . A method of modulating an activity of a GTPase comprising, administering an effective amount of a guanine nucleotide exchange factor or a biologically-active fragment thereof, or an effective amount of a compound which modulates the activity of the guanine nucleotide exchange factor.  
     
     
         45 . A method of  claim 44 , wherein the guanine nucleotide exchange factor, or biologically-active fragment thereof, comprises an amino acid sequence which has a specific binding activity for a guanine nucleotide depleted state of said GTPase.  
     
     
         46 . A method of testing for an agent which modulates the guanine nucleotide exchange activity of a guanine nucleotide exchange factor comprising: 
 contacting a mixture of (a) a polypeptide comprising a guanine nucleotide exchange factor, or a biologically-active fragment thereof, and (b) a polypeptide comprising a GTPase, or a biologically-active fragment thereof, to which the exchange factor can bind, with an agent; and    assaying for the presence or amount of guanine nucleotide exchange activity in the presence or absence of a GEF enhancer.    
     
     
         47 . A method of  claim 46 , wherein the GTPase is Rac.  
     
     
         48 . A method of  claim 47 , wherein the guanine nucleotide exchange factor is Rac GEF and paid GEF enhancer is ascorbyl stearate.  
     
     
         49 . An agent identified by the method of  claim 48 .  
     
     
         50 . A method of testing for an agent which modulates the binding between a guanine nucleotide exchange factor and a GTPase comprising: 
 contacting a mixture of (a) a polypeptide comprising a guanine nucleotide exchange factor, or a biologically-active fragment thereof, and (b) a polypeptide comprising a GTPase, or a biologically-active fragment thereof, to which the exchange factor can bind, with an agent; and    detecting the presence or amount of binding between the guanine nucleotide exchange factor polypeptide, or the biologically-active fragment thereof, and the GTPase.    
     
     
         51 . A method of  claim 50 , wherein the GTPase is Rac.  
     
     
         52 . A method of  claim 50 , wherein the guanine nucleotide exchange factor is Rac-GEF.  
     
     
         53 . An isolated agent identified by the method of  claim 50 .  
     
     
         54 . An isolated antibody which is specific for a Rac-GEF or a peptide comprising a sequence present therein.  
     
     
         55 . An isolated antibody of  claim 54 , which binds to an amino acid sequence selected from the group consisting of  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   H 2 NAFRELIAOLELDPKCOOH 
                     
                 
                     
                     
                 
                     
                   H 2 NYQERTYKLPFSSFLCOOH 
                 
                     
                     
                 
                     
                   H 2 NPQRSQNKDRRKLGSRNRQCOOH 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         56 . A method of increasing the guanine nucleotide exchange activity of a guanine nucleotide exchange factor, or a biologically-active fragment thereof, said factor capable of acting on a member of the Ras superfamily of GTPases, comprising the steps of: 
 contacting said guanine nucleotide exchange factor, or a biologically-active fragment thereof, with said member of the Ras superfamily of GTPases, or a biologically-active fragment thereof; and    assaying for guanine nucleotide exchange activity under appropriate conditions in the presence of a guanine nucleotide exchange factor enhancer.    
     
     
         57 . A method as described in  claim 56  wherein said member of the Ras superfamily of GTPases is Rac and said guanine nucleotide exchange factor is Tiam-1.  
     
     
         58 . A method as described in  claim 57  wherein said GEF enhancer is selected from the group consisting of ascorbyl stearate, ascorbyl palmitate, and phosphoinositol.  
     
     
         59 . A method as described in  claim 58  wherein said phosphoinositol is selected from the group consisting of PI3,4,5P 3 , PI4,5P 2  and PI4P.  
     
     
         60 . A method of assaying for a compound to treat disease resulting from increased guanine nucleotide exchange activity of a guanine nucleotide exchange factor, or a biologically-active fragment thereof, said factor acting on a member of the Ras superfamily of GTPases, comprising the steps of: 
 contacting said guanine nucleotide exchange factor, or a biologically-active fragment thereof, with said member of the Ras superfamily of GTPases, or a biologically-active fragment thereof;    assaying for guanine nucleotide exchange activity under appropriate conditions in the presence of a guanine nucleotide exchange factor enhancer, and in the presence and absence of said compound; and    determining if said compound decreases said guanine nucleotide exchange activity.    
     
     
         61 . A method as described in  claim 60  wherein said member of the Ras superfamily of GTPases is Rac.  
     
     
         62 . A method as described in  claim 61  wherein said guanine nucleotide exchange factor is Tiam-1.  
     
     
         63 . A method as described in  claim 62  wherein said GEF enhancer is selected from the group consisting of ascorbyl stearate, ascorbyl palmitate, and phosphoinositol.  
     
     
         64 . A method as described in  claim 63  wherein said phosphoinositol is selected from the group consisting of PI3,4,5P 3 , PI4,5P 2  and PI4P.  
     
     
         65 . Compounds of  claim 60  that decrease said guanine nucleotide exchange activity.  
     
     
         66 . Ligands that bind to the Src homology 3 domain on Rac-GEF.  
     
     
         67 . Ligands that bind to the Src homology 3 domain on Rac-GEF identified by the methods of claims  46  or  50 .