Translocation Assays of Protein Kinase C Activation
The protein kinase C (PKC) family members include at least 11 different isoforms that, based on their different requirements for activation, have been divided into three subfamilies, the Ca2+-dependent (cPKCα, (βI, βII, and γ), the Ca2+-independent (nPKCδ,ɛ,η,θ, and μ), and phorbol ester-insensitive (aPKCζ and ι, the human counterpart of mouse PKCλ) subfamilies. Much research on this growing family of protein kinases has concentrated on the possibility that these enzymes may have assumed distinct responsibilities for the control of complex and diverse cellular functions. The current working hypothesis is that the differential sensitivity of PKC isoforms to endogenous agonists and their differential targeting to discrete subcellular domains may dictate what substrates are phosphorylated by a given isoform. For this reason, an important initial goal in the analysis of the connection between PKC activation and a cellular response, such as neurodegeneration, is to identify the endogenous PKC isoforms that become “membrane-associated” in response to an appropriate stimulus. Translocation assays have classically been used to screen the individual isoforms of PKC for the production of active PKC-membrane complexes. The principal criteria used to distinguish between inactive and active forms of PKC are that, in the later case, the solubility of the enzyme is reduced when intact cells are treated with an appropriate agonist (e.g., phorbol esters) and that the association of a given PKC isoform with the particulate fraction resists extraction with Ca2+-chelators, but not nonionic detergents.
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