Protein kinase CK2 differentially phosphorylates maize chromosomal high mobility group B (HMGB) proteins modulating their stability and DNA interactions.
The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins.
Stemmer
Christian
C
Department of Life Science, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Schwander
Andrea
A
Bauw
Guy
G
Fojan
Peter
P
Grasser
Klaus D
KD
D000595:Amino Acid Sequence; D000818:Animals; D047390:Casein Kinase II; D002478:Cells, Cultured; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D024002:HMGB Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D003313:Zea mays / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D024002:HMGB Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D004247:DNA; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases