At least three major mitotic processes are regulated by the PP2A-B56 phosphatase complex: the Spindle Assembly Checkpoint (SAC), kinetochore-microtubule attachments and sister chromatid cohesion. We show here that these key functions of PP2A-B56, which require its localization to either the kinetochore or centromere, are split between distinct subsets of B56 isoforms. PP2A-B56γ and PP2A-B56δ localize to the outer kinetochore (via BUBR1), whereas PP2A-B56α and PP2A-B56ε localize to the centromere (via Sgo2). The differential localization observed is due to a difference in affinity for the receptors: PP2A-B56γ has a reduced affinity for Sgo2 compared to PP2A-B56α and, vice versa, PP2A-B56α has a reduced affinity for BUBR1 compared to PP2A-B56γ. Given that the known binding interfaces for both BUBR1 and Sgo2 are highly conserved in all B56 isoform, we generated a series of chimeras between B56α and B56γ to uncover isoform specific interactions. This led to the identifications of two distinct regions within B56α and B56γ that regulate the binding to Sgo2 and BUBR1. Furthermore, site directed mutagenesis has revealed that proper holoenzyme assembly has a role in regulating the localization of B56: it is needed for centromeric accumulation and it interferes with kinetochore accumulation of B56α. We will present a model to explain how this differential localization could be linked to post-translational modifications of PP2AC. Together, these results help to clarify how individual PP2A-B56 isoforms achieve subcellular specificity during mitosis.

Dictyostelium discoideum is a soil dwelling amoeba which has been widely used as a model organism to study cellular processes such as signal transduction, chemotaxis, endocytosis and exocytosis. The process of phagocytosis in Dicytostelium is largely comparable to that of neutrophils and macrophages in the mammalian system. Neutrophils and macrophages are cells of the innate immune system and they engulf infectious bacteria through phagocytosis. Dictyostelium cells uptake yeast and bacteria for their nutrition through phagocytosis, which is an actin dependent mechanism and is a target of multiple signaling inputs. Recent studies have uncovered different proteins involved in the signaling of particle and further studies are required to decipher the intricate mechanism leading to the F-actin rearrangement. Two of the proteins have previously known to be involved in the pathways regulating the F- actin rearrangement name PP2A phosphatase and LKB1 kinase The main objective of this project was to determine how these proteins are affecting the two actin driven particle uptake processes, phagocytosis and fluid uptake. We showed that ablation of PsrA gene which codes the regulatory subunit of PP2A resulted in a defective phagocytosis, whereas the fluid uptake was normal. We also showed for the first time that there was an increase in the phosphorylation of some of the PKB substrate proteins in wild type cell. Cells lacking PsrA gene displayed an aberrant phosphorylation of PKB substrate protein when compared to the wild type cells further confirming the involvement of PKB substrate in phagocytosis. Further, we looked at the effects of LKB1 kinase on phagocytosis by using a LKB1 knockdown construct introduced into wild type cells. The knock down of LKB1 resulted in a higher rate of phagocytosis while introduction of a LKB1 over expressing construct severally decreased the rate of phagocytosis indicating an inhibitory effect of LKB1. Furthermore there was an increase in the PKB substrate protein but a different pattern compared to the psrA- cells. We also carried out adhesion assays on LKB1 knockdown cells and the results showed a higher substrate adhesion as compared to the wild type cells, while psrA- cells had no adhesion defect.