Compositions and methods for monitoring the phosphorylation of natural binding partners

Abstract

This invention relates to methods and compositions for monitoring the interaction of binding partners as a function of the addition or subtraction of a phosphate group to or from one of the binding partners by a protein kinase or phosphatase.

What is claimed is:  
     
         1 . An isolated natural binding domain and a binding partner therefor, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation or dephosphorylation of said site.  
     
     
         2 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein said phosphorylation or dephosphorylation is performed by an enzyme which is a kinase or a phosphatase, respectively.  
     
     
         3 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein phosphorylation of said site prevents binding of said isolated natural binding domain to said binding partner.  
     
     
         4 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein phosphorylation of said site promotes binding of said isolated natural binding domain to said binding partner.  
     
     
         5 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein dephosphorylation of said site prevents binding of said isolated natural binding domain to said binding partner.  
     
     
         6 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein dephosphorylation of said site promotes binding of said isolated natural binding domain to said binding partner.  
     
     
         7 . The isolated natural binding domain and binding partner therefor of  claim 1 , wherein at least one of said isolated natural binding domain and said binding partner comprises a detectable label.  
     
     
         8 . The isolated natural binding domain and binding partner therefor of  claim 7 , wherein said detectable label emits light.  
     
     
         9 . The isolated natural binding domain and binding partner therefor of  claim 8 , wherein said light is fluorescent.  
     
     
         10 . The isolated natural binding domain and binding partner therefor of  claim 9 , wherein one of said isolated natural binding domain and said binding partner comprises a quencher for said detectable label.  
     
     
         11 . A kit comprising an isolated natural binding domain and a binding partner therefor, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and packaging material therefor.  
     
     
         12 . The kit of  claim 11 , wherein said kit further comprises a buffer which permits phosphorylation-dependent binding of said isolated natural binding domain and said binding partner.  
     
     
         13 . The kit of  claim 12 , wherein said buffer permits phosphorylation or dephosphorylation of said site by a kinase or a phosphatase, respectively.  
     
     
         14 . The kit of  claim 11 , wherein said kit further comprises one or both of a kinase and a phosphatase.  
     
     
         15 . The kit of  claim 14 , wherein said kit further comprises a substrate for said phosphatase or kinase, said substrate being MgATP.  
     
     
         16 . The kit of  claim 14 , wherein said kit further comprises a cofactor for one or both of said kinase and phosphatase.  
     
     
         17 . The kit of  claim 11 , wherein at least one of said isolated natural binding domain and said binding partner comprises a detectable label.  
     
     
         18 . The kit of  claim 17 , wherein said detectable label emits light.  
     
     
         19 . The kit of  claim 18 , wherein said light is fluorescent.  
     
     
         20 . A method for monitoring activity of an enzyme comprising performing a detection step to detect binding of an isolated natural binding domain and a binding partner therefor as a result of contacting one or both of said isolated natural binding domain and said binding partner with said enzyme, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of enzyme activity.  
     
     
         21 . A method for monitoring activity of an enzyme comprising performing a detection step to detect dissociation of an isolated natural binding domain from a binding partner therefor as a result of contacting one or both of said isolated natural binding domain and said binding partner with said enzyme, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site and wherein detection of dissociation of said isolated natural binding domain from said binding partner as a result of said contacting is indicative of enzyme activity.  
     
     
         22 . The method of  claim 20  or  21 , wherein at least one of said isolated natural binding domain and said binding partner is labeled with a detectable label.  
     
     
         23 . The method of  claim 22 , wherein said label emits light.  
     
     
         24 . The method of  claim 23 , wherein said light is fluorescent.  
     
     
         25 . The method of  claim 22  wherein said detection step is to detect a change in signal emission by said detectable label.  
     
     
         26 . The method according to  claim 25 , wherein said method further comprises exciting said detectable label and monitoring fluorescence emission.  
     
     
         27 . The method according to  claim 25 , wherein said method further comprises the step, prior to or after said detection step, of contacting said isolated natural binding domain and said binding partner with an agent which modulates the activity of said enzyme.  
     
     
         28 . A method for monitoring the activity of a modulator of the activity of an enzyme comprising: 
 a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said isolated natural binding domain, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs prior to said mixing step; and    b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.    
     
     
         29 . A method for monitoring the activity of a modulator of the activity of an enzyme comprising: 
 a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said isolated natural binding domain, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs during said mixing step; and    b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.    
     
     
         30 . A method for monitoring the activity of a modulator of the activity of an enzyme comprising: 
 a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said binding partner, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs prior to said mixing step; and    b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.    
     
     
         31 . A method for monitoring the activity of a modulator of the activity of an enzyme comprising: 
 a) mixing an isolated natural binding domain, a binding partner of said isolated natural binding domain, said enzyme, and a candidate modulator which binds to said binding partner, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner dependent upon phosphorylation of said site, and wherein the combination of said isolated natural binding domain and said binding partner comprises detection means for monitoring association or dissociation of said isolated natural binding domain and said binding partner, and wherein detection of binding of said isolated natural binding domain and said binding partner as a result of said mixing is indicative of enzyme activity, and wherein said phosphorylation of said site occurs during said mixing step; and    b) monitoring association or dissociation of said isolated natural binding domain and said binding partner, said association or dissociation being indicative of modulation by said candidate modulator of said activity, wherein said modulator reduces binding of said isolated natural binding domain and said binding partner and wherein a reduction in binding is detected by said detection means.    
     
     
         32 . The method of  claim 28 ,  29 ,  30  or  31 , wherein at least one of said isolated natural binding domain and said binding partner is labeled with a detectable label.  
     
     
         33 . The method of  claim 32 , wherein said label emits light.  
     
     
         34 . The method of  claim 33 , wherein said light is fluorescent.  
     
     
         35 . The method of  claim 34 , wherein said detection step is to detect a change in signal emission by said detectable label.  
     
     
         36 . The method of  claim 35 , wherein said method further comprises exciting said detectable label and monitoring fluorescence emission.  
     
     
         37 . The method of  claim 35 , wherein said method further comprises the step, prior to or after said detection step, of contacting said isolated natural binding domain and said binding partner with an agent which modulates the activity of said enzyme.  
     
     
         38 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said isolated natural binding domain, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and    b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.    
     
     
         39 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds to said isolated natural binding domain, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs during said mixing, and    b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.    
     
     
         40 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said binding partner, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs prior to mixing, and    b) monitoring the binding of said isolated natural binding domain to said binding partner, wherein binding or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.    
     
     
         41 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an isolated natural binding domain, a binding partner therefor and an enzyme with a candidate modulator of said kinase or phosphatase which binds said binding partner, wherein said natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by said kinase or phosphatase and wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner, and wherein said phosphorylation or dephosphorylation occurs during said mixing, and    b) monitoring the association or dissociation of said isolated natural binding domain to said binding partner, wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of said kinase or phosphatase.    
     
     
         42 . The method of  claim 38 ,  39 ,  40  or  41 , wherein said detectable label emits light.  
     
     
         43 . The method of  claim 42 , wherein said light is fluorescent.  
     
     
         44 . The method of  claim 43 , wherein said monitoring comprises measuring a change in energy transfer between a detectable label present on said isolated natural binding domain and a detectable label present on said binding partner.  
     
     
         45 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an assay system comprising an isolated natural binding domain and a binding partner for said isolated natural binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said isolated natural binding domain, and    b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.    
     
     
         46 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an assay system comprising an isolated natural binding domain and a binding partner for said isolated natural binding domain with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said isolated natural binding domain, and    b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs during said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.    
     
     
         47 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an assay system comprising an isolated natural binding domain and a binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said binding partner, and    b) monitoring association or dissociation of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs prior to said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.    
     
     
         48 . A method of screening for a candidate modulator of enzymatic activity of a kinase or a phosphatase, the method comprising 
 a) mixing an assay system comprising an isolated natural binding domain and a binding partner with a candidate modulator of enzymatic activity of a said kinase or phosphatase which binds to said binding partner, and    b) monitoring binding of an isolated natural binding domain and a binding partner therefor in said assay system, wherein said isolated natural binding domain includes a site for post-translational phosphorylation and binds said binding partner in a manner that is dependent upon phosphorylation or dephosphorylation of said site by a said kinase or phosphatase in said assay system, wherein the combination of said isolated natural binding domain and said binding partner comprises a detection means for monitoring association or dissociation between said isolated natural binding domain and said binding partner and wherein said phosphorylation or dephosphorylation occurs during said mixing, and wherein association or dissociation of said isolated natural binding domain and said binding partner as a result of said contacting is indicative of modulation of enzymatic activity by said candidate modulator of a said kinase or phosphatase.    
     
     
         49 . The method of  claim 28 ,  29 ,  30 ,  31 ,  38 ,  39 ,  40 ,  41 ,  45 ,  46 ,  47 , or  48 , wherein said method comprises real-time observation of association or dissociation of a said isolated natural binding domain and its binding partner.