The mechanism of protein kinase C's nuclear uptake

Contact: Stefan Wagner

Protein kinase C (PKC) is redistributed from the cytosol into the nucleus upon various stimuli. Since PKC does not contain any of the canonical nuclear localization signals (NLS), the exact mechanism of the translocation into the nucleus is still not known. Due to difficulties in purifying large amounts of single PKC isoforms, which are necessary for investigating nuclear transport in permeabilized cells, expression of a PKC/GFP fusion protein in recombinant baculovirus-infected cells was carried out. GFP serves as an easily detectable reporter for PKC. Comparing with the PKC a protein I was able to show that GFP does not influence the kinase activity.With the PKC/GFP fusion protein we are establishing an in vitro nuclear transport assay, which will enable us to study the mechanism of nuclear PKC transport in detail. Furthermore, I transfect fibroblasts with expression vectors containing an enhanced variant of GFP and PKC generating GFP/PKC and PKC/GFP fusion proteins. Future experiments will deal with the construction of PKC mutants to determine sequences which are necessary for PKC translocation to the nucleus.

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