Inventors:
Jonathan Violin - Durham NC, US
Alexandra Newton - San Diego CA, US
Roger Tsien - La Jolla CA, US
Jin Zhang - Baltimore MD, US
International Classification:
C12Q001/68
C12Q001/42
C07K014/47
Abstract:
A chimeric phosphorylation indicator (CPI) as provided herein can contain a donor molecule, a phosphorylatable domain, a phosphoaminoacid binding domain (PAABD), and an acceptor molecule. Where the phosphorylatable domain is phosphorylatable by protein kinase C (PKC), the CPI is a c-kinase activity reporter (CKAR). Donor and acceptor molecules may be, independently, fluorescent proteins such as non-oligomerizing fluorescent proteins. A CPI can contain a phosphorylatable polypeptide and a fluorescent protein; the phosphorylatable polypeptide may be contained within the sequence of the fluorescent protein, or the fluorescent protein may be contained within the sequence of the phosphorylatable polypeptide. The spatiotemporal properties of the PKC signal pathway may be tested with CKAR, calcium-sensing fluorophores and FRET-based translocation assays. Polynucleotides encoding such CPIs, and kits containing the indicators and/or the polynucleotides, are provided. A method of using the chimeric phosphorylation indicators to detect a kinase or phosphatase in a sample is provided.
