PKN Associates and Phosphorylates the Head-Rod Domain of Neurofilament Protein
Autor: | Hideyuki Mukai, Yoshitaka Ono, Hiroko Sunakawa, Hideki Shibata, Masako Miyahara, Masaki Shimakawa, Michinori Kitagawa, Masanao Toshimori |
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Rok vydání: | 1996 |
Předmět: |
Neurofilament
Macromolecular Substances Recombinant Fusion Proteins Protein subunit Protein Serine-Threonine Kinases Biology Polymerase Chain Reaction Biochemistry Substrate Specificity Serine Neurofilament Proteins Two-Hybrid System Techniques Humans Phosphorylation Binding site Molecular Biology Protein Kinase C Protein kinase C Glutathione Transferase Binding Sites Kinase Cell Biology Protein-Tyrosine Kinases Fusion protein Molecular biology Kinetics Spinal Cord Protein Biosynthesis Protein Multimerization |
Zdroj: | ResearcherID |
ISSN: | 0021-9258 |
Popis: | PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament. |
Databáze: | OpenAIRE |
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