Dynamic and Differential in Vivo Modifications of the Isoform HMGA1a and HMGA1b Chromatin Proteins

Abstract

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between nonmalignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from nonmetastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.Overexpression of the HMGA1 gene (formerly known as HMGIY (1)) is such a consistent feature of tumors that it has been suggested to be a "diagnostic" biochemical marker of both neoplastic transformation and cancer progression (2-8). Elevated levels of HMGA1 gene products and HMGA1 proteins have been observed in almost every cancer type investigated and are correlated with increasing degrees of malignancy and metastatic potential of a number of cancer types (6, 9, 10).Additionally, it was demonstrated that overexpression of HMGA1 proteins in "normal" rat 1a cells and a human breast adenocarcinoma cell line caused neoplastic transformation and malignant metastatic progression of these cells, respectively (11,12). Together, these results demonstrate that overexpression of full-length HMGA1 proteins in tumor cells is extremely widespread and biologically important.The HMGA1a (formerly known as HMG-I) and HMGA1b (formerly known as HMG-Y) isoform proteins are architectural transcription factors that are derived from alternatively spliced mRNA transcripts coded for by the HMGA1 gene located on human chromosome 6 (locus 6p21) with HMGA1b (95 amino acids; ϳ10.6 kDa) containing an internal 11-amino acid deletion compared with HMGA1a (106 amino acids; ϳ11.5 kDa) (13). Both isoforms contain three independent DNA-bin...

High mobility group proteins and their post-translational modifications

“…Methylation-Among the PTMs of HMGA proteins, methylation was the most recently reported [81,[86][87][88][89] and it was strictly related to the execution of programmed cell death [87,88]. In the studied cell systems, including human leukemia, human prostate tumor and rat thyroid transformed cells, only HMGA1a was found to be methylated and the methylation level was increased during apoptosis [88].…”

“…Acetylation-Other than phosphorylation, HMGA1 proteins are also post-translationally acetylated on a couple of lysine residues in vivo [80][81][82][83], and the N-termini of HMGA1a and HMGA1b proteins are constitutively acetylated in vivo [80]. The ordered acetylation of lysine residues in HMGA1 proteins governed the accurate execution of the interferon-β transcriptional switch [84].…”

The HMGA proteins: A myriad of functions (Review)

“…This allows the different HMGA proteins to establish interactions with alternatively spaced AT-rich DNA stretches. From studies concerning possible differential in vivo modifications, it is also clear that HMGA1a and HMGA1b proteins might participate in different cellular processes based on their unique collections of in vivo post-translational modifications (56,64). In the next section it will become clear that these differences in target gene selection and/or binding affinity by the HMGA family members result in different cellular functions.…”

“…Methylation of this residue appears to be modulated during apoptosis, reaching highest levels at later stages of the apoptotic process (63). Dimethylation of arginine and lysine residues on both HMGA1a and HMGA1b was found to be increased in breast cancer cells with higher metastatic potential (64,65). Sgarra et al identified HMGA1a as a target of the protein arginine methyltransferase PRMT6, which specifically methylates HMGA1a on Arg 56 and Arg 58 within the second AT-hook domain (66).…”

A Mass Spectrometric Study on the in Vitro Methylation of HMGA1a and HMGA1b Proteins by PRMTs: Methylation Specificity, the Effect of Binding to AT-Rich Duplex DNA, and the Effect of C-Terminal Phosphorylation

“…Considering that HMGA1 proteins can bind to AT-rich DNA stretches in vitro and undergo conformational change upon DNA binding (2,9), we also investigated the in vitro methylation of rhHMGA1 proteins in the presence of duplex DNA. Multiple forms of PTMs, such as phosphorylation, acetylation, and methylation, are often observed simultaneously in HMGA1 proteins under various physiological conditions in vivo (13,39,40). However, the correlation among different PTMs of HMGA1 proteins remains unknown, though the existence of a PTM "code" for HMGA1 proteins has been proposed (38).…”