Differential in vivo modifications of the HMGI(Y) nonhistone chromatin proteins modulate nucleosome and DNA interactions

Abstract

The HMGI(Y) family of "high mobility group" nonhistone proteins are architectural transcription factors whose overexpression is highly correlated with both cancerous transformation and increased malignancy and metastatic potential of tumors in vivo. Here we report on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. The MCF-7 cell line exhibits many characteristics of normal breast epithelial cells and does not form tumors when injected into nude mice, whereas the MCF-7/PKC-alpha cell line, a derivative of MCF-7 that expresses a transgene coding for the enzyme protein kinase C-alpha (PKC-alpha), is both malignant and highly metastatic. Using MALDI mass spectrometry, we show that the HMG-Y protein is more highly modified than the HMG-I protein in both the MCF-7 and the MCF-7/PKC-alpha cells. Significantly, the HMG-Y protein isolated from the highly metastatic MCF-7/PKC-alpha cells possesses a unique constellation of phosphorylations, methylations, and acetylations not found on the HMG-I protein isolated from either the MCF-7 or MCF-7/PKC-alpha cells. We further demonstrate that some of the same amino acid residues phosphorylated on recombinant HMGI(Y) proteins by purified PKC in vitro are also phosphorylated on the HMG-I(Y) proteins isolated from MCF-7/PKC-alpha cells, suggesting that PKC phosphorylates these proteins in vivo. Quantitative substrate binding analyses indicate that the biochemical modifications present on the HMG-I and HMG-Y proteins differentially influence the ability of these proteins to interact with both A.T-rich DNA substrates and nucleosome core particles in vitro, suggesting a similar modulation of such binding affinities in vivo. To our knowledge, this is the first demonstration of differences in the types of in vivo biochemical modifications found on the HMG-I and HMG-Y proteins in cells and also the first experimental evidence suggesting a possible linkage between such posttranslational modifications and the neoplastic potential of cells.