Anti-UBR5 (aa 2677-2774) polyclonal antibody

Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5, with the majority of mutations within the HECT domain of the protein that can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B-cell maturation and activation, we generated a conditional mutant disrupting UBR59s C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and upregulation of proteins associated with messenger RNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B-cell maturation by stabilization of spliceosome components during B-cell development and suggests UBR5 mutations play a role in MCL transformation.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs most of the serine- and threonine-dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of serine and threonine (Ser-Thr) protein kinases. PP1 gains substrate specificity through binding to a large number (>200) of regulatory proteins that control PP1 localization, activity, and interactions with substrates. PP1 recognizes the well-characterized RVxF binding motif that is present in many of these regulatory proteins, thus generating a multitude of distinct PP1 holoenzymes. We showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), was phosphorylated specifically during mitosis and that this phosphorylation event abrogated the interaction of PP1 with the regulatory protein. We determined that this phosphorylation was primarily governed by the mitotic protein kinase Aurora B and that high phosphorylation site stoichiometry of these sites maintained the phosphorylation of PP1 substrates during mitosis by disrupting the assembly of PP1 holoenzymes. We generated an antibody that recognizes the phosphorylated form of the RV[S/T]F motif (RVp[S/T]F) and used it to identify known PP1 regulatory proteins (KNL1, CDCA2, and RIF1) and multiple proteins that could potentially act as PP1 binding partners (UBR5, ASPM, SEH1, and ELYS) governed by this mechanism. Together, these data suggest a general regulatory mechanism by which the coordinated activities of Aurora B and PP1 control mitotic progression.