Methods and kits for detecting protein kinases

Abstract

Methods and kits for detecting kinase activity A method for measuring protein kinase activity comprising: (a) providing a first solution comprising ATP and a protein kinase to be tested, and a second solution comprising ATP in the absence of said kinase to be tested; (b) adding a substrate capable of being phosphorylated by the protein kinase to be tested to the first and second solutions of step (a); (c) measuring the concentration of ATP and/or ADP, or the rate of change thereof with respect to time, in each of the reaction mixtures formed in step (b) using a bioluminescence reaction; and (d) using the information about the concentration of ATP and/or ADP to determine the activity of the protein kinase to be tested.

1 - 42 . (Canceled)  
     
     
         43 . A method for detecting protein kinase activity comprising 
 (a) establishing a reaction mixture comprising ATP, a protein kinase to be tested and a substrate capable of being phosphorylated by the protein kinase; and    (b) using a bioluminescence reaction to detect whether any change in ATP concentration occurs.    
     
     
         44 . A method for determining protein kinase activity comprising 
 (a) establishing a reaction mixture comprising ATP, a protein kinase to be tested and a substrate capable of being phosphorylated by the protein kinase;    (b) using a bioluminescence reaction to detect whether any change in ATP concentration occurs, and    (c) using the detection of step (b) to obtain information for determining protein kinase activity.    
     
     
         45 . A method for identifying a compound which modulates the activity of a protein kinase, said method comprising: 
 (a) establishing a reaction mixture comprising ATP, a protein kinase to be tested, a substrate capable of being phosphorylated by the protein kinase and a compound to be tested for an ability to modulate the activity of the protein kinase;    (b) using a bioluminescence reaction to detect whether any change in ATP concentration occurs;    (c) using the detection of step (b) to obtain information for identifying whether the compound modulates the activity of the protein kinase.    
     
     
         46 . The method of  claim 43 ,  44  or  45  wherein the reaction mixture is substantially cell-free.  
     
     
         47 . The method of  claim 45  wherein the compound to be tested is identified as a protein kinase inhibitor if the activity of the kinase is lower in the presence of the compound.  
     
     
         48 . The method of  claim 45  wherein the compound to be tested is identified as a protein kinase activator if the activity of the kinase is higher in the presence of the compound.  
     
     
         49 . The method of  claim 43 ,  44 , or  45  wherein the kinase is activated prior to step (a).  
     
     
         50 . The method of  claim 43 ,  44  or  45  wherein the reaction mixture comprises a buffer.  
     
     
         51 . The method of  claim 50  wherein the buffer is Hepes buffer.  
     
     
         52 . The method of  claim 43 ,  44 , or  45  wherein phosphorylation is allowed to proceed at room temperature prior to step (b).  
     
     
         53 . The method of  claim 52  comprising a further step (a′), carried out after step (a) and before step (b), of adding a reagent to the reaction mixture for stopping phosphorylation of the substrate.  
     
     
         54 . The method of  claim 53  wherein the reagent for stopping phosphorylation is selected from the group consisting of an acid, EGTA and EDTA.  
     
     
         55 . The method of  claim 54  comprising a further step (a″), carried out after step (a′) and before step (b), of adjusting the pH of the reaction mixture formed in step (a∝) to pH 7.0.  
     
     
         56 . The method of  claim 55  wherein step (a″) comprises adding Hepes buffer.  
     
     
         57 . The method of  claim 43 ,  44  or  45  wherein step (b) comprises: 
 (i) adding a bioluminescent reagent comprising luciferin or a derivative thereof and a luciferase to said reaction mixture, said luciferin or a derivative thereof emitting light in a bioluminescent reaction with the luciferase in the presence of ATP; and  
 (ii) detecting a light intensity, or a change of light intensity with time, emitted by the bioluminescent reaction.  
 
     
     
         58 . The method of  claim 57  wherein step (b) further comprises the following steps carried out after the light intensity detected in step (ii) has reached a substantially constant level: 
 (iii) adding a reagent that converts ADP to ATP;  
 (iv) adding a bioluminescent reagent comprising luciferin or a derivative thereof and luciferase to said reaction mixture of step (iii), said luciferin or a derivative thereof emitting light in a bioluminescent reaction with the luciferase in the presence of ATP; and  
 (v) detecting light intensity emitted by the bioluminescent reaction  
 wherein the difference in the intensity of light in step (v) and the steady state intensity of light in step (ii) is a measure of ADP concentration in the reaction mixture of step (ii).  
 
     
     
         59 . The method of  claim 45  wherein the kinase is JNK-1 and the substrate is GST-c-jun.  
     
     
         60 . The method of  claim 45  wherein the kinase is MAP Kinase-1 (ERK-1) and the substrate is myelin basic protein.  
     
     
         61 . The method of  claim 45  wherein the kinase is MAP Kinase-2 (ERK-2) and the substrate is myelin basic protein.  
     
     
         62 . The method of  claim 45  wherein the kinase is PKA and the substrate is Kemptide.  
     
     
         63 . The method of  claim 45  wherein the kinase is JNK-2 and the substrate is GST-c-jun.  
     
     
         64 . The method of  claim 45  wherein the kinase is MEK-1 and the substrate is inactive MAP Kinase-2 (ERK-2).  
     
     
         65 . The method of  claim 45  wherein the kinase is JNKα and the substrate is ATF-2.  
     
     
         66 . The method of  claim 45  wherein the kinase is JNKα and the substrate is c-jun.  
     
     
         67 . The method of  claim 45  wherein the kinase is SAPK-3 and the substrate is myelin.  
     
     
         68 . A kit for detecting protein kinase activity comprising: 
 (a) a bioluminescent reagent comprising luciferin or a derivative thereof and a luciferase, said luciferin or a derivative thereof emitting light in a bioluminescent reaction with the luciferase in the presence of ATP;    (b) a kinase;    (c) a substrate capable of being phosphorylated by said kinase; and    (d) ATP.    
     
     
         69 . The kit of  claim 68  further comprising one or more buffers for reconstituting, diluting or dissolving the bioluminescent reagent, kinase, substrate and/or ATP.  
     
     
         70 . The kit of  claim 68  further comprising a reagent capable of stopping the reaction of said kinase with said substrate.  
     
     
         71 . The kit of  claim 68  further comprising one or more reagent(s) which converts ADP to ATP.  
     
     
         72 . The kit of  claim 71 , wherein the reagent which converts ADP to ATP comprises pyruvate kinase and phosphoenol pyruvate.  
     
     
         73 . The kit of  claim 68  further comprising two or more different kinases and corresponding substrates.  
     
     
         74 . The kit of  claim 68  wherein the reagent or reagents is or are provided in lyophilised form.  
     
     
         75 . The kit of  claim 68  further comprising a multiwell microtitre plate.  
     
     
         76 . The kit of  claim 75  wherein the multiwell microtitre plate contains 96 wells or more.  
     
     
         77 . A compound identified using a method according to  claim 45.