Dataset Information

Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.

ABSTRACT: Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day-specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.

SUBMITTER: Baker CL

PROVIDER: S-EPMC2711022 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.

Baker Christopher L CL Kettenbach Arminja N AN Loros Jennifer J JJ Gerber Scott A SA Dunlap Jay C JC

Molecular cell 20090501 3

Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hr rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct posttranslational modifications (PTMs). To better understand how PTMs impact a circadian oscillator, we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantit ...[more]

PMID: 19450533

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Project description:Ca(2+)/calmodulin-dependent protein kinase II? (CaMKII?) autophosphorylation at Thr286 and Thr305/Thr306 regulates kinase activity and modulates subcellular targeting and is critical for normal synaptic plasticity and learning and memory. Here, a mass spectrometry-based approach was used to identify Ca(2+)-dependent and -independent in vitro autophosphorylation sites in recombinant CaMKII? and CaMKII?. CaMKII holoenzymes were then immunoprecipitated from subcellular fractions of forebrains isolated from either wild-type (WT) mice or mice with a Thr286 to Ala knock-in mutation of CaMKII? (T286A-KI mice) and analyzed using the same approach in order to characterize in vivo phosphorylation sites in both CaMKII isoforms and identify CaMKII-associated proteins (CaMKAPs). A total of six and seven autophosphorylation sites in CaMKII? and CaMKII?, respectively, were detected in WT mice. Thr286-phosphorylated CaMKII? and Thr287-phosphorylated CaMKII? were selectively enriched in WT Triton-insoluble (synaptic) fractions compared to Triton-soluble (membrane) and cytosolic fractions. In contrast, Thr306-phosphorylated CaMKII? and Ser315- and Thr320/Thr321-phosphorylated CaMKII? were selectively enriched in WT cytosolic fractions. The T286A-KI mutation significantly reduced levels of phosphorylation of CaMKII? at Ser275 across all subcellular fractions and of cytosolic CaMKII? at Ser315 and Thr320/Thr321. Significantly more CaMKAPs coprecipitated with WT CaMKII holoenzymes in the synaptic fraction compared to that in the membrane fraction, with functions including scaffolding, microtubule organization, actin organization, ribosomal function, vesicle trafficking, and others. The T286A-KI mutation altered the interactions of multiple CaMKAPs with CaMKII, including several proteins linked to autism spectrum disorders. These data identify CaMKII isoform phosphorylation sites and a network of synaptic protein interactions that are sensitive to the abrogation of Thr286 autophosphorylation of CaMKII?, likely contributing to the diverse synaptic and behavioral deficits of T286A-KI mice.

Dataset Information

Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.

Publications

Quantitative proteomics reveals a dynamic interactome and phase-specific phosphorylation in the Neurospora circadian clock.

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