Diversity of Human Clock Genotypes and Consequences - Progress in Molecular Biology and Translational Science - page 60

from this family. Sleep-wake and temperature rhythms of one FASP subject
were monitored in time isolation and show a circadian period of 23.3 h
( Fig. 3.1 ) , which is substantially shorter than that of control subjects
(24.2 h) and is consistent with the advanced phase of sleep-wake cycle.
In order to identify the mutation that leads to FASP in the subjects in this
family, linkage analysis was performed, which mapped the allele to chromo-
some 2qter. 76 Further physical mapping was carried out and led to identi-
fication of

40 cDNAs localized to this region. The only coding
mutation identified is in the PER2 cDNA at position 2106 (A-G), which
results in substitution of a serine at amino acid 662 with a glycine
(S662G). Functional characterization was subsequently carried out to estab-
lish whether the S662G mutation causes FASP. In vitro study using PER2
truncation mutants demonstrates that S662 is located within CK1-binding
region and the S662G mutation causes hypophosphorylation by CK1.
Sequence analysis of PER2 reveals four additional serine residues that are
C-terminal to S662 and each with two amino acids in between (i.e.,
S665, S668, S671, and S674), consistent with the CK1 recognition consen-
sus motif. Furthermore, mutating S662 to aspartate (S662D), which mimics
a phosphoserine, restores CK1-dependent phosphorylation, suggesting that
S662 is a phosphorylation site on PER2. Similarly, in vitro phosphorylation
assays using PER2 peptides that encompass residues from 660 to 674 dem-
onstrate that PER2 peptide with a phosphate covalently linked to S662 is
phosphorylated at the other residues by CK1, whereas PER2 peptide with-
out a phosphate at S662 is not phosphorylated by CK1. 77 A quantitative
assay using PER2 peptides shows that an additional 4 mol of phosphate were
incorporated per mole of the PER2 peptide, corresponding to the four ser-
ine residues C-terminal to S662. Subsequent phosphoamino acid analysis
revealed that the threonine and tyrosine residues on the peptide are not
phosphorylated, implying that phosphorylation occurs at the serine residues.
Taken together, these results suggest that phosphorylation at S662 of PER2
serves as a priming event that is critical for a cascade of phosphorylations
downstream of S662 by CK1.
To investigate the functional consequences of the S662G mutation
in vivo , transgenic mice carrying wild-type h PER2 and h PER2 with
S662G or S662Dmutations were generated using a human bacterial artificial
chromosome (BAC) which carries the cis -acting genomic regulatory ele-
ments that can faithfully recapitulate endogenous PER2 expression. 77
Behavior analysis shows that the S662G transgenic mice exhibit

2h

shorter free-running period, whereas the S662D mice exhibit 0.5 h