Diversity of Human Clock Genotypes and Consequences - Progress in Molecular Biology and Translational Science - page 66

12. CK1d

Exon sequencing of circadian genes for individuals that belong to a
moderate-sized family with FASP led to the identification of a second muta-
tion that causes FASP. The mutation is a threonine-to-alanine alteration at
amino acid 44 of CK1 d (CK1 d -T44A), and this threonine is conserved in
other mammalian CK1s and Drosophila CK1 (dDBT). 14

In vitro kinase assay
demonstrates that this mutation results in decreased phosphorylation of both
exogenous substrates (phosvitin and alpha-casein) and circadian substrates
(PER1-3). To examine the effects of this mutation on circadian rhythms
in vivo , BAC transgenic mice carrying either the wild type (h CK1d-WT )
or the mutant (h CK1d-T44A )h CK1d were generated. The behavioral

period under free-running condition is significantly shorter in the mutant
transgenic mice compared to wild type, consistent with the phase-advanced
phenotype of human subjects carrying this mutation. Neither CK1d

þ/ nor
h CK1d-WT transgenic mice exhibit altered period, suggesting that the
period is not affected by wild-type CK1d

gene dosage. Thus, the shorter
period observed in h CK1d-T44A transgenic mice is likely due to the
T44Amutation and not altered CK1d gene dosage. Interestingly, expression

of h CK1d-T44A in Drosophila circadian neurons results in longer period
compared to expression of h CK1d-WT . This may reflect differences in
the regulatory mechanism of the mammalian clock versus invertebrate clock.
The aforementioned h PER2-S662G and h CK1d-T44A mutations indi-
cate that phosphorylation of PER2 by CK1 is critical for circadian timing in
humans. To characterize the functional relevance of the interaction between
PER2 and CK1 in vivo ,h PER2 transgenic mice were crossed with both
h CK1d-WT transgenic and CK1d knockout mice. 77 As described earlier

in this chapter, h PER2-S662G transgenic mice exhibit a short period of
22 h, whereas neither h CK1d-WT transgenic nor CK1d þ/ exhibits
altered circadian period. However, in mice carrying both h PER2-S662G
and h CK1d-WT transgenes, the period is shorter than h PER2-S662G single
transgenic animals by over 1 h. Consistently, expressing h PER2-S662G on
the CK1d

þ/ background slightly lengthens the period compared to
expressing h PER2-S662G on a wild-type background. On the other hand,
h PER2-S662D transgenic mice, which show long period on wild-type
background, exhibit even longer period in CK1d

þ/ background and a
shorter period in h CK1d-WT background. Therefore, decreasing CK1d
dosage lengthens period for both h PER2-S662G and h PER2-S662D