[
EMBO J,
2011]
Calcium/calmodulin-dependent protein kinase II (CaMKII) interprets information conveyed by the amplitude and frequency of calcium transients by a controlled transition from an autoinhibited basal intermediate to an autonomously active phosphorylated intermediate (De Koninck and Schulman, 1998). We used spin labelling and electron paramagnetic resonance spectroscopy to elucidate the structural and dynamic bases of autoinhibition and activation of the kinase domain of CaMKII. In contrast to existing models, we find that autoinhibition involves a conformeric equilibrium of the regulatory domain, modulating substrate and nucleotide access. Binding of calmodulin to the regulatory domain induces conformational changes that release the catalytic cleft, activating the kinase and exposing an otherwise inaccessible phosphorylation site, threonine 286. Autophosphorylation at Thr286 further disrupts the interactions between the catalytic and regulatory domains, enhancing the interaction with calmodulin, but maintains the regulatory domain in a dynamic unstructured conformation following dissociation of calmodulin, sustaining activation. These findings support a mechanistic model of the CaMKII holoenzyme grounded in a dynamic understanding of autoregulation that is consistent with a wealth of biochemical and functional data.
Schedl, Tim, Audhya, Anjon, Niessen, Sherry, Desai, Arshad, Swathi, Arur, Laband, Kimberley, Piano, Fabio, Mayers, Jonathan, Green, Rebecca A., Wang, Shaohe, Fridolfsson, Heidi, Gunsalus, Kristin, Schulman, Monty, Oegema, Karen, Kao, Huey-Ling, Starr, Daniel, Schloissnig, Siegfried, Hyman, Anthony
[
International Worm Meeting,
2011]
High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach- profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for 106 uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes- pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.