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Imaging PKC activity at MAGUK protein scaffolds
Author(s) -
Gallegos Lisa L,
Garcia Erin,
Hall Randy,
Newton Alexandra C
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1048.9
Subject(s) - pdz domain , protein kinase c , microbiology and biotechnology , diacylglycerol kinase , cytosol , guanylate kinase , chemistry , scaffold protein , signal transduction , biology , membrane protein , biochemistry , membrane , enzyme
The protein kinase C (PKC) family of Ser/Thr kinases relays extracellular signals to intracellular substrates involved in processes such as cell growth and division, memory and learning, and the immune response. PKC isoforms typically reside in the cytosol in a mature but catalytically‐inactive conformation. Generation of the second messengers diacylglycerol and, for conventional PKCs, calcium causes translocation of PKC to cellular membranes, a hallmark of PKC activation. This activation can be precisely visualized using a genetically‐encoded reporter for PKC activity, CKAR (C kinase activity reporter) (Violin, et al. 2003). We have previously tethered CKAR to various intracellular membranes and analyzed the spatiotemporal dynamics of signaling at the plasma membrane, Golgi, mitochondria, nucleus and cytosol (Gallegos, et al. 2006). In this study, we take our reporters one step further by tethering them to protein scaffolds to visualize agonist‐evoked PKC signaling localized at discrete signaling complexes. Specifically, we used a PDZ array to identify potential scaffolds for the PDZ ligand on PKC and found a strong interaction with members of the membrane‐associated guanylate kinase (MAGUK) family of protein scaffolds. This interaction was confirmed by immunoprecipitation of PKC with two MAGUK family members, SAP97 and PSD95. We show that CKAR can be targeted to MAGUK scaffolds, and that this scaffold‐targeted reporter reads out a different profile of PKC activity compared to untargeted CKAR. The ability to visualize PKC activity at a signaling complex provides insight into the efficiency and specificity of compartmentalized signaling.