The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNAdependent 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. High mobility group (HMG)1 proteins represent a heterogeneous class of small and relatively abundant chromatin-associated proteins of eukaryotes (1, 2). Proteins belonging to the subgroup of the HMGB proteins 2 (previously termed HMG1/2 proteins (3)) have in common a distinctive DNA-binding motif, termed the HMG-box domain, in which the global fold is well conserved and consists essentially of three ␣-helices arranged in an L-shape (1, 4). The HMG-box domain mediates both non-sequence-specific binding of these proteins to the minor groove of linear DNA and high affinity interactions with distorted DNA structures such as four-way junctions, minicircles, and cis-platinated DNA (2, 4, 5). In complexes with B DNA, a hydrophobic wedge on the concave surface of the HMG-box domain is inserted into the minor groove of the DNA, which contributes to the extent of DNA bending induced by the protein (5). The DNA interactions of the HMG-box domains, which occur in different plant, vertebrate, insect, and yeast HMGB proteins, are modulated by basic and acidic domains flanking the DNA-binding motif (4). HMGB proteins act as architectural components in chromatin facilitating the assembly of nucleoprotein complexes, which are involved, for instance, in the regulation of transcription and recombination (2, 4).In contrast to other eukaryotes, which usually have two or three different HMGB proteins, (higher) plants contain several HMGB proteins (Ն5 family members). The plant HMGB proteins have a single HMG-box domain, which is flanked by a basic N-terminal domain and an acidic C-terminal domain (6). Although the amino acid sequences of the HMG-box domains o...

Components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex and other nucleic acid-binding proteins are subject to methylation on specific arginine residues by the catalytic activity of arginine methyltransferases. The methylation has been implicated in transcriptional regulation and RNA and protein trafficking and signal transduction, but the mechanism by which these functions are achieved has remained undetermined. We show here that the predominant arginine methyltransferase in human cells, protein arginine methyltransferase 1 (PRMT1), is associated with hnRNP complexes, dependent on the methylation status of the cell, and that it methylates its preferred substrates in situ. Binding of PRMT1 occurs through physical interaction with scaffold attachment factor A (SAF-A), also known as hnRNP-U, which is quantitatively methylated by PRMT1 in all investigated cell lines as determined by a novel, highly specific, methylation-sensitive antibody.