In Vivo Posttranslational Modifications of the High Mobility Group A1a Proteins in Breast Cancer Cells of Differing Metastatic Potential

Abstract

The high mobility group (HMG) proteins are important modulators of chromatin structure and gene transcription. Overexpression of HMGA1 proteins in vivo induces neoplastic transformation and promotes a highly metastatic cellular phenotype. This study focuses on characterization of HMGA1a in vivo posttranslational modification (PTM) patterns found in a nonmetastatic and two metastatic lines of MCF-7 human breast cancer cells of differing tumorigenic potential. PTM types and the amino acids on which they occur were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Mass analysis was restricted to MALDI peaks having less than +/-150 parts per million (ppm) error, thereby holding our analysis to a more stringent criterion than previously published work with the HMG proteins. Validation of MALDI-TOF MS analysis was accomplished utilizing electrospray ionization tandem mass spectrometry (ESI MS/MS) and manual analysis of ion fragmentation spectra. Patterns and sites of PTMs identified in this study suggest that HMGA1a proteins, like the histones, exhibit a biochemical modification "code" that relates to cellular function. For example, both increased levels of acetylation and a previously unidentified dimethylation of both lysine and arginine residues were found on HMGA1a proteins from metastatic cells compared to proteins found in their nonmetastatic precursors. Additionally, the types of modification present on lysine-45 (e.g., unmodified, acetylation, or dimethylation) varied, depending on the metastatic potential of cells. These findings suggest that examination of the PTM patterns on HMGA1 proteins may provide valuable information concerning the physiological and phenotypic state of mammalian cells.

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

“…Arg 25 , localized within the first AT-hook of HMGA1a, has been shown to be a major site of modification in tumor cells (11), but other Arg residues, in addition to those reported in this paper, have been found methylated in vivo (9,10). In this study, we were unable to identify other PRMTs, except PRMT6, that modify HMGA1a protein.…”

“…More recently, dimethylation on the same Arg 25 residue has been reported by another group (21). An extensive study on both HMGA1a and HMGA1b proteins reported a high level of PTMs, and in particular, dimethylation of arginine and lysine residues was increased in breast cancer cells with higher metastatic potential (9,10). Altogether, these data lead authors to suggest the existence of a PTM "code" for HMGA proteins similar to that reported for histones (10,11).…”

“…An extensive study on both HMGA1a and HMGA1b proteins reported a high level of PTMs, and in particular, dimethylation of arginine and lysine residues was increased in breast cancer cells with higher metastatic potential (9,10). Altogether, these data lead authors to suggest the existence of a PTM "code" for HMGA proteins similar to that reported for histones (10,11). Although the kinases and acetyltransferases that are able to modify HMGA proteins have been identified, enzymes responsible for arginine and lysine methylation are just starting to be discovered (22).…”

“…Indeed, it has been shown that acetylation of Lys 59 in HMGA1b (which corresponds to Lys 70 of the isoform HMGA1a) was required for HMGA1b-induced apoptosis in normal rat thyroid cells (47). Arginine methylation in HMGA1a has been found to correlate with apoptosis and tumor progression (10,11). It is therefore possible that this PTM can play a direct role in modulating the HMGA1a function in these two processes.…”

A quantitative study on the in vitro and in vivo acetylation of high mobility group A1 proteins

“…Reversible acetylation of Lys-64 and Lys-70 modulates the transcription of IFN-␤ gene by regulating the destabilization and formation, respectively, of an enhanceosome on the promoter region of the°IFN-␤°gene° [26,°27].°Lys-64°and°Lys-70°in°HMGA1a were found to be acetylated in nonmetastatic and moderately metastatic breast cancer cells such as MCF-7/Tet-off cells and HA7C cells, but not in highly metastatic°HA8A°breast°cancer°cells° [19].°In°addition,°Lys-14 in HMGA1 proteins was observed to be acetylated in PC-3° [28]°and°HA7C°cells° [19],°whereas°the°same residue was monomethylated in HMGA1a in nonmetastatic MCF-7/Tet-off cells. The biological implications of the acetylation of these lysine residues remain to be established.…”