FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF PALB2-ROCK2 INTERACTION

INTRODUCTION AND OBJECTIVES: PALB2 is a tumor suppressor gene that encodes for a homonymous protein with a pivotal role in the homologous recombination (HR) DNA repair pathway, being essential for genomic integrity maintenance. PALB2 acts as a scaffold connecting BRCA1 and BRCA2 to promote the recruitment of RAD51 recombinase to double-strand break (DSB) sites, allowing the DNA’s faithful repair. Previously, our group identified ROCK2, a serine/threonine kinase protein, as a new PALB2 interactor (data not published). Among other functions, ROCK2 acts together with BRCA2 in regulating centrosome duplication, a cellular process in which BRCA1 participates through another protein complex. Our group also identified the presence of PALB2 at centrosomes using confocal microscopy, suggesting a novel function for this protein. Therefore, this study aims to map and characterize the PALB2-ROCK2 interaction and to evaluate the PALB2 putative role in the centrosome’s biology. MATERIAL AND METHODS: Different regions (fragments) of both PALB2 and ROCK2 proteins are under investigation for their relevance to the PALB2-ROCK2 interaction through GST-pulldown routines in HEK293FT cell line. We will also evaluate whether DNA damage and phosphorylation regulate PALB2-ROCK2 interaction through GST-pulldown. PALB2 and ROCK2 colocalization will be assessed in MCF7 cell line using confocal microscopy. Furthermore, to investigate the role of PALB2 in centrosome duplication, the PALB2 expression will be silenced and its impact will be assessed by centrosomes numbers quantification throughout cell cycle phases and examining mitotic spindle integrity using confocal microscopy. RESULTS AND CONCLUSION: As preliminary results, we have already generated constructs that encode four distinct fragments of PALB2 and two different variants, to be used in PALB2-ROCK2 interaction mapping. First experiments showed that PALB2 interacts with ROCK2 full length (ROCK2_FL) through its N and C-terminus. Moreover, both PALB2 p.L35P (a missense variant positioned in the coiled-coil (CC) motif with dysfunctional BRCA1 interaction) and a CC deleted variant (Δcc) showed increased interaction with ROCK2_FL when compared with the wild-type protein. To complete the set of constructs to be used in PALB2-ROCK2 interaction mapping approach, coding constructs for ROCK2 fragments are being generated. The full scope of PALB2’s involvement in cellular processes, particularly beyond the homologous recombination pathway, remains incompletely understood. Identifying novel interaction partners of PALB2 represents a strategic approach toward understanding its function more comprehensively, potentially unveiling its involvement in additional cellular mechanisms.