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Isolation and characterisation of protein kinase C interacting proteins in the yeast Saccharomyces cerevisiae

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posted on 2014-12-15, 10:38 authored by William John. Richards
The activation of the protein kinase C (PKC) signalling pathway has been implicated in the control of many diverse cellular events. PKC is in fact a family of isoforms, each individual isoform having discrete biological role(s) in vivo. The functional speciation of the PKC family is thought to be mediated at least in part by the different subcellular distributions of the various activated PKC isoforms. This translocation of PKC from the cytosol to assorted subcellular compartments following activation is mediated by protein-protein interactions. Clearly, these PKC-binding proteins (termed RACKs for receptors of activated C-kinase) are central to the process of signal transduction via PKC. Both PKC activity and PKC-homologous genes are present in the yeast S. cerevisiae. In order to learn more about RACKs and their role in the PKC signal transduction pathway, a molecular screen for yeast genes encoding PKC-binding proteins was conducted. One of the ORFs identified by this screen corresponded to the GCD6 gene which encodes a translation initiation factor, Gcd6p. Polyclonal antibodies were raised against recombinant Gcd6p and its expression in yeast cells was demonstrated by western blotting. Gcd6p was shown to bind mammalian PKC in vitro, and was also shown to bind endogenous yeast proteins which cross-react with anti-mammalian PKC antibodies. A yeast-based two-hybrid screen was conducted to isolate proteins interacting with Gcd6p in vivo. The possible cellular role of Gcd6p is discussed with respect to its probable biological partners, and the involvement of PKC or PKC-homologous enzymes.

History

Date of award

1997-01-01

Author affiliation

Genetics

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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