| Cell signaling networks are highly complex. Cells modulate their functions precisely through dynamic controls over intracellular proteins. The capability of one single protein regulating multiple cellular processes relies on the posttranslational modification responsive to specific external stimuli. Reversible phosphorylation plays a key role in cell signaling pathways. Errors in kinase or phosphatase activities are involved in human diseases, including cancers, heart disease, diabetes and Alzheimer's Disease. Developing new probes that enable the detection of specific kinase activity in a time-efficient manner is critical for understanding fundamental mechanisms in signal transductions, providing useful pharmaceutical targets in disease pathology. In this work, the design of an expressible protein kinase-inducible domain (pKID), whose metal binding affinity and fluorescence are switchable according to its phosphorylation state, is described. The design is based on EF-hand motif, containing a loop and a connecting alpha-helix, which binds Ca2+ with either side-chain or backbone oxygen. The key design element uses phosphoserine, phosphothreonine or phosphotyrosine as an inducible mimic of a structurally important glutamate residue in the EF hand. Due to the similar electronics and ionic radii of calcium and lanthanides, lanthanides coordinate to the peptides, thereby yielding EF hands with luminescent properties. Fluorescence emission spectra revealed that the nonphosphorylated peptides bound Tb 3+ poorly, displaying weak terbium luminescence. In contrast, phosphorylated peptides bound Tb3+ well and displayed strong luminescence. This approach was applied to the development of several kinase sensors, including PKA, PKC, Erk2, Akt, Pim1, AMPK, cdk5, GSK-3beta, MARK, Fyn, Able, and ErbB2. pKID is a general approach for a variety of Ser/Thr/Tyr kinases with direct fluorescence readout of kinase activity in vitro and in cells. |
