Yasuhito Shirai, Norio Sakai and Naoaki Saito (*)
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe
University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan
(*) To whom correspondence should be addressed.
Abstract: To clarify the subspecies-specific functions of protein
kinase C (PKC), we constructed cDNAs encoding gamma-, epsilon- and delta-PKC
fused with green fluorescent protein (GFP). All fusion proteins had enzymological
and immunological characteristics similar to those of native PKCs. When
expressed in CHO-K1 cells, each fusion protein showed a specific subcellular
localization. Their translocations induced by various stimulation were also
diverse. For example, ATP translocated gamma-, epsilon- and delta-PKC-GFP
in the cytoplasm to the plasma membrane within 30 sec with a return to the
cytoplasm in 3 min, whereas TPA induced slow and irreversible translocation
of all subspecies to the plasma membrane. Fatty acids also induced the translocation
of gamma- and epsilon-PKC-GFP, but the two PKC subspecies showed distinct
translocation and sensitivity to various fatty acids. Furthermore, we revealed
that the PKC translocation requires neither the kinase activity of PKC nor
its association with cytoskeletal proteins such as F-actin. These results
indicate that each subspecies has a spatially and temporally different targeting
mechanism that depends on the extracellular and intracellular signals, contributing
to the subspecies-specific functions of PKC. These remarkable findings also
indicate that a system for monitoring the PKC translocation is a powerful
tool for investigating the subspecies-specific functions of PKCs and mechanism
of its translocation.
Keywords: Protein kinase C, Translocation, Targeting, Green fluorescent
protein