Development of fluorescence-based techniques to study protein kinase activity and subunit interactions

The transduction of extracellular signals to produce intracellular responses is involved in the action of most drugs, hormones, and neurotransmitters. Phosphorylation of proteins by protein kinases is involved in many signal transduction pathways. The involvement of protein kinases in signal transduction and drug action makes it imperative to understand their functions as well as their involvement in various signaling cascades. To achieve this goal, methods utilizing fluorescence were developed to study subunit interactions and catalytic activity.; Previous studies have shown that the δ-subunit of phosphorylase kinase (calmodulin) interacts with two noncontiguous calmodulin-binding domains in the regulatory domain of the γ-subunit. In this study, a 65-residue regulatory domain peptide containing both calmodulin-binding domains was obtained. A mutant calmodulin with cysteine at position 146 was fluorescently labeled and used to determine the dissociation constant (K_D) of the peptide-calmodulin by fluorescence anisotropy. In the presence of Ca2+, the K _D was approximately 0.25 nM, indicating that this peptide has higher affinity for calmodulin than each of the two segments alone. Removal of Ca 2+ resulted in a drop in fluorescence anisotropy, indicating that this interaction is calcium-dependent. Therefore, other factors must be involved in the calcium-independent interaction between the γ-subunit and δ-subunit.; To study protein kinase activity, a fluorescence-based protein kinase assay was developed that utilizes a short peptide substrate labeled with two fluorescent dyes. The two fluorescent dyes form an intramolecular dye dimer once attached and this interaction leads to ground-state fluorescence quenching of both dyes. Fluorescence is restored by dissociating the dye dimer. The cAMP-dependent protein kinase (PKA) was used as the model system for assay development. Substrate peptides were synthesized initially using sequences based on Kemptide and later based on the kinase-inducible domain (KID) of the cAMP-response element binding (CREB) protein. The double-labeled peptides were readily phosphorylated by PKA; however, phosphorylation alone did not induce dye dimer dissociation with either the Kemptide or the KID-based peptides. Antibody that binds to phosphorylated KID peptides was then added to facilitate dissociation the dye dimer. The antibody caused a modest increase in fluorescence, probably by trapping a fraction of the phosphorylated peptide in the dissociated state. These results demonstrate a novel fluorescence-based homogeneous protein kinase assay. With refinement, such an assay could be used as a general method for assaying protein kinase activities.