Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity

Abstract

The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook.

Figure 1

Product-ion spectra of the ESI-produced [M + 2H]²⁺ ions of tryptic peptides of (a) KTTTT₇₇PGR (m/z 472.5), (b) EPSEVPT₅₂PK (m/z 532.4), and (c) KQPPVS₃₅PGTALVGSQK (m/z 837.5) in HMGA1a that was phosphorylated by HIPK2. The phosphorylated residues are highlighted in gray italic fonts in the peptide sequences. An asterisk (*) indicates that a fragment ion bears a phosphate group. A scheme summarizing the observed fragment ions for each peptide is shown on the top right of each panel.

Figure 2

Histograms showing that HIPK2 and cdc2 exhibit different site preferences on the in-vitro phosphorylation of HMGA1a. Phosphorylations mediated by HIPK2 (“pA1a_HIPK2”) and cdc2 (“pA1a_cdc2”) are represented by black and shaded bars, respectively. The level of phosphorylation of each modified residue was quantified by using LC-MS/MS (see Materials and Methods). The data represent the means and standard deviations of results from three independent measurements.

Figure 3

HIPK2 modulates HMGA1a’s binding affinity towards human germ line promoter. (a) EMSA of HMGA1a (control, lanes 2-4), HIPK2-phosphorylated HMGA1a (lanes 6-8) and HMGA1a in the presence of HIPK2 but not ATP (lanes 9-11). “A1a” and “pA1a_HIPK2” represent recombinant and HIPK2-phosphorylated HMGA1a proteins, respectively. (b) Quantification results based on triplicate measurements of the control recombinant HMGA1a (in shaded bar) and HMGA1a in the presence of HIPK2 (in black bar) at different protein concentrations. For the EMSA results of pA1a_HIPK2 shown in lanes 9-11, HIPK2 was also present in the binding reaction mixture.

Figure 4

(a) EMSA for the binding of recombinant HMGA1a (lanes 1-2), cdc2-phosphorylated HMGA1a (lanes 3-4) and HIPK2-phosphorylated HMGA1a (lanes 5-6) to human germ line ε promoter. (b) Quantitative results from triplicate measurements from (a) of HMGA1a (“A1a” in white bar), HIPK2-phosphorylated HMGA1a (“pA1a_HIPK2” in black bar) and cdc2-phosphorylated HMGA1a (“pA1a_cdc2” in shaded bar) at different protein concentrations. To exclude the interference introduced by the presence of either HIPK2 or cdc2, these kinases were removed prior to carrying out the EMSA experiments.

Figure 5

Diagram of human HMGA1a and HMGA1b showing the phosphorylated residues (in bold) that have been detected in vivo in human cells and the corresponding kinases that could be responsible for the phosphorylation of these residues in-vivo. The three AT-hooks, functional motifs binding to DNA, are highlighted in black.