The Circadian tau Mutation in Casein Kinase 1 Is Part of a Larger Domain That Can Be Mutated to Shorten Circadian Period.

Anandakrishnan Venkatesan,Samuel Bouyain,Jeffrey Price,Jin-Yuan Fan

doi:10.3390/ijms20040813

Anandakrishnan Venkatesan, Samuel Bouyain + Show 2 more

Open Access

https://doi.org/10.3390/ijms20040813

Copy DOI

Abstract

Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIε) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. We mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.

Highlights

Circadian rhythms are biological processes that are controlled by environmental cues and exhibit oscillations of 24 h during a typical diurnal cycle
The period-altering mutants of CKI/DBT that do not completely lose kinase activity have more diverse effects on period; DBTS, CKIεtau and CKIδT44A shorten period whereas DBTL, DBTAR, DBTG and DbtH lengthen period
Mutations affecting lysines or arginines in either the phosphate recognition triad, the nuclear localization signal (NLS) or a lysine outside either the phosphate recognition triad or the NLS all shortened circadian period, while mutations of internally located arginines lengthened circadian period. These results suggest that a surface domain including the NLS and phosphate recognition motif can be mutated to shorten circadian period and raise the possibility that disruption of a DBT protein-protein interaction produces the effect

Summary

Introduction

Circadian rhythms are biological processes that are controlled by environmental cues and exhibit oscillations of 24 h during a typical diurnal cycle. The shorter periods of the tau and dbtS mutations have been shown to be evolutionarily conserved in transgenic flies expressing either Drosophila DBT or vertebrate CKIδ [21] It has been shown in both mammals and flies that complete loss of kinase activity of CKI through mutations of DBT or CKI (e.g., DBTK/R) [10,22] and tissue specific knockout or pharmacological inhibition of CKIδ [22,23] lengthen period and cause arrhythmicity, demonstrating the vital role for DBT/CKI in the clock process. The period-altering mutants of CKI/DBT that do not completely lose kinase activity have more diverse effects on period; DBTS, CKIεtau and CKIδT44A shorten period (only the dbtS mutation found in Drosophila) whereas DBTL, DBTAR, DBTG and DbtH lengthen period (all of these identified in Drosophila). Only some of the mutations disrupted the previously demonstrated DBT/BDBT interaction [29] while the tau mutation did not, so the domain is predicted to disrupt interactions with another protein to produce the short-period rhythms

Results

Mutations that Affected Arginines Localized Internally Lengthened Period

Site-Directed Mutagenesis

Analysis of Circadian Locomotor Activity Rhythms

Analysis of Transgenic and Endogeonous DBT Levels