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.