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 non-malignant 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 non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.
Highlights
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]
We report the first observed differential in vivo modifications on the HMGA1a and HMGA1b proteins isolated from the same cell type, and we demonstrate that the post-translational modifications (PTMs) patterns found on these isoforms differ in non-metastatic and metastatic MCF-7 cells
Diverse Post-translationally Modified HMGA1 Isoform Protein Populations within Human MCF-7 Mammary Epithelial Cells—As illustrated in Fig. 1, the endogenous HMGA1a and HMGA1b proteins were isolated from the MCF-7/Tet-Off cells and scanned with MALDI-TOF MS to examine their PTMs
Summary
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]. 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]. Direct confirmation that HMGA1a and HMGA1b could have different functions in vivo was subsequently obtained when one or the other of these isoforms was overexpressed as a tetracycline-regulated transgene in human MCF-7 mammary epithelial tumor cells [12]. Western blot analysis of proteins being expressed in these cells further confirmed the microarray results, demonstrating that the HMGA1a and HMGA1b isoforms differentially regulate specific genes in the transgenic MCF-7 mammary epithelial cells [12]. The present knowledge of the types and patterns of in vivo PTMs found on HMGA1 proteins in different cell types and in the same cells under different physiological conditions is quite limited, and must be better characterized before the biological significance of such modifications can be fully and critically assessed
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