Identification of protein-protein interactions at the nuclear envelope

Abstract

The nuclear envelope (NE) is involved in numerous biological processes, including cellular compartmentalization, control of nucleocytoplasmic transport, genome organization, DNA replication, repair and transcription. Reflecting the diversity and complexity of these processes, several hundreds proteins are reported to accumulate at the NE. Interactions between NE components are incompletely mapped, which hinders full understanding of NE biogenesis and functions. Phosphorylation of NE proteins is critical for NE breakdown at entry to mitosis. We previously described VRK1 (Vaccinia-Related Kinase 1) to phosphorylate BAF, which is involved in anchoring of chromatin to the NE via nuclear lamins and inner nuclear membrane proteins, such as emerin and LEM2. VRK1 is known to phosphorylate other chromatin proteins, like histones H2A and H3 as well as the transcription factors p53, c-Jun, ATF2 and CREB. In the absence of VRK1, BAF remains chromosome-bound upon mitotic entry and defects in chromosome segregation are observed. VRK1 is also expressed in postmitotic cells and to identify novel partners we immunoprecipitated VRK1 from human cells followed by mass spectrometry analysis. The top candidates include several NE and chromatin-associated proteins, including BAF and emerin. Currently, we are applying a variety of methods, such as yeast two-hybrid assays and surface plasmon resonance technology to verify these interactions. In addition, we developed and compared mono- and bicistronic systems to study protein-protein interactions in vivo by protein fragment complementation assays based on BiFC and NanoLuc technologies. This system enables us to test for VRK1 interactions at physiological expression levels and subcellular resolution, but can be adapted to any other protein pair.