Phosphorylation on Ser106 modulates the cellular functions of the SHOX homeodomain protein.
Mutations within the homeobox SHOX gene have been associated with short stature and the skeletal deformities found in Léri-Weill, Turner and Langer syndromes implying an involvement of SHOX in growth and bone formation. Despite its clinical significance, the precise role of SHOX and the mechanisms that modulate its functions remain unknown. We reported previously that SHOX is a nuclear protein that specifically binds DNA and acts as a transcriptional activator. We have shown that ectopic expression of SHOX leads to cell-cycle arrest and apoptosis in osteosarcoma and primary cells. To further characterize SHOX, we investigated whether the protein could be a target for phosphorylation. Here, we report that SHOX is phosphorylated exclusively on serine residues in vivo. Two-dimensional phospho-peptide mapping showed that SHOX is phosphorylated to various extents on multiple sites. Site-directed mutagenesis demonstrated that serine 106 is the major SHOX phosphorylation site. We show also that casein kinase II phosphorylates SHOX on serine 106 efficiently in vitro and specific casein kinase II inhibitors reduce SHOX phosphorylation strongly in vivo. Finally, we provide evidence that phosphorylation may play an important role in modulating SHOX biological activities, since a S106A SHOX mutant, defective in phosphorylation, does not activate transcription and fails to induce cell-cycle arrest and apoptosis.
Marchini
Antonio
A
Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. antonio_marchini@med.uni-heidelberg.de
Daeffler
Laurent
L
Marttila
Tiina
T
Schneider
Katja U
KU
Blaschke
Rüdiger J
RJ
Schnölzer
Martina
M
Rommelaere
Jean
J
Rappold
Gudrun
G
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism*; D000074122:Short Stature Homeobox Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018398:Homeodomain Proteins; C106071:SHOX protein, human; D000074122:Short Stature Homeobox Protein; D012694:Serine; D047390:Casein Kinase II