A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling

A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis

A complex of BRCA2 and PP2A-B56 is required for DNA repair by homologous recombination

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers

Regulation of Protein Phosphatase One During Cell Cycle

Protein phosphatase 1 (PP1) is a highly conserved enzyme that controls the majority of serine/threonine (Ser/Thr) dephosphorylation reactions in eukaryotes. PP1 gains substrate specificity through binding to a large number (> 200) of regulatory proteins, which control PP1 localization, activity, and substrate interaction. PP1 recognizes the majority of these regulatory proteins via well-characterized RVxF binding motif generating hundreds of distinct PP1 holoenzymes. The main objective of this research was to uncover the regulatory mechanisms that govern the interaction of PP1 with its regulatory proteins during the cell cycle. The progression of cell cycle is largely governed by reversible protein phosphorylation. I showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), are phosphorylated specifically during mitosis and that this phosphorylation event abrogates the interaction of PP1 with the regulatory protein. This phosphorylation is primarily governed by mitotic protein kinase Aurora B and is crucial to maintain phosphorylation of PP1 substrates during mitosis. In addition, I showed that PP1 itself dephosphorylates RVp[S/T]F motifs during mitotic exit, which allows the phosphatase to re-associate with the regulatory proteins and dephosphorylate other mitotic substrates. To gain further insight into the regulation of PP1 function in cell cycle, I characterized the novel cell cycle dependent interactome of PP1. Using quantitative mass spectrometry, I identified 113 novel RVxF containing potential PP1 binding partners including 17 mitosis-specific partners. Furthermore, using immunoblotting, I validated 9 of the PP1 interactions both in asynchronous and mitotic populations with proteins involved in cell cycle regulation (Aurora B, Aurora A, TPX2, CDCA2 (RM), TACC3, GCN2, DBC1, BRCA1 and RIF1). In addition, I demonstrated a novel interaction of PP1 with centrosomal protein, CEP192 via its ‘KHVTF’ motif. The work presented here expands our understanding of the regulation of PP1 in the cell cycle, and also suggests a novel regulatory mechanism by which the coordinated activities of Aurora B kinase and PP1 drive mitotic progression, which is crucial to maintain the genomic stability

Coordination of Protein Kinase and Phosphoprotein Phosphatase Activities in Mitosis

Dynamic changes in protein phosphorylation govern the transitions between different phases of the cell division cycle. A “tug of war” between highly conserved protein kinases and the family of phosphoprotein phosphatases (PPP) establishes the phosphorylation state of proteins, which controls their function. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells, with the highest occupancy of phosphorylation sites seen in mitosis. Spatial and temporal regulation of opposing kinase and PPP activities is crucial for accurate execution of the mitotic program. The role of mitotic kinases has been the focus of many studies, while the contribution of PPPs was for a long time underappreciated and is just emerging. Misconceptions regarding the specificity and activity of protein phosphatases led to the belief that protein kinases are the primary determinants of mitotic regulation, leaving PPPs out of the limelight. Recent studies have shown that protein phosphatases are specific and selective enzymes, and that their activity is tightly regulated. In this review, we discuss the emerging roles of PPPs in mitosis and their regulation of and by mitotic kinases, as well as mechanisms that determine PPP substrate recognition and specificity

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset