Emission ratiometric indicators of phosphoinositides

Abstract

Emission ratiometric indicators of phosphoinositides comprise a fusion protein comprising a pleckstrin homology (PH) domain of Akt (also known as protein kinase B) and a “pseudoligand” containing acidic amino acid residues, which is sandwiched between resonance energy transfer (RET) pairs, such as cyan and yellow mutants of GFP (a FRET pair). Such indicators can be used, inter alia, to monitor spatiotemporal dynamics of phosphoinositides and in high throughput assays for inhibitors of PI3K, including drug screening assays.

1 . A phosphoinositol (PI) indicator, wherein the PI indicator comprises:
 (a) a first polypeptide comprising a polypeptide donor moiety of a FRET pair;   (b) a second polypeptide comprising a pleckstrin homology domain of protein kinase B (SEQ ID NO:2), wherein the N terminus of the second polypeptide is linked to the C terminus of the first polypeptide;   (c) a third polypeptide comprising a pseudoligand peptide sequence (SEQ ID NO:3) for the protein kinase B, wherein the N terminus of the third polypeptide is linked to the C terminus of the second polypeptide; and   (d) a fourth polypeptide comprising a polypeptide acceptor moiety of the FRET pair, wherein the N terminus of the fourth polypeptide is linked to the C terminus of the third polypeptide and wherein the polypeptide donor moiety and the polypeptide acceptor moiety exhibit a detectable resonance energy transfer when the donor moiety is excited.   
     
     
         2 . The PI indicator of  claim 1  wherein the FRET pair is selected from the group consisting of (1) a cyan fluorescent protein and a yellow fluorescent protein; and (2) a green fluorescent protein and a red fluorescent protein. 
     
     
         3 . The PI indicator of  claim 1 , further comprising (e) a fifth polypeptide comprising a subcellular targeting peptide sequence linked to either the N terminus or the C terminus of the PI indicator. 
     
     
         4 . A nucleic acid molecule encoding the PI indicator of  claim 1 . 
     
     
         5 . A cell comprising the PI indicator of  claim 1 . 
     
     
         6 . A method for measuring spatiotemporal phosphoinositide dynamics, comprising:
 (i) detecting a first resonance energy transfer of the PI indicator of  claim 1  at a first time point or place;   (ii) detecting a second resonance energy transfer of the PI indicator at a second time point or place; and   (iii) comparing the first and the second resonance energy transfers, wherein a difference between the first and the second resonance energy transfers reflects a change in spatiotemporal phosphoinositide dynamics.