A phospho-proteomic characterization of the cellular binding partners of the HIV tether LEDGF/p75 and their regulation by CK2

Lens Epithelium Derived Growth Factor/p75 (LEDGF/p75) is a transcriptional co-activator tethering proteins and protein complexes to active chromatin. The C-terminus of LEDGF/p75 comprises the Integrase Binding Domain or the IBD that facilitates its tethering activity. This domain is seized by the Human Immunodeficiency Virus (HIV) to allow the integration of the virus into active chromatin. Our lab developed the first-in-class small molecules, termed LEDGINs, inhibiting the LEDGF/p75-HIV interaction which are currently in early clinical development (Christ et. al., 2010). In addition to HIV integrase, LEDGF/p75 also tethers several disease-specific and cell cycle-oriented factors in a similar fashion. Using NMR spectroscopy and protein interaction assays, we defined the interaction profile of LEDGF/p75 with its cellular binding partners MLL, JPO2, IWS1, MED1 and PogZ. Association of LEDGF/p75 with JPO2 (involved in Medulloblastoma) and MLL (mixed-lineage leukemia) makes it an interesting target of study from a therapeutic perspective. These data show that a very short intrinsically disordered but conserved IBD-binding motif (IBM), present in these unrelated proteins, is responsible for bringing about the interaction with LEDGF/p75. We also show that this interaction can be modulated by post-translational modifications, specifically CK2-driven phosphorylation. The importance of these phospho-sites were verified in vitro through mutational analysis. Based on IBM conservation, we validated the effect of phosphorylation on LEDGF/p75 and its cellular binding partners.status: publishe