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Biochemical assays for multiple activation states of protein kinase C

Nature Protocols volume 1, pages 2791–2795 (2006)Cite this article

Abstract

This protocol describes biochemical procedures to monitor the activation of the protein kinase C (PKC) family using PKCδ as the representative. The PKC family is composed of ten isoforms divided into cPKC, nPKC and aPKC groups, and their catalytic activity is regulated by multiple mechanisms. For example, PKCδ in the nPKC group is activated by diacylglycerol as a second messenger in the receptor-coupled manner, through tyrosine phosphorylation and protein complex formation in stress-stimulated cells, and by the caspase-catalyzed cleavage during apoptosis. The isoform is immunoprecipitated from cultured cells, the protein kinase activity is measured by in vitro kinase assay and the tyrosine phosphorylation and protein complex formation are characterized by immunoblot, whereas the generation of the catalytic fragment is detected by immunoblot in the cell extract. The combination of these procedures is useful to evaluate the activation states of the PKC family in cells. This protocol can be completed in 3–5 d.

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Figure 1: Activation mechanisms of PKCδ.
Figure 2: Activation of PKCδ.
Figure 3: Association of PKCδ.
Figure 4: The catalytic fragment formation of PKCδ.

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Authors and Affiliations

  1. Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, 657-8501, Japan

    Toshiyoshi Yamamoto, Hidenori Matsuzaki, Shinji Kamada, Yoshitaka Ono & Ushio Kikkawa

Authors
  1. Toshiyoshi Yamamoto
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  2. Hidenori Matsuzaki
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  3. Shinji Kamada
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  4. Yoshitaka Ono
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  5. Ushio Kikkawa
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Correspondence to Ushio Kikkawa.

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The authors declare no competing financial interests.

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Yamamoto, T., Matsuzaki, H., Kamada, S. et al. Biochemical assays for multiple activation states of protein kinase C. Nat Protoc 1, 2791–2795 (2006). https://doi.org/10.1038/nprot.2006.420

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