SUMOylation of the hepatoma-derived growth factor negatively influences its binding to chromatin.

Hepatoma-derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma-derived growth factor, its routes of secretion and internalization or post-translational modifications. In the present study, we show for the first time that hepatoma-derived growth factor is modified by the covalent attachment of small ubiquitin-related modifier 1 (SUMO-1), a post-translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI-TOF-MS based peptide analysis, we identified the lysine residue SUMOylated located in the N-terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma-derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma-derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma-derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma-derived growth factor.

Ubc9 fusion-directed SUMOylation identifies constitutive and inducible SUMOylation.

Constitutive and induced protein SUMOylation is involved in the regulation of a variety of cellular processes, such as regulation of gene expression and protein transport, and proceeds mainly in the nucleus of the cell. So far, several hundred SUMOylation targets have been identified, but presumably they represent only a part of the total of proteins which are regulated by SUMOylation. Here, we used the Ubc9 fusion-dependent SUMOylation system (UFDS) to screen for constitutive and induced SUMOylation of 46 randomly chosen proteins with proven or potential nuclear localization. Fourteen new UFDS-substrate proteins were identified of which eight could be demonstrated to be SUMOylated in a UFDS-independent manner in vivo. Of these, three were constitutively SUMOylated (FOS, CRSP9 and CDC37) while the remaining five substrates (CSNK2B, TAF10, HSF2BP, PSMC3 and DRG1) showed a stimulation-dependent SUMOylation induced by the MAP3 kinase MEKK1. Hence, UFDS is appropriate for the identification and characterization of constitutive and, more importantly, induced protein SUMOylation in vivo.

Ubc9 fusion-directed SUMOylation (UFDS): a method to analyze function of protein SUMOylation.

Although small ubiquitin-like modifier (SUMO) is conjugated to proteins involved in diverse cellular processes, the functional analysis of SUMOylated proteins is often hampered by low levels of specific SUMOylated proteins in the cell. Here we describe a SUMO-conjugating enzyme (Ubc9) fusion-directed SUMOylation (UFDS) system, which allows efficient and selective in vivo SUMOylation of proteins. Although SUMOylation of overexpressed p53 and STAT1 was difficult to detect in HEK293 cells, up to 40% of p53 and STAT1 were conjugated with endogenous SUMO when fused to Ubc9. We verified the specificity of UFDS using SUMOylation-site mutants and showed that the method is not dependent on SUMO ligases. Using UFDS we demonstrated that SUMOylation of STAT1 inhibits its phosphorylation at Tyr701 and discovered p53 multi-SUMOylation in vivo. We propose that UFDS will be useful for the analysis of function of SUMOylation in protein interactions, subcellular localization as well as enzymatic activity.