Construction of fluorescent protein expression system to study intramolecular interaction of protein kinase C isoforms

초록

Activation and inactivation of protein kinase C (PKC) isoforms in mammalian cells control various signaling pathways including cell proliferation, differentiation, and apoptosis. There are at least 10 PKC isoforms in mammalian cells with overlapping and distinct functions. Different PKC isoforms are known to be expressed depending on tissue types. All of PKC isoforms have regulatory domain for cofactor binding and catalytic domain for substrate phosphorylation. Activation of PKC isoforms requires binding of specific cofactors and phosphorylation by other kinases in isoform-specific manner. PKC isoforms also have pseudosubstrate sequences in the regulatory domain. PKC isoforms have been reported to maintain their inactive states by binding between regulatory domain and catalytic domain through pseudosubstrate sequence. We generated various fluorescent protein expression system to study intramolecular interaction between regulatory domain and catalytic domain using fluorescence resonance energy transfer (FRET) in vivo and in vitro. Bacterial expression systems for Cerulean, YPet and Cherry were generated using pET28-based expression vectors. Expression systems for YPet-mCerulean, YPet-mCherry were also generated for FRET positive control. Fluorescent proteins were expressed in BL21(DE3) E.coli strain and tested for fluorescence induced by excitation light (365nm). Histidine-tagged fluorescent proteins were also purified by Ni-NTA column.