Modification by SUMOylation Controls Both the Transcriptional Activity and the Stability of Delta-Lactoferrin

Abstract

Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability.

Regulation of transcription factors by sumoylation

“…Acetylation at this residue was shown to preclude sumoylation and thereby promote transcriptional activation on a delta-lactoferrin responsive reporter gene. 27 Competition between SUMO and Lys-based ubiquitin modifications has also been observed, usually influencing TF stability (discussed below), but the competition between sumoylation and acetylation often represents a switch between active and inactive forms of modified TFs, with SUMO typically acting as the “off” switch. 26-28 …”

“… 55 For delta-lactoferrin and the homeodomain TF ZHX1, sumoylation and ubiquitination are also thought to compete for modification of the same Lys residues, with sumoylation favoring stability of the TFs by blocking ubiquitin-mediated proteolysis. 27,56 In other cases, TF sumoylation inhibits ubiquitination with a consequential increase in TF stability, but whether the modifications compete for the same Lys residues is not yet known (e.g., refs. 57-59 ).…”

Dual Regulation of Host TRAIP Post-translation and Nuclear/Plasma Distribution by Porcine Reproductive and Respiratory Syndrome Virus Non-structural Protein 1α Promotes Viral Proliferation

“…There is crosstalk between SUMOylation and other post-translational modifications (56), and SUMOylation can compete with ubiquitination for substrate lysine residues to prevent proteasome degradation and ensure its stability (57, 58). However, some studies have shown that substrate proteins modified by SUMO as labeled molecules can recruit SUMO-targeted ubiquitin ligases (STUbL) to mediate subsequent ubiquitination degradation (50, 59, 60).…”

The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97

“…Negative crosstalk may result from the direct competition of two PTMs for the same amino acid or from indirect effects due to one specific PTM masking the recognition site of a second PTM (Hunter, 2007 ). For example, direct competition exists between SUMOylation, ubiquitylation, phosphorylation, and acetylation with ubiquitylation/SUMOylation and SUMOylation/acetylation being mutually exclusive, while SUMOylation/phosphorylation can be agonistic or antagonistic depending on the substrate in question (Escobar-Ramirez et al, 2015 ). Furthermore, the combination of different PTMs on a protein generates a highly regulated interface which may be recognized by specific effector proteins resulting in the controlled initiation of downstream signaling events and facilitating the interactions with diverse binding partners (Sims and Reinberg, 2008 ), thus explaining, for example, how p97 can participate in such a large variety of different cellular functions.…”