Mutation in the Q(28)SDD(31)SD site, but not in the two SQ sites of the survival of motor neuron protein, affects its foci formation

作者：Aoki Yusuke^*; Fukao Toshiyuki; Zhang Gaixiu; Ohnishi Hidenori; Kondo Naomi

来源：International Journal of Molecular Medicine, 2010, 26(5): 667-671.

DOI：10.3892/ijmm_00000512

• survival motor neuron
• spinal muscular atrophy
• phosphorylation
• foci
• mutation

The survival of motor neuron (SMN) protein forms a multiprotein complex (SMN complex) with Gemin proteins. The complex is known to play a crucial role in RNA metabolism. Several lines of evidence show that SMN is phosphorylated at serine and/or threonine residues. In this study, we hypothesized that SMN is phosphorylated at two kinds of serine residues, the Q(28)SDD(31)SD site and two SQ sites ((80)SQ and (163)SQ). A FLAG-tagged wild-type construct (SMNfull) and three FLAG-tagged mutant constructs were made: an SMNAQ mutant with two AQ sites instead of two SQ sites at residues 80 and 163, an SMNQADDAD mutant with QADDAD instead of Q(28)SDD(31)SD, and an SMNAQ/QADDAD mutant with the two AQ sites and QADDAD. We expressed these mutants in He La cells and analyzed their phosphorylated bands by immunoblotting, the protein stability using cycloheximide, binding to Gemin 2 and foci formation. Mutations in Q(28)SDD(31)SD, but not in two SQ sites reduced the intensity of phosphorylation bands, indicating that Q(28)SDD(31)SD is the major phosphorylation site in SMN. Mutations in the two SQ sites and Q(28)SDD(31)SD did not affect protein stability and binding to Gemin 2. Whereas mutations in the two SQ sites did not cause apparent changes in foci formation, mutations in Q(28)SDD(31)SD resulted in a reduced number of large foci in the cytosol. We demonstrated that phosphorylation in Q(28)SDD(31)SD may be important in cytosolic foci formation.

• 出版日期2010-11