title: Scaffold state switching amplifies, accelerates, and insulates protein kinase C signaling.
abstract: Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction.
authors.affiliation: From the Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908 and.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Greenwald
authors.forename: John M
authors.initials: JM
authors.lastname: Redden
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Dodge-Kafka
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Saucerman
mesh_terms: D054758:A Kinase Anchor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002227:Carbazoles / Q000737:chemistry; D002522:Chlorocebus aethiops; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D009281:Naphthalenes / Q000737:chemistry; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014709:Vero Cells
chemical_list: D054758:A Kinase Anchor Proteins; C112596:AKAP7 protein, human; D002227:Carbazoles; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009281:Naphthalenes; C081021:Go 6976; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; C058819:calphostin C
keywords: AKAP; Anchoring; Cell Signaling; Enzyme Kinetics; Mathematical Modeling; Protein Kinase C (PKC); Scaffold Proteins