Use of the regulatory subunit of the cAMP dependent protein kinase (PKA) from dictyostelium for cAMP measurements

Abstract

The invention relates to the use of the regulatory subunit (R) of the CAMP dependent protein kinase (PKA) from Dictyostelium discoideum for cAMP detection. It includes constructs for expression of the R-subunit in E. coli and fusion to green fluorescent proteins (GFP). Fluorescence energy transfer is used as a way to monitor cAMP binding, either by using fluorescently labelled cAMP or cGMP, or by using mutant GFPs with modified absorption and emission spectra. FRET changes upon cAMP binding will allow measurement of cAMP level either in vitro or within living cells.

1 . A DNA construct for the preparation of a fusion product, which construct comprises the coding sequence of at least one CAMP binding site of the regulatory subunit (R) of a cAMP dependent protein kinase that is unable to dimerise, which coding sequence is operably linked to a DNA sequence encoding a reporter polypeptide, wherein the fusion product is for use in the measurement of cAMP concentration.  
     
     
         2 . The DNA construct of  claim 1 , wherein the CAMP dependent protein kinase is from  Dictyostelium discoideum.    
     
     
         3 . The DNA construct of  claim 1  or  2 , wherein the DNA sequence encoding the reporter protein is inserted in frame within said regulatory subunit.  
     
     
         4 . The DNA construct of  claim 2  or  3 , wherein the DNA sequence encoding the reporter protein is inserted at base 510 within the R subunit, resulting in the production of an R-protein that is truncated after amino acid 170 and fused to the reporter protein.  
     
     
         5 . The DNA construct of  claim 4 , which in the construct R26 of FIG. 1.  
     
     
         6 . The DNA construct of  claim 2  or  3 , wherein the DNA sequence encoding the reporter protein is inserted in frame after base 147 within the R subunit DNA sequence.  
     
     
         7 . The DNA construct of  claim 6 , which le the construct R28 of FIG. 1.  
     
     
         8 . The DNA construct of claims  2  or  3 , wherein the DNA sequence encoding the reporter protein is inserted in frame after base 792 within the R subunit DNA sequence.  
     
     
         9 . The DNA construct of  claim 8 , which is the construct R33 of FIG. 1.  
     
     
         10 . The DNA construct of  claims 1  to  9 , wherein a second DNA sequence encoding a reporter protein is inserted in frame within said R subunit DNA sequence.  
     
     
         11 . The DNA construct of  claim 10 , wherein both DNA sequences encoding a reporter protein are located outside one CAMP binding site on the R subunit DNA sequence.  
     
     
         12 . The DNA construct of claims  10  or  11 , wherein the DNA sequence encoding the first reporter protein is inserted at position 147 within the R subunit DNA sequence, and the DNA sequence encoding the second reporter protein is inserted at position 792 within the R subunit DNA sequence.  
     
     
         13 . The DNA construct of  claim 12 , which is the construct Double of FIG. 1.  
     
     
         14 . The DNA construct of claims  1 - 13 , wherein at least one of the DNA sequences encoding a reporter protein encodes a fluorescent protein.  
     
     
         15 . The DNA construct of  claim 14 , wherein the DNA sequence encoding the fluorescent reporter protein encodes a green fluorescent protein (GFP) from  Aeguorea victoria.    
     
     
         16 . The DNA construct of  claim 14  or  15 , wherein the fluorescent protein is a GFP mutant w7.  
     
     
         17 . The DNA construct of  claim 14  or  15 , wherein the fluorescent protein is a GFP mutant S65T.  
     
     
         18 . The DNA construct of claims  10 - 17 , wherein the location of the DNA sequences encoding the fluorescent proteins within the construct leads to the capability of fluorescence energy transfer (FRET) between the fluorescent proteins in the fusion product.  
     
     
         19 . The DNA construct of claims  10 - 18 , wherein the location of the DNA sequences encoding the fluorescent proteins within the construct is such that in the fusion product the fluorescent proteins will be located on the same face of the regulatory subunit tertiary structure.  
     
     
         20 . The DNA construct of claims  10 - 19 , wherein the location of the DNA sequences encoding the fluorescent proteins within the construct is such that in the fusion product the fluorescent proteins are placed in such locations that FRET changes upon binding to the catalytic subunit (C).  
     
     
         21 . The DNA construct of claims  10 - 20 , wherein the location of the DNA sequences encoding the fluorescent proteins within the construct is such that in the fusion product the fluorescent proteins are placed in such locations that FRET changes upon CAMP binding.  
     
     
         22 . The DNA construct of claims  10 - 21 , wherein the location of the DNA sequences encoding the fluorescent proteins within the construct is such that in the fusion product the distance between the two fluorescent proteins is about 4 Å.  
     
     
         23 . Method for the preparation of a measuring tool for measurement of cAMP concentration, comprising 
 a) introducing a DNA construct as claimed in claims  1 - 22  in a suitable host cell;    b) expressing the fusion protein encoded by the DNA construct in the host cell; and    c) isolating the fusion protein, which is the tool for measurement of cAMP concentration.    
     
     
         24 . Method as claimed in  claim 23 , wherein the host is a bacterial host, in particular  Escherichia coli.    
     
     
         25 . Method as claimed in  claim 23  or  24 , wherein the fusion protein is isolated by means of Ni- and cAMP-affinity and size fractionation.  
     
     
         26 . Fusion protein for measuring the cAMP concentration encoded by a construct as claimed in claims  1 - 22 .  
     
     
         27 . Fusion protein as claimed in  claim 26  obtainable by a method as claimed in claims  23 - 25 .  
     
     
         28 . Method for measuring the CAMP concentration in a biological fluid, comprising 
 a) adding a fusion protein as claimed in claims  26  or  27  together with a defined concentration of fluorescent cyclic nucleotide to the biological fluid,    b) recording fluorescence emission and determining CAMP concentration in the biological fluid by comparing the values of the fluorescence optimum with a standard curve obtained with defined concentrations of cAMP.    
     
     
         29 . Method as claimed in  claim 28 , in which the fluorescent nucleotide is ( 8 -{{2-{(Fluoresceinylthioureido)amino}ethyl}thio} guanosine-3′,5′-cyclic monophosphate (cGMP).  
     
     
         30 . Method as claimed in  claim 28 , in which the fluorescent nucleotide is ( 8 -{{2-{(Fluoresceinylthioureido)amino}ethyl}thio} adenosine-3′,5′-cyclic monophosphate (cAMP).  
     
     
         31 . Method for inserting fluorescence donor and acceptor proteins in a cAMP dependent protein kinase regulatory subunit in order to obtain Fluorescence energy transfer (FRET), which method comprises: 
 a) placing a DNA construct as claimed in claims  1 - 22  in a suitable expression vector,    b) transforming either procaryotic or eukaryotic cells with the suitable expression vector containing the DNA construct;    c) measuring FRET in living cells or extracts using the ratio of emission peaks from acceptor and donor fluorescent proteins.    
     
     
         32 . Method as in  claim 31 , in which the DNA construct is designed such that the fluorescence donor and acceptor proteins encoded by it are placed on the same side of the regulatory subunit.  
     
     
         33 . Method as in  claim 31  or  32 , in which the DNA construct is designed such that the fluorescence donor and acceptor proteins encoded by it are placed on both sides of a cAMP binding domain.  
     
     
         34 . Method as in claims  31 - 33 , in which the fluorescence donor and acceptor proteins are GFPs  
     
     
         35 . Method as in  claim 34 , in which the fluorescence donor and acceptor proteins are mutants w7 and S65T.  
     
     
         36 . Method as claimed in claims  31 - 35 , in which the FRET is modified by binding of the catalytic subunit to the regulatory subunit.  
     
     
         37 . Method as claimed in claim  31 - 36 , in which the FRET is modified by binding of cAMP to the regulatory subunit.