real - time monitoring of the circadian gene expression was performed using rat-1 cells expressing a luciferase reporter under the control of the upstream region of bmal1 gene .
rat-1 cells were treated with 0.1  m dexamethasone for 2  h for the rhythm synchronization , and the bioluminescence signals from the cells were continually recorded at 37c under air with dish - type bioluminescence detector , kronos ( atto , ab-2500 ) or lumicycle ( actimetrics ) .
the in vitro kinase assay was carried out at 30c in a reaction mixture ( 10  l ) consisting of 1  mm dithiothreitol , 100  m [ -p]atp , 10  ng 30k - camkii and 100  ng substrate protein .
the substrate proteins , gst - sp , mbp - nt , gst or mbp ( each 100  ng ) were prepared as described previously .
after incubation for 30  min , the reaction was stopped by the addition of 10  l of 2  sds sample buffer .
real - time monitoring of the circadian gene expression was performed using rat-1 cells expressing a luciferase reporter under the control of the upstream region of bmal1 gene .
rat-1 cells were treated with 0.1  m dexamethasone for 2  h for the rhythm synchronization , and the bioluminescence signals from the cells were continually recorded at 37c under air with dish - type bioluminescence detector , kronos ( atto , ab-2500 ) or lumicycle ( actimetrics ) .
the in vitro kinase assay was carried out at 30c in a reaction mixture ( 10  l ) consisting of 1  mm dithiothreitol , 100  m [ -p]atp , 10  ng 30k - camkii and 100  ng substrate protein .
the substrate proteins , gst - sp , mbp - nt , gst or mbp ( each 100  ng ) were prepared as described previously .
after incubation for 30  min , the reaction was stopped by the addition of 10  l of 2  sds sample buffer .
the animal experiments were conducted in accordance with the guidelines of the university of tokyo .
the mice ( c57bl/6 background , male , 10-week - old ) were housed individually at 23c in cages with food and water available ad libitum .
this work was supported in part by grants - in - aid for scientific research from jsps and mext , japan .
   molecular oscillation of the circadian clock is based on e - box - mediated transcriptional feedback loop formed with clock genes and their encoding products , clock proteins . 
 the clock proteins are regulated by post - translational modifications such as phosphorylation . 
 we investigated the effects of a series of kinase inhibitors on gene expression rhythms in rat-1 fibroblasts . 
 the period of the cellular circadian rhythm in culture was lengthened by treatment with sb203580 ( p38 mapk inhibitor ) , sp600125 ( jnk inhibitor ) , ic261 ( cki inhibitor ) and roscovitine ( cdk inhibitor ) . on the other hand , 
 the period was shortened by sb216763 ( gsk-3 inhibitor ) or kn93 ( camkii inhibitor ) treatment . 
 application of 20  m kn93 completely abolished the rhythmic gene expression . 
 the activity of camkii exhibited circadian variation in a phase close to the e - box - mediated transcriptional rhythms . in vitro kinase assay revealed that camkii directly phosphorylates n - terminal and ser / pro - rich domains of clock , an activator of e - box - mediated transcription . 
 these results indicate a phosphorylation - dependent tuning of the period length by a regulatory network of multiple kinases and reveal an essential role of camkii in the cellular oscillation mechanism . 