Since the discovery that protein kinase C (PKC) transduces the ab- dance of signals that result in phospholipid hydrolysis, this enzyme has been at the forefront of research in signal transduction. Protein Kinase C Protocols covers fundamental methods for studying the structure, function, regulation, subcellular localization, and macromolecular interactions of PKC. Protein Kinase C Protocols is divided into 11 sections representing the major aspects of PKC regulation and function. Part I contains an introduction and a historical perspective on the discovery of PKC by Drs. Yasutomi Nishizuka and Ushio Kikkawa. Part II describes methods to purify PKC. Part III describes the standard methods for measuring PKC activity: its enzymatic activity and its stimulus-dependent translocation from the cytosol to the membrane. Part IV describes methods for measuring the membrane interaction of PKC in vivo and in vitro. Part V provides methodologies and techniques for measuring the ph- phorylation state of PKC, including a protocol for measuring the activity of PKC¿s upstream kinase, PDK-1. Novel methods for identifying substrates are described in Part VI. Part VII presents protocols for expressing and analyzing the membrane targeting domains of PKC. Part VIII provides a comprehensive c- pilation of methods used to identify binding partners for PKC. Part IX describes pharmacological probes used to study PKC. The book ends with a presentation of genetic approaches to study PKC (Part X) and a discussion of approaches used to study PKC in disease (Part XI).

The Ins and Outs of Protein Kinase C.- Early Studies of Protein Kinase C.- Expression and Purification.- Expression and Purification of Protein Kinase C from Insect Cells.- Expression and Purification of Protein Kinase C? from Bacteria.- Measuring the Activity of Protein Kinase C.- Complexities in Protein Kinase C Activity Assays.- Enzyme Assays for Protein Kinase C Activity.- Subcellular Translocation of Protein Kinase C.- Measuring the Interaction of Protein Kinase C with Membranes.- Measuring the Interaction of Protein Kinase C with Membranes.- Fluorescence Imaging of Protein Kinase C Translocation in Living Cells.- Measuring the Binding of Protein Kinase C to Sucrose-Loaded Vesicles.- Use of Stopped-Flow Fluorescence Spectroscopy to Measure Rapid Membrane Binding by Protein Kinase C.- [3H]Phorbol 12,13-Dibutyrate Binding Assay for Protein Kinase C and Related Proteins.- Measuring the Phosphorylation of Protein Kinase C.- Protein Kinase C Phosphorylation.- Pulse-Chase Analysis of Protein Kinase C.- PDK-1 and Protein Kinase C Phosphorylation.- In Vitro Autophosphorylation of Protein Kinase C Isozymes.- Tyrosine Phosphorylation of Protein Kinase C.- Methods to Study Dephosphorylation of Protein Kinase C In Vivo.- Phosphopeptide-Specific Antibodies to Protein Kinase C.- Identifying Protein Kinase C Substrates.- Identifying Protein Kinase C Substrates.- A Chemical Genetic Approach for the Identification of Direct Substrates of Protein Kinases.- Studying the Optimal Peptide Substrate Motifs of Protein Kinase C Using Oriented Peptide Libraries.- Structural Analysis of Protein Kinase C.- Structural Analysis of Protein Kinase C.- Bacterial Expression and Purification of C1 and C2 Domains of Protein Kinase C Isoforms.- Crystallization of the Protein Kinase C? C1B Domain.-Methods for Detecting Binding Proteins.- Methods for Detecting Binding Proteins.- Detection of Protein Kinase-Binding Partners by the Yeast Two-Hybrid Analysis.- Glutathione S-Transferase Pull-Down Assay.- Overlay Method for Detecting Protein-Protein Interactions.- An Overlay Assay for Detecting Protein Kinase C-Binding Proteins and Substrates.- Functional Proteomic Analysis of the Protein Kinase C Signaling System.- Pharmacological Probes for Protein Kinase C.- Pharmacological Probes for Protein Kinase C.- Applications of Inhibitors for Protein Kinase C and Their Isoforms.- Phorbol Esters as Probes for the Study of Protein Kinase C Function.- Irreversible Inactivation of Protein Kinase C Isozymes by Thiol-Reactive Peptide Substrate Analogs.- Genetic Approaches to Studying Protein Kinase C.- Genetic Approaches to Studying Protein Kinase C.- Animal Models in the Study of Protein Kinase C Isozymes.- Genetic Manipulation of Protein Kinase C In Vivo.- Yeast as a Host to Screen for Modulators and Regulatory Regions of Mammalian Protein Kinase C Isoforms.- Protein Kinase C in Disease.- Protein Kinase C in Disease.- Characterization of the Role of Protein Kinase C Isozymes in Colon Carcinogenesis Using Transgenic Mouse Models.- Alcohol Addiction.