Mutation in the Q28SDD31SD site, but not in the two SQ sites of the survival of motor neuron protein, affects its foci formation.
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 Q28SDD31SD site and two SQ sites (80SQ and 163SQ). 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 Q28SDD31SD, and an SMNAQ/QADDAD mutant with the two AQ sites and QADDAD. We expressed these mutants in HeLa cells and analyzed their phosphorylated bands by immunoblotting, the protein stability using cycloheximide, binding to Gemin 2 and foci formation. Mutations in Q28SDD31SD, but not in two SQ sites reduced the intensity of phosphorylation bands, indicating that Q28SDD31SD is the major phosphorylation site in SMN. Mutations in the two SQ sites and Q28SDD31SD 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 Q28SDD31SD resulted in a reduced number of large foci in the cytosol. We demonstrated that phosphorylation in Q28SDD31SD may be important in cytosolic foci formation.
Aoki
Yusuke
Y
Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu 501-1194, Japan. y-a@xg7.so-net.ne.jp
Fukao
Toshiyuki
T
Zhang
Gaixiu
G
Ohnishi
Hidenori
H
Kondo
Naomi
N
D001665:Binding Sites; D003513:Cycloheximide / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D055532:SMN Complex Proteins / Q000235:genetics / Q000378:metabolism; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics* / Q000378:metabolism
D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D003513:Cycloheximide