3.2 Analyses of the serine sites essential for phosphorylation
in the accessory domain
To elucidate why the phosphorylation of the accessory domain would lead
to its dislocation from the NES, the contribution of the three
phosphorylation sites were examined separately by changing the
phosphorylation patterns in additional MD simulations. The two
phosphorylation scenarios studied were (1) single-site phosphorylation
at Ser 140 and (2) double-site phosphorylation at Ser 159 and Ser 177.
For scenario (1), Ser 140 was identified through searches of multiple
phosphorylation sites.7-11 For scenario (2), Ser 159
and Ser 177 were identified experimentally, where they were
phosphorylated by CK2 as reported in a previous
study.8 The validity of the phosphorylation sites was
confirmed using a phosphorylation prediction server
(NetPhos).25 According to the MD simulations, neither
the single phosphorylation (p-Ser 140) nor the double phosphorylation
(p-Ser 159 and p-Ser 177) resulted in any significant increase in the
SASA of the NES (Figure S1 in Supporting Information), indicating that
the phosphorylation of all three sites at once will contribute equally
to the increase in the SASA; that is, all the serine residues (Ser 140,
Ser 159, and Ser 177) might play an important role in exposing the NES
via their phosphorylation.